RESUMEN
Neuroinflammation is the hallmark of several infectious and neurodegenerative diseases. Synthetic glucocorticoids (GCs) are the first-line immunosuppressive drugs used for controlling neuroinflammation. A delayed diffusion of GCs molecules and the high systemic doses required for brain-specific targeting lead to severe undesirable effects, particularly when lifelong treatment is required. Therefore, there is an urgent need for improving this current therapeutic approach. The intranasal (i.n.) route is being employed increasingly for drug delivery to the brain via the olfactory system. In this study, the i.n. route is compared to the intravenous (i.v.) administration of GCs with respect to their effectiveness in controlling neuroinflammation induced experimentally by systemic lipopolysaccharide (LPS) injection. A statistically significant reduction in interleukin (IL)-6 levels in the central nervous system (CNS) in the percentage of CD45+ /CD11b+ /lymphocyte antigen 6 complex locus G6D [Ly6G+ and in glial fibrillary acidic protein (GFAP) immunostaining was observed in mice from the i.n.-dexamethasone (DX] group compared to control and i.v.-DX-treated animals. DX treatment did not modify the percentage of microglia and perivascular macrophages as determined by ionized calcium binding adaptor molecule 1 (Iba1) immunostaining of the cortex and hippocampus. The increased accumulation of DX in brain microvasculature in DX-i.n.-treated mice compared with controls and DX-IV-treated animals may underlie the higher effectiveness in controlling neuroinflammation. Altogether, these results indicate that IN-DX administration may offer a more efficient alternative than systemic administration to control neuroinflammation in different neuropathologies.
Asunto(s)
Corteza Cerebral , Hipocampo , Lipopolisacáridos/toxicidad , Microglía , Enfermedades Neurodegenerativas , Administración Intranasal , Animales , Antígenos Ly/inmunología , Antígeno CD11b/inmunología , Proteínas de Unión al Calcio/inmunología , Corteza Cerebral/inmunología , Corteza Cerebral/patología , Proteína Ácida Fibrilar de la Glía/inmunología , Hipocampo/inmunología , Hipocampo/patología , Interleucina-6/inmunología , Antígenos Comunes de Leucocito/inmunología , Masculino , Ratones , Proteínas de Microfilamentos/inmunología , Microglía/inmunología , Microglía/patología , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/inmunología , Enfermedades Neurodegenerativas/patologíaRESUMEN
BACKGROUND: Neuroinflammation (NI) is a key feature in the pathogenesis and progression of infectious and non-infectious neuropathologies, and its amelioration usually improves the patient outcome. Peripheral inflammation may promote NI through microglia and astrocytes activation, an increased expression of inflammatory mediators and vascular permeability that may lead to neurodegeneration. Several anti-inflammatory strategies have been proposed to control peripheral inflammation. Among them, electrical stimulation of the vagus nerve (VNS) recently emerged as an alternative to effectively attenuate peripheral inflammation in a variety of pathological conditions with few side effects. Considering that NI underlies several neurologic pathologies we explored herein the possibility that electrically VNS can also exert anti-inflammatory effects in the brain. METHODS: NI was experimentally induced by intraperitoneal injection of bacterial lipopolysaccharide (LPS) in C57BL/6 male mice; VNS with constant voltage (5 Hz, 0.75 mA, 2 ms) was applied for 30 s, 48 or 72 h after lipopolysaccharide injection. Twenty four hours later, pro-inflammatory cytokines (IL-1ß, IL-6, TNFα) levels were measured by ELISA in brain and spleen extracts and total brain cells were isolated and microglia and macrophage proliferation and activation was assessed by flow cytometry. The level of ionized calcium binding adaptor molecule (Iba-1) and glial fibrillary acidic protein (GFAP) were estimated in whole brain extracts and in histologic slides by Western blot and immunohistochemistry, respectively. RESULTS: VNS significantly reduced the central levels of pro-inflammatory cytokines and the percentage of microglia (CD11b/CD45low) and macrophages (CD11b/CD45high), 24 h after the electrical stimulus in LPS stimulated mice. A significantly reduced level of Iba-1 expression was also observed in whole brain extracts and in the hippocampus, suggesting a reduction in activated microglia. CONCLUSIONS: VNS is a feasible therapeutic tool to attenuate the NI reaction. Considering that NI accompanies different neuropathologies VNS is a relevant alternative to modulate NI, of particular interest for chronic neurological diseases.
RESUMEN
N-truncated and N-modified forms of amyloid beta (Abeta) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Abeta is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant N-truncated/modified Abeta peptide bearing amino-terminal pyroglutamate at position 3 (AbetaN3(pE)). We demonstrated that AbetaN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AbetaN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AbetaN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AbetaN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Abeta, which is absent in N-amino truncated peptides.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Vacunas contra el Alzheimer/inmunología , Péptidos beta-Amiloides/inmunología , Anticuerpos/inmunología , Epítopos Inmunodominantes/inmunología , Ácido Pirrolidona Carboxílico/inmunología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Vacunas contra el Alzheimer/química , Vacunas contra el Alzheimer/farmacología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Animales , Anticuerpos/farmacología , Especificidad de Anticuerpos/inmunología , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/patología , Humanos , Estructura Molecular , Peso Molecular , Péptidos/química , Péptidos/inmunología , Péptidos/toxicidad , Fosfatidilserinas/metabolismo , Estructura Terciaria de Proteína/fisiología , Ácido Pirrolidona Carboxílico/química , ConejosRESUMEN
Active and passive immunotherapy targeted at the amyloid-beta (Abeta) peptide has been proposed as therapeutic approach against Alzheimer's disease (AD), and efforts towards the generation and application of antibody-based reagents that are capable of preventing and clearing amyloid aggregates are currently under active investigation. Previously, we selected and characterized a new anti-Abeta1-42 phage-displayed scFv antibody, designated clone b4.4, using a non-immune human scFv antibody library and demonstrated that a peptide based on the sequence of the Ig heavy chain (VH) complementarity-determining region (HCDR3) of this antibody fragment bound to Abeta1-42)and had neuroprotective potential against Abeta1-42 mediated neurotoxicity in rat hippocampal cultured neurons. In the present study, using novel computational methods and in vitro experiments we demonstrated that b4.4 binds to the central region of Abeta1-42. We also demonstrated that this scFv antibody binds to Abeta-derived diffusible ligands (ADDLs) and neutralizes the toxicity of both fibrillar and oligomeric forms of Abeta1-42 tested in vitro in SH-SY5Y cell cultures.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Regiones Determinantes de Complementariedad/farmacología , Fragmentos de Inmunoglobulinas/farmacología , Cadenas Pesadas de Inmunoglobulina/farmacología , Fármacos Neuroprotectores/farmacología , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/inmunología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regiones Determinantes de Complementariedad/química , Mapeo Epitopo , Humanos , Fragmentos de Inmunoglobulinas/química , Cadenas Pesadas de Inmunoglobulina/química , Modelos Moleculares , Fármacos Neuroprotectores/química , Oligopéptidos/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Unión ProteicaRESUMEN
Taenia solium cysticercosis is a parasitic disease frequently affecting human health and the pig industry in many developing countries. A synthetic peptide vaccine (designated S3Pvac) against porcine cysticercosis has been developed previously as an aid to interrupt transmission and has been shown to be effective. The results of the present study support the effectiveness of the vaccine under endemic field conditions. However, given the time-frame of the vaccination trial, no changes in the local levels of transmission were detectable before and after vaccination using sentinel pigs. Thus, this investigation shows the limited usefulness of single vaccination as the sole means of interrupting Taenia solium transmission in an endemic region.
Asunto(s)
Cisticercosis/veterinaria , Enfermedades de los Porcinos/prevención & control , Taenia solium/inmunología , Vacunas de Subunidad/inmunología , Animales , Cisticercosis/epidemiología , Cisticercosis/inmunología , Cisticercosis/prevención & control , Enfermedades Endémicas/veterinaria , Femenino , Masculino , México/epidemiología , Vigilancia de Guardia , PorcinosRESUMEN
A library of phage-displayed human single-chain Fv (scFv) antibodies was selected against the human amyloid-beta peptide (Abeta42). Two new anti-Abeta42 phage-displayed scFvs antibodies were obtained, and the sequences of their V(H) and Vkappa genes were analyzed. A synthetic peptide based on the sequence of Ig heavy chain (V(H)) complementarity-determining region (HCDR3) of the clone with the highest recognition signal was generated and determined to bind to Abeta42 in ELISA. Furthermore, we showed for the first time that an HCDR3-based peptide had neuroprotective potential against Abeta42 neurotoxicity in rat cultured hippocampal neurons. Our results suggest that not only scFvs recognizing Abeta42 but also synthetic peptides based on the V(H) CDR3 sequences of these antibodies may be novel potential candidates for small molecule-based Alzheimer's disease (AD) therapy.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Regiones Determinantes de Complementariedad/metabolismo , Región Variable de Inmunoglobulina/metabolismo , Fragmentos de Péptidos/farmacología , Animales , Anticuerpos/análisis , Anticuerpos/metabolismo , Células Cultivadas , Regiones Determinantes de Complementariedad/análisis , Humanos , Región Variable de Inmunoglobulina/análisis , Unión Proteica/fisiología , Ratas , Ratas WistarRESUMEN
The Cytochrome b (Cyt b) gene has proved to be useful for identification and classification of many mammals and plants. In order to evaluate the utility of this gene for discrimination of Leishmania parasites as well as for exploring their phylogenetic relationships, we determined the nucleotide sequences of the Cyt b gene from 13 human-infecting Leishmania species (14 strains) from the New and Old Worlds. The Cyt b genes, approximately 1080 base pairs, were found to be A/T rich, and their 5' terminal-editing regions were highly conserved. The nucleotide sequence variation among them was enough to discriminate parasite species; 245 nucleotide positions were polymorphic and 190 positions were parsimony informative. The phylogenetic relationships based on this gene, showed good agreement with the classification of Lainson & Shaw (1987) except for the inclusion of L. (L.) major in the L. (L.) tropica complex and the placement of L. tarentolae in another genus. These data show that the Cyt b gene is useful for phylogenetic study of Leishmania parasites.
Asunto(s)
Citocromos b/genética , Leishmania/enzimología , Leishmania/genética , Secuencia de Aminoácidos , Animales , Composición de Base , Secuencia de Bases , Secuencia Conservada , Citocromos b/química , ADN Protozoario/química , ADN Protozoario/genética , Variación Genética , Humanos , Leishmania/clasificación , Leishmaniasis/parasitología , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Alineación de SecuenciaRESUMEN
A single-chain fragment variable (scFv) antibody library displayed on phage was constructed using spleen cells from mice immunized with human amyloid-beta peptide (Abeta42). This first anti-Abeta42 scFv immune antibody library was selected against human Abeta42. A number of positive clones were obtained, and sequences of VH and Vkappa genes were analyzed using ExPASy and BLAST computer tools. This analysis revealed that only two unique clones with identical VH and Vkappa complementarity determining region (CDR) (except HCDR2) and identical germline genes were selected, indicating that oligoclonal immune response was occurring in Abeta42-immunized mice. Abeta42-specific scFv antibodies selected from this first immune anti-Abeta42 phage antibody library may be an important tool for the development of therapeutic molecules for Alzheimer's disease (AD).
Asunto(s)
Péptidos beta-Amiloides/inmunología , Bacteriófago M13/inmunología , Epítopos/inmunología , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Región Variable de Inmunoglobulina/aislamiento & purificación , Fragmentos de Péptidos/inmunología , Biblioteca de Péptidos , Animales , Bacteriófago M13/genética , Ensayo de Inmunoadsorción Enzimática , Genes de Inmunoglobulinas , Vectores Genéticos , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Ratones , Análisis de Secuencia de ADNRESUMEN
Three random phage display peptide libraries were screened with sera from human papillomavirus (HPV)-infected patients to characterize the specificities of antibodies present in patients' sera and to identify molecules that correspond to or mimic natural epitopes; 141 phage clones were randomly selected in three rounds of bioselection and their binding properties were analyzed in ELISA using sera from 36 patients with confirmed HPV 16 infection and 24 healthy control female blood donors. Sixteen of 36 (44%) patients' sera reacted with at least 1 phage clone, and only 2 of 24 female donors' sera showed positive reaction with 1 of the selected clones. We conclude that the combination of various disease-specific epitopes generated by screening of phage display peptide libraries may potentially lead to a multicomponent diagnostic assay for the early detection of HPV infection and precancerous cervical lesions, making possible the prevention of one of the most common cancers in women.
Asunto(s)
Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Papillomaviridae/genética , Papillomaviridae/inmunología , Infecciones por Papillomavirus/inmunología , Infecciones Tumorales por Virus/inmunología , Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Infecciones por Papillomavirus/sangre , Biblioteca de Péptidos , Infecciones Tumorales por Virus/sangreRESUMEN
Taenia solium cysticercosis seriously affects human health when localised in the central nervous system (CNS) and causes great economic loss in pig husbandry in rural areas of endemic countries. Increasing the resistance to the parasite in the obligatory host pig may help in curbing transmission. Three synthetic peptides based on protein sequences of the murine parasite Taenia crassiceps, which had previously been shown to induce protection in mice against homologous challenge, were tested as a vaccine against T. solium cysticercosis in pigs. Vaccinated and unvaccinated piglets (240 in all) were distributed in pairs among the peasants' households of two rural villages in Mexico in which 14% of the native pigs were cysticercotic. Ten to twelve months later, the effect of vaccination was evaluated at necropsy. Vaccination decreased the total number of T. solium cysticerci (98.7%) and reduced the prevalence (52.6%). The natural challenge conditions used in this field trial strengthen the likelihood of successful transmission control to both pig and human through a large-scale pig vaccination program. We believe this is a major contribution in anticysticercosis vaccine development as these rather simple yet protective peptides are potentially more cost-effective to produce and less variable in results than antigens that are more complex.
Asunto(s)
Cisticercosis/veterinaria , Enfermedades de los Porcinos/prevención & control , Taenia/inmunología , Vacunación/veterinaria , Vacunas/inmunología , Secuencia de Aminoácidos , Animales , Cisticercosis/economía , Cisticercosis/epidemiología , Cisticercosis/inmunología , Cisticercosis/prevención & control , Exposición a Riesgos Ambientales , Femenino , México/epidemiología , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Proyectos Piloto , Embarazo , Prevalencia , Salud Rural , Porcinos , Enfermedades de los Porcinos/economía , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/inmunología , Vacunación/economía , Vacunas de Subunidad/inmunología , ZoonosisRESUMEN
Taenia crassiceps recombinant antigens KETc1 and KETc12 have been shown to induce high level of protection against experimental murine T. crassiceps cysticercosis, an experimental model successfully used to test candidate antigens for use in vaccination against porcine Taenia solium cysticercosis. Based on the deduced amino acid sequence, KETc1 and KETc12 were chemically synthesized in linear form. Immunization with KETc1 induced 66.7 to 100% protection against murine cysticercosis, and immunization with KETc12 induced 52.7 to 88.1% protection. The elicited immune response indicated that both peptides contain at least one B-cell epitope (as demonstrated by their ability to induce specific antibodies) and one T-cell epitope that strongly stimulated the proliferation of T cells primed with either the free peptide or total cysticercal T. crassiceps antigens. The high percentage of spleen cells expressing inflammatory cytokines points to the likelihood of a T1 response being involved in protection. The protective capacity of the peptides and their presence in all developmental stages of T. solium point to these two epitopes as strong candidates for inclusion in a polyepitopic synthetic vaccine against T. solium pig cysticercosis.
Asunto(s)
Antígenos Helmínticos/uso terapéutico , Cisticercosis/prevención & control , Taenia/inmunología , Vacunación , Vacunas/uso terapéutico , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/inmunología , Antígenos Helmínticos/aislamiento & purificación , Cisticercosis/inmunología , Epítopos/aislamiento & purificación , Epítopos/uso terapéutico , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/uso terapéutico , Especificidad de la Especie , Bazo/citología , Bazo/inmunología , Linfocitos T/inmunologíaRESUMEN
A new type of immunogenic molecule was engineered by replacing all three complementarity-determining-region (CDR) loops of the human immunoglobulin (Ig) heavy-chain variable (V(H)) domain with the Taenia crassiceps epitope PT1 (PPPVDYLYQT) and by displaying this construct on the surfaces of M13 bacteriophage. When BALB/c mice were immunized with such phage particles (PIgphage), a strong protection against challenge infection in very susceptible female hosts was obtained. When specifically stimulated, the in vivo-primed CD4(+) and CD8(+) T cells isolated from mice immunized with PT1, both as a free peptide and as the PIgphage construct, proliferated in vitro, indicating efficient epitope presentation by both major histocompatibility complex class II and class I molecules in the specifically antigen-pulsed macrophages used as antigen-presenting cells. These data demonstrate the immunogenic potential of recombinant phage particles displaying CDR epitope-grafted Ig V(H) domains and establish an alternative approach to the design of an effective subunit vaccine for prevention of cysticercosis. The key advantage of this type of immunogen is that no adjuvant is required for its application. The proposed strategy for immunogen construction is potentially suitable for use in any host-pathogen interaction.
Asunto(s)
Antígenos Helmínticos/inmunología , Cisticercosis/prevención & control , Epítopos de Linfocito T/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Taenia/inmunología , Vacunas Sintéticas/inmunología , Animales , Antígenos Helmínticos/genética , Bacteriófago M13 , Cisticercosis/inmunología , Modelos Animales de Enfermedad , Epítopos de Linfocito T/genética , Femenino , Ingeniería Genética , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos BALB C , Biblioteca de Péptidos , Vacunación , ViriónRESUMEN
The Taenia crassiceps recombinant antigen KETc7 has been shown to be effective as a vaccine against experimental murine cysticercosis, a laboratory model used to test potentially promising molecules against porcine Taenia solium cysticercosis. Based on the deduced amino acid sequence of this proline-rich polypeptide, three fragments, GK-1, GK-2, and GK-3, were chemically synthesized in linear form. Of the three peptides, only GK-1 induced sterile protection against T. crassiceps cysticercosis in 40 to 70% of BALB/cAnN male mice. GK-1 is an 18-amino-acid peptide which contains at least one B-cell epitope, as demonstrated by its ability to induce an antibody response to the peptide and T. crassiceps antigen without need of a carrier protein. Immunofluorescence studies revealed that anti-GK1 antibodies strongly react with the native protein in the tegument of T. crassiceps and also with anatomical structures of T. solium eggs, oncospheres, cysticercus, and tapeworm. GK-1 also contains at least one T-cell epitope, capable of stimulating the proliferation of CD8(+) and to a lower extent CD4(+) T cells primed either with the free peptide or T. crassiceps total antigen. The supernatant of the stimulated cells contained high levels of gamma interferon and low levels of interleukin-4. Similar results were obtained with T cells tested for intracellular cytokine production, an indication of the peptide's capacity to induce an inflammatory response. The remarkable protection induced by GK-1 immunization, its physicochemical properties, and its presence in all developmental stages of T. solium point to this synthetic peptide as a strong candidate in the construction of a synthetic vaccine against T. solium pig cysticercosis.
Asunto(s)
Cisticercosis/inmunología , Cisticercosis/prevención & control , Taenia/inmunología , Vacunas Sintéticas/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/química , Antígenos Helmínticos/genética , Linfocitos B/inmunología , Epítopos/química , Epítopos/genética , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Inmunización , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Especificidad de la Especie , Linfocitos T/inmunología , Taenia/genética , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Seven patients with a presumptive diagnosis of osteoid osteoma located at the hip were treated with percutaneous resection of the nidus through computed tomography guidance. Histologic confirmation was obtained in 5 of the 7 patients. The average hospital stay was 27 hours. At followup, from 12 to 40 months, all patients remain asymptomatic. This procedure presents potential advantages that traditional open surgery techniques do not have.