RESUMEN
AIM: To use radioreceptor analysis for comparing substance P (SP) receptor expression in human pulp tissue samples collected from teeth having a clinical diagnosis of acute irreversible pulpitis, healthy pulps and teeth with induced inflammation. METHODOLOGY: Five pulp samples were obtained from teeth having a clinical diagnosis of acute irreversible pulpitis. Another 10 pulp samples were obtained from healthy premolars where extraction was indicated for orthodontic purposes. In five of these premolars inflammation was induced prior to pulp collection. All of the samples were processed and labelled with 125I-SP. Binding sites were identified by 125I-SP and standard SP competition assays. Kruskal-Wallis and Mann-Whitney (post-hoc) tests were used to establish statistically significant differences between the groups. RESULTS: Substance P receptor expression was found in all human pulp tissue samples. Most receptors were found in the group of pulps from teeth having a clinical diagnosis of acute irreversible pulpitis, followed by the group of pulps having induced inflammation. The least number of receptors was expressed in the group of healthy pulps. Statistical analysis revealed significant differences between the group of healthy pulp and both inflamed pulp groups (P < 0.01). CONCLUSION: Substance P receptor expression in human pulp tissue is significantly increased during inflammatory phenomena such as acute irreversible pulpitis.
Asunto(s)
Pulpitis/metabolismo , Receptores de Neuroquinina-1/biosíntesis , Adolescente , Adulto , Estudios de Casos y Controles , Pulpa Dental/metabolismo , Femenino , Humanos , Radioisótopos de Yodo , Masculino , Ensayo de Unión Radioligante , Receptores de Neuroquinina-1/análisisRESUMEN
The hepatitis B virus (HBV) core antigen (HBcAg) is a potent immunogen in animal models and humans and has been used as a carrier for several antigens; however, the mucosal immunogenicity of HBcAg has been poorly studied. In this study, we explored the immunogenicity and the immunoenhancing effect elicited by two different variants of the recombinant complete nucleocapside of HBV in mice by intranasal route. For this purpose, we used as co-administered antigen, the HBV surface protein (HBsAg) and the antibody response in sera was evaluated after each dose. To analyze the specificity of the generated antibody response, the recognition of lineal epitopes was evaluated on a cellulose membrane bearing 12 mer peptides covering the HBcAg sequence. The obtained results evidenced that the intranasal immunogenicity of both variants of HBcAg was similar and high, developing early responses of IgG. The immunoenhancing effect on the HBsAg-specific antibody response was also similar for both variants. The results of the recognition of lineal epitopes study evidenced a similar recognition pattern to all sera and vaginal lavages samples generated by the immunization of mice with the two variants of HBcAg, and also similar to a pool of human anti-HBcAg positive sera samples.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Epítopos/inmunología , Femenino , Hepatitis B/inmunología , Antígenos del Núcleo de la Hepatitis B/administración & dosificación , Antígenos de Superficie de la Hepatitis B/administración & dosificación , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/administración & dosificación , Humanos , Inmunoglobulina G/sangre , Ratones , Garrapatas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vagina/inmunologíaRESUMEN
The hepatitis B virus (HBV) core and surface antigens are potent immunogens in animal models and humans. They have been used in vaccine studies for prevention or therapy of HBV diseases and also as carrier molecules in new developments. In this study we explored the nasal immunogenicity of two different variants of the recombinant complete nucleocapsid (HBcAg) as well as their adjuvant effect on hepatitis B surface antigen (HBsAg). To characterize the immune response, the serum IgG antibody response was tested during one year against both antigens, and the serum and vaginal secretions were tested for recognition of linear epitopes of HBcAg for both HBcAg variants. The results obtained evidenced that the intranasal immunogenicity of both HBcAg variants was similar and high, developing early and long lasting IgG responses. A similar recognition pattern to all sera and vaginal washes samples was generated by the two variants of HBcAg, also similar to a pool of human anti-HBcAg positive sera. A synergistic effect in the enhancement of the immunogenicity for both antigens was evidenced in the combined formulation after nasal administration. Taken together, these results would be of interest in the design of more potent therapeutic and preventive vaccines complementing systemic and mucosal responses.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos contra la Hepatitis B/sangre , Humanos , Inmunoglobulina G/sangre , Ratones , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/inmunología , Mapeo PeptídicoRESUMEN
There are estimated to be 350 million chronic carriers of hepatitis B infection worldwide. Patients with chronic hepatitis B are at risk of liver cirrhosis with associated mortality because of hepatocellular carcinoma and other complications. An important goal, therefore, is the development of an effective therapeutic vaccine against chronic hepatitis B virus (HBV). A major barrier to the development of such a vaccine is the impaired immune response to HBV antigens observed in the T cells of affected patients. One strategy to overcome these barriers is to activate mucosal T cells through the use of nasal vaccination because this may overcome the systemic immune downregulation that results from HBV infection. In addition, it may be beneficial to present additional HBV epitopes beyond those contained in the traditional hepatitis B surface antigen (HbsAg) vaccine, for example, by using the hepatitis B core antigen (HBcAg). This is advantageous because HBcAg has a unique ability to act as a potent Th1 adjuvant to HbsAg, while also serving as an immunogenic target. In this study we describe the effect of coadministration of HBsAg and HBcAg as part of a strategy to develop a more potent and effective HBV therapeutic vaccine.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/inmunología , Hepatitis B/terapia , Células TH1/inmunología , Administración Intranasal , Animales , Formación de Anticuerpos , Enfermedad Crónica , Femenino , Antígenos del Núcleo de la Hepatitis B/administración & dosificación , Antígenos de Superficie de la Hepatitis B/administración & dosificación , Inmunoglobulina G/análisis , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/inmunologíaRESUMEN
The nasal mucosa may provide a simple, non-invasive route to deliver DNA encoding genes that stimulate a specific immune response. Based on this, a new approach using pCMVbeta-gal plasmid DNA complexed to the Opc meningococcal outer membrane protein was assayed for. Optimal conditions of interaction were established between recombinant Opc protein and pCMVbeta-gal plasmid DNA. Complexes were fully characterized by electrophoresis analysis, DNAse resistance assay and transmission electron microscopy. DNA-protein complexes were also evaluated in in vitro transfection experiments. After the characterisation of complexes, Balb/c mice were intranasal (i.n.) and intramuscularly (i.m.) immunized. The humoral immune response against beta-galactosidase was measured by ELISA. The proliferative response in the spleen lymph nodes was also measured. Complexes administered by i.n. route induced both systemic and mucosal antibody responses. This behavior was not observed with the naked DNA. Finally, a lymphoproliferative response specific to beta-galactosidase induced by DNA-protein complexes was also detected.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Plásmidos/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Administración Intranasal , Animales , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/genética , Células COS , Línea Celular , Esquemas de Inmunización , Ratones , Ratones Endogámicos BALB C , Plásmidos/administración & dosificación , Plásmidos/metabolismo , Plásmidos/ultraestructura , TransfecciónRESUMEN
DNA immunization is a promising approach in generating immune responses to infectious pathogens in many different animal models. In an effort to augment the anti-[hepatitis C virus (HCV) core] immune response, generated after DNA immunization, the importance of vaccination regimen regarding dose and boosting was investigated in the present study. Balb/c mice were intramuscularly injected with an expression plasmid encoding a truncated variant comprising amino acids 1-176 of the HCV core protein. The highest anti-core antibody titres (1:3700) were detected in mice inoculated with 50-100 microg of core-encoding plasmid. Additionally, we demonstrated that antibody levels induced by a single injection of DNA could be further increased by boosting with a second injection of DNA three weeks after primary immunization. However, administration of additional doses or lengthening of the resting period between inoculations resulted in similar or even weaker anti-core antibody responses. A similar anti-(HCV core) lymphoproliferative response was also detected in animals that had the highest level of anti-core antibodies. These results indicate that, in clinical trials, vaccination regimen might be a critical factor in generating optimal anti-(HCV core) immune responses after genetic immunization.
Asunto(s)
Hepacivirus/inmunología , Antígenos de la Hepatitis C/genética , Plásmidos , Animales , División Celular/inmunología , Femenino , Anticuerpos contra la Hepatitis C/biosíntesis , Antígenos de la Hepatitis C/inmunología , Inmunidad Celular , Ratones , Ratones Endogámicos BALB C , Linfocitos T/citología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunologíaRESUMEN
Vaccination of BALB/c mice with pIDKCo, a plasmid containing the coding sequence for the first 176 amino acids of the hepatitis C virus (HCV) core protein, induced both humoral and cellular specific immune responses. Particularly, the level of anti-core antibodies increased slowly with time up to a mean value above 1:8000 that was generally superior than that found in anti-HCV positive individuals. Six out of nine anti-HCV positive human sera were able to inhibit at different extent the binding of mouse anti-core sera to a recombinant capsid protein. Our results show that it is possible to elicit a potent humoral and cellular immune response against the HCV core antigen in mice following DNA immunization.
Asunto(s)
Hepacivirus/inmunología , Vacunas de ADN/farmacología , Proteínas del Núcleo Viral/inmunología , Vacunas contra Hepatitis Viral/farmacología , Animales , Unión Competitiva , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/biosíntesis , Humanos , Inmunidad Celular , Técnicas In Vitro , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Plásmidos/genética , Linfocitos T/inmunología , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Proteínas del Núcleo Viral/genética , Vacunas contra Hepatitis Viral/genética , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
Immunological response against envelope protein E1 is very important in natural hepatitis C virus (HCV) infection, although it is insufficient to clear the viraemia. The HCV genomic region encoding the first 149 amino acids of the envelope E1 protein (E1(340), amino acids 192-340) was expressed in Escherichia coli (to a level of 30% of the whole cellular proteins) and purified to 85%. We measured the immune response in rabbits and mice as well as the reactivity against 37 human sera raised against the whole recombinant protein and E1-encoding peptides. From this, 51.1% of human sera were found to react with E1(340). High-level antibodies against E1(340) were obtained in rabbits and mice when immunized. These antibodies had a similar peptide-recognition pattern to that described previously for human sera. The most reactive region was located at the N-terminus of the E1 protein. Cellular immunity in mice was evaluated by delayed-type hypersensitivity assay. It revealed the induction of a CD4+ T-cell-mediated response by this protein. This E1(340) protein and the animal-derived anti-E1 sera are immunological tools that could aid in the monitoring and development of anti-HCV therapies.