RESUMEN
Tuberculosis is transmitted by inhalation of Mycobacterium tuberculosis-containing aerosols; 75â% of all patients show pulmonary manifestation. Immune responses after exposure that lead to clinical symptoms occur mainly in the respiratory tract and are only poorly understood. In most cases, cells of the innate immune system are believed to control the growth of or eradicate inhaled mycobacteria. However, this cannot be verified in vivo using standard methods. Subsequently, CD4+ and CD8+ T cell-driven adaptive immune responses are induced that attempt to control bacterial growth. The humoral defence appears to be less important. This article gives an overview of the current understanding of pulmonary immune mechanisms during exposure, latent infection, active disease and therapy of tuberculosis.
Asunto(s)
Pulmón/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , HumanosRESUMEN
The use of monoclonal antibodies has led to new therapeutic possibilities for many clinical conditions. However, their application also bears risks, as demonstrated by the acute occurrence of a cytokine storm following administration of TGN1412, an anti-CD28 superagonist, in March 2006. This article highlights the principles of the Paul-Ehrlich-Institut (PEI) for the scientific assessment of first-in-man clinical trial applications for monoclonal antibodies. These principles are implemented as a standard operating procedure in the PEI Quality Management System and are intended as a supplement specific to monoclonal antibodies to the published general guideline issued by the Committee for Medicinal Products for Human Use (CHMP). Central aspects are the identification of risk factors for monoclonal antibodies based on defined criteria, since not every novel monoclonal antibody represents a risk per se. Furthermore, a PEI expert group that supports the scientific assessment procedure has been founded.
Asunto(s)
Anticuerpos Monoclonales/toxicidad , Ensayos Clínicos Fase I como Asunto/normas , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Evaluación Preclínica de Medicamentos/normas , Alemania , Humanos , Selección de Paciente , Medición de Riesgo , Gestión de la Calidad Total/normasRESUMEN
This review summarizes scientific, ethical and regulatory aspects of Phase I clinical trials with monoclonal antibodies. The current standard requirements for pre-clinical testing and for clinical study design are presented. The scientific considerations discussed herein are generally applicable, the view on legal requirements for clinical trials refer to the German jurisdiction only. The adverse effects associated with the TGN1412 Phase I trial indicate that the predictive value of pre-clinical animal models requires reevaluation and that, in certain cases, some issues of clinical trial protocols such as dose fixing may need refinement or redesign. Concrete safety measures, which have been proposed as a consequence of the TGN1412 event include introduction of criteria for high-risk antibodies, sequential inclusion of trial participants and implementation of pre-Phase I studies where dose calculation is based on the pre-clinical No Effect Level instead of the No Observed Adverse Effect Level. The recently established European clinical trials database (EUDRACT Database) is a further safety tool to expedite the sharing of relevant information between scientific authorities.
Asunto(s)
Anticuerpos Monoclonales/efectos adversos , Antineoplásicos/efectos adversos , Ensayos Clínicos Fase I como Asunto/legislación & jurisprudencia , Aprobación de Drogas/legislación & jurisprudencia , Experimentación Humana/legislación & jurisprudencia , Legislación de Medicamentos , Proyectos de Investigación , Sistemas de Registro de Reacción Adversa a Medicamentos , Animales , Anticuerpos Monoclonales/toxicidad , Anticuerpos Monoclonales Humanizados , Antineoplásicos/toxicidad , Ensayos Clínicos Fase I como Asunto/ética , Ensayos Clínicos Fase I como Asunto/métodos , Evaluación Preclínica de Medicamentos/métodos , Revisión Ética , Alemania , Guías como Asunto , Experimentación Humana/ética , Humanos , Nivel sin Efectos Adversos Observados , Valores de Referencia , Medición de Riesgo , Pruebas de ToxicidadAsunto(s)
Proteínas PrPC/inmunología , Enfermedades por Prión/prevención & control , Enfermedades por Prión/terapia , Animales , Anticuerpos/uso terapéutico , Bovinos , Síndrome de Creutzfeldt-Jakob/prevención & control , Síndrome de Creutzfeldt-Jakob/terapia , Encefalopatía Espongiforme Bovina/prevención & control , Encefalopatía Espongiforme Bovina/terapia , Epítopos , Humanos , Fragmentos Fab de Inmunoglobulinas , Inmunoterapia , Scrapie/inmunología , Scrapie/prevención & controlAsunto(s)
Anticuerpos Antivirales/inmunología , Receptores Fc/inmunología , Acrecentamiento Dependiente de Anticuerpo , Reacciones Antígeno-Anticuerpo/inmunología , Antígenos Virales/ultraestructura , Linfocitos B/inmunología , Proteínas del Sistema Complemento/inmunología , Humanos , Memoria Inmunológica , Cooperación Linfocítica , Linfocitos T/inmunología , Vacunas/inmunología , Virosis/inmunología , Virus/inmunologíaRESUMEN
Variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy are initiated by extracerebral exposure to prions. Although prion transmission from extracerebral sites to the brain represents a potential target for prophylaxis, attempts at vaccination have been limited by the poor immunogenicity of prion proteins. To circumvent this, we expressed an anti-prion protein (anti-PrP) mu chain in Prnp(o/o) mice. Transgenic mice developed sustained anti-PrP titers, which were not suppressed by introduction of Prnp+ alleles. Transgene expression prevented pathogenesis of prions introduced by intraperitoneal injection in the spleen and brain. Expression of endogenous PrP (PrP(C)) in the spleen and brain was unaffected, suggesting that immunity was responsible for protection. This indicates the feasibility of immunological inhibition of prion disease in vivo.
Asunto(s)
Anticuerpos/inmunología , Proteínas PrPSc/inmunología , Priones/inmunología , Scrapie/prevención & control , Amiloide/genética , Animales , Anticuerpos/sangre , Linfocitos B/inmunología , Western Blotting , Química Encefálica , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Cadenas mu de Inmunoglobulina/sangre , Cadenas mu de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas PrPC/genética , Proteínas PrPSc/análisis , Proteínas Priónicas , Priones/genética , Precursores de Proteínas/genética , Bazo/química , Bazo/inmunologíaRESUMEN
New-variant Creutzfeldt-Jakob disease and scrapie are typically initiated by extracerebral exposure to the causative agent, and exhibit early prion replication in lymphoid organs. In mouse scrapie, depletion of B-lymphocytes prevents neuropathogenesis after intraperitoneal inoculation, probably due to impaired lymphotoxin-dependent maturation of follicular dendritic cells (FDCs), which are a major extracerebral prion reservoir. FDCs trap immune complexes with Fc-gamma receptors and C3d/C4b-opsonized antigens with CD21/CD35 complement receptors. We examined whether these mechanisms participate in peripheral prion pathogenesis. Depletion of circulating immunoglobulins or of individual Fc-gamma receptors had no effect on scrapie pathogenesis if B-cell maturation was unaffected. However, mice deficient in C3, C1q, Bf/C2, combinations thereof or complement receptors were partially or fully protected against spongiform encephalopathy upon intraperitoneal exposure to limiting amounts of prions. Splenic accumulation of prion infectivity and PrPSc was delayed, indicating that activation of specific complement components is involved in the initial trapping of prions in lymphoreticular organs early after infection.
Asunto(s)
Proteínas del Sistema Complemento/metabolismo , Enfermedades por Prión/etiología , Enfermedades por Prión/inmunología , Animales , Secuencia de Bases , Encéfalo/metabolismo , Encéfalo/patología , Proteínas del Sistema Complemento/deficiencia , Proteínas del Sistema Complemento/genética , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Enfermedades por Prión/patología , Priones/metabolismo , Receptores de Complemento/deficiencia , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Scrapie/etiología , Scrapie/inmunología , Scrapie/patología , Bazo/inmunología , Bazo/metabolismo , Factores de TiempoRESUMEN
Mice infected with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, mount a virus-neutralizing antibody response protecting against lethal disease. VSVneutralizing monoclonal IgGs isolated from primary immune responses were devoid of somatic mutations, whereas most secondary and all hyperimmune response IgGs tested were hypermutated. A comparative analysis of recombinant single-chain antibody fragments (scFv-Ckappa) revealed that even the germ-line precursor of one hypermutated antibody bound and neutralized VSV. Four somatic amino acid substitutions in V(H) increased by 300-fold the binding strength of monovalent scFv-Ckappa. The multivalent binding avidity of germ-line scFv-Ckappa was increased by more than 10-fold compared with the monovalent binding strength. In contrast, hypermutated scFv-Ckappa did not show such avidity effects. Thus the overall binding difference between the germ-line and the hypermutated VSV-neutralizing antibody was only 10- to 15-fold. This may explain why primary germ-line antibodies and secondary hypermutated antibodies directed against pathogens such as viruses and bacteria expressing repetitive antibody determinants show rather similar binding qualities, whereas monovalently binding hapten-specific antibodies can show "affinity maturation" effects of up to 1000-fold.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Mutación de Línea Germinal , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Región Variable de Inmunoglobulina/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Secuencia de Bases , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/genética , Ratones , Ratones SCID , Ácaros/inmunología , Ácaros/virología , Mutagénesis Sitio-Dirigida , Pruebas de Neutralización , Proteínas Recombinantes/inmunologíaRESUMEN
Poorly cytopathic or noncytopathic viruses can escape immune surveillance and establish a chronic infection. Here we exploited the strategy of combining antiviral drug treatment with the induction of a neutralizing antibody response to avoid the appearance of neutralization-resistant virus variants. Despite the fact that H25 immunoglobulin transgenic mice infected with lymphocytic choriomeningitis virus mounted an early neutralizing antibody response, the virus escaped from neutralization and persisted. After ribavirin treatment of H25 transgenic mice, the appearance of neutralization-resistant virus was prevented and virus was cleared. Thus, the combination of virus-neutralizing antibodies and chemotherapy efficiently controlled the infection, whereas each defense line alone did not. Similar additive effects may be unexpectedly efficient and beneficial in humans after infections with persistent viruses such as hepatitis C virus and hepatitis B virus and possibly human immunodeficiency virus.
Asunto(s)
Anticuerpos Antivirales/inmunología , Antivirales/uso terapéutico , Cadenas mu de Inmunoglobulina/inmunología , Coriomeningitis Linfocítica/tratamiento farmacológico , Coriomeningitis Linfocítica/inmunología , Ribavirina/uso terapéutico , Latencia del Virus , Animales , Secuencia de Bases , Línea Celular , Cricetinae , ADN Viral , Femenino , Variación Genética , Cadenas mu de Inmunoglobulina/genética , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/genética , Virus de la Coriomeningitis Linfocítica/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Pruebas de NeutralizaciónRESUMEN
TNFR1-/- mice have been shown to lack networks of mature follicular dendritic cells (FDCs) and they do not form germinal centers. With nonreplicating Ags, IgG titers were inefficiently induced and not maintained. In this study, the neutralizing Ab response and the establishment of B cell memory in TNFR1-/- mice after infection with vesicular stomatitis virus (VSV) were analyzed histologically and functionally. Immunization with VSV-derived protein Ags without adjuvant induced only IgM but no IgG Abs in TNFR1-/- mice, whereas VSV glycoprotein emulsified in CFA or IFA induced IgM and IgG responses that were short-lived and of moderate titer. However, infection with live VSV induced excellent neutralizing IgM and IgG responses in TNFR1-/- mice, and adoptively transferable B cell memory was generated and persisted for more than 300 days. In contrast, IgG levels and Ab-forming cells in the bone marrow declined within 300 days by 90-95% compared with controls. These findings suggest that 1) increased Ag dose and time of Ag availability can substitute for FDC-stored Ab-complexed Ag in the induction of efficient IgG responses in TNFR1-/- mice devoid of classical germinal centers; 2) the induction and maintenance of adoptively transferable B cell memory can occur in the absence of Ag bound to mature FDCs; and 3) the long-term maintenance of elevated IgG titers is largely dependent on FDC-associated persisting Ag. However, about 5-10% of the Ab production remained in the absence of detectable persisting Ag in TNFR1-/- mice, probably either due to immature FDCs being partially functional and/or due to long-lived plasma cells.
Asunto(s)
Antígenos CD/genética , Linfocitos B/inmunología , Linfocitos B/virología , Comunicación Celular/inmunología , Células Dendríticas Foliculares/inmunología , Memoria Inmunológica/genética , Receptores del Factor de Necrosis Tumoral/genética , Virus de la Estomatitis Vesicular Indiana/inmunología , Traslado Adoptivo , Animales , Afinidad de Anticuerpos , Antígenos CD/metabolismo , Antígenos Virales/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Médula Ósea/inmunología , Médula Ósea/metabolismo , Médula Ósea/patología , Comunicación Celular/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Cricetinae , Células Dendríticas Foliculares/metabolismo , Células Dendríticas Foliculares/patología , Adyuvante de Freund/administración & dosificación , Adyuvante de Freund/inmunología , Cambio de Clase de Inmunoglobulina/genética , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Inmunoglobulina M/biosíntesis , Inyecciones Subcutáneas , Cinética , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Mesenterio , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral , Bazo/inmunología , Bazo/metabolismo , Bazo/patología , Virus de la Estomatitis Vesicular Indiana/fisiología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
Variable (V) region gene replacement was recently implicated in B cell repertoire diversification, but the contribution of this mechanism to antibody responses is still unknown. To investigate the role of V gene replacements in the generation of antigen-specific antibodies, we analyzed antiviral immunoglobulin responses of "quasimonoclonal" (QM) mice. The B cells of QM mice are genetically committed to exclusively express the anti-(4-hydroxy-3-nitrophenyl) acetyl specificity. However, approximately 20% of the peripheral B cells of QM mice undergo secondary rearrangements and thereby potentially acquire new specificities. QM mice infected with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus, or poliovirus mounted virus-specific neutralizing antibody responses. In general, kinetics of the antiviral immunoglobulin responses were delayed in QM mice; however, titers similar to control animals were eventually produced that were sufficient to protect against VSV-induced lethal disease. VSV neutralizing single-chain Fv fragments isolated from phage display libraries constructed from QM mice showed VH gene replacements and extensive hypermutation. Thus, our data demonstrate that secondary rearrangements and hypermutation can generate sufficient B cell diversity in QM mice to mount protective antiviral antibody responses, suggesting that these mechanisms might also contribute to the diversification of the B cell repertoire of normal mice.
Asunto(s)
Diversidad de Anticuerpos , Linfocitos B/inmunología , Reordenamiento Génico de Linfocito B , Mutación , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/genética , Secuencia de Bases , ADN/genética , Marcación de Gen , Ratones , Ratones Endogámicos C57BL , Pruebas de Neutralización , Homología de Secuencia de Ácido Nucleico , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
B cell tolerance is maintained by active deletion and functional anergy of self-reactive B cells depending on the time, amount, and site of the self-antigen expression. To study B cell tolerance toward a transplacentally transmitted viral Ag, we crossed transgenic mice expressing the mu heavy and the kappa light chain of the lymphocytic choriomeningitis virus (LCMV)-neutralizing mAb KL25 (HL25-transgenic mice) with persistently infected LCMV carrier mice. Although HL25-transgenic LCMV carrier mice exhibited the same high virus titers as nontransgenic LCMV carrier mice, no evidence for B cell tolerance was found. In contrast, enhanced LCMV-neutralizing Ab titers were measured that, however, did not clear the virus. Instead, LCMV isolates from different tissues turned out to be neutralization resistant Ab escape variants expressing different substitutions of amino acid Asn119 of the LCMV-glycoprotein 1 that displays the neutralizing B cell epitope. Virus variants with the same mutations were also selected in vitro in the presence of the transgenic mAb KL25 confirming that substitutions of Asn119 have been selected by LCMV-neutralizing Abs. Thus, despite abundant expression of viral neo-self-antigen in HL25-transgenic LCMV carrier mice, transgenic B cells expressing LCMV-neutralizing Abs were rather stimulated than tolerized and neutralization resistant Ab escape variants were selected in vivo.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Linfocitos B/inmunología , Portador Sano/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Autotolerancia/genética , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Animales , Anticuerpos Antivirales/genética , Asparagina/genética , Linfocitos B/metabolismo , Linfocitos B/virología , Portador Sano/virología , Inmunidad Innata , Coriomeningitis Linfocítica/genética , Virus de la Coriomeningitis Linfocítica/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Pruebas de NeutralizaciónRESUMEN
We present a mathematical model to simulate the kinetics of B-cell activation and the virus-neutralizing immunoglobulin response in the spleen of mice after infection with vesicular stomatitis virus (VSV). Our model combines data from in vitro experiments and in vivo kinetic observations. A system of eight nonlinear differential equations was used in the computer experiments and numerically solved. The isotype switch from IgM to IgG in the presence of T-cell help was modelled by a time variable function, used as a parameter. The model solutions indicate fast kinetics of the generation of VSV-neutralizing IgM antibodies within 2-3 days post immunization peaking on day 5 at a serum concentration of approximately 80 microgram ml-1 IgM, which is the equivalent to about 10% of the total IgM serum concentration. The frequency of virus-specific B cells increases about 1000-fold within the first 4 days after immunization. Protective levels of VSV-neutralizing IgG antibodies (>/=10 microgram ml-1) are reached within 5 to 6 days post immunization. Fitting the model solution to the experimentally observed neutralizing serum titers suggests an increase in the neutralizing activity of IgGs occurring between days 5 and 8 post-infection. The model indicates that less than 10 VSV-specific B cells have to be triggered daily to maintain protective IgG serum titers during the memory phase.
Asunto(s)
Inmunoglobulinas/inmunología , Modelos Inmunológicos , Infecciones por Rhabdoviridae/inmunología , Bazo/inmunología , Virus de la Estomatitis Vesicular Indiana , Animales , Anticuerpos Antivirales/sangre , Linfocitos B/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Activación de Linfocitos , Ratones , Linfocitos T/inmunología , Factores de TiempoRESUMEN
Following infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin mu heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host's capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin mu heavy and the kappa light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.
Asunto(s)
Anticuerpos Antivirales/inmunología , Inmunoglobulina M/inmunología , Coriomeningitis Linfocítica/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/genética , Acrecentamiento Dependiente de Anticuerpo/inmunología , Pruebas Inmunológicas de Citotoxicidad , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/genética , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/inmunología , Cadenas mu de Inmunoglobulina/biosíntesis , Cadenas mu de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/inmunología , Coriomeningitis Linfocítica/prevención & control , Coriomeningitis Linfocítica/virología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pruebas de Neutralización , Factores de TiempoRESUMEN
Although prion proteins are most efficiently propagated through intracerebral inoculation, peripheral administration has caused the diseases kuru, iatrogenic Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE) and new-variant CJD. The development of neurological disease after peripheral inoculation depends on prion expansion within cells of the lymphoreticular system. Here we investigate the identity of these cells by using a panel of immune-deficient mice inoculated with prions intraperitoneally: we found that defects affecting only T lymphocytes had no apparent effect, but that all mutations that disrupted the differentiation and response of B lymphocytes prevented the development of clinical scrapie. As an absence of B cells and of antibodies correlates with severe defects in follicular dendritic cells, a lack of any of these three components may prevent the development of clinical scrapie. However, we found that scrapie developed after peripheral inoculation in mice expressing immunoglobulins that were exclusively of the M subclass and without detectable specificity for the normal form of the prion PrPC, and in mice which had differentiated B cells but no functional follicular dendritic cells. We conclude that differentiated B cells are crucial for neuroinvasion by scrapie, regardless of the specificity of their receptors.
Asunto(s)
Linfocitos B/inmunología , Scrapie/inmunología , Animales , Western Blotting , Encéfalo/patología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Priones/inmunología , Scrapie/patología , Scrapie/fisiopatologíaRESUMEN
We describe a patient with severe generalized dystrophic epidermolysis bullosa (EBD) and a novel combination of compound heterozygous mutations in the COL7A1 gene. The maternal mutation was an A-to-G transition (425-A --> G) at position -2 of the donor splice site within exon 3 that causes aberrant splicing of two abnormal transcripts. One includes intron 3, and one excludes both exon 3 and intron 3. Both splice variants contained a premature termination of the translation. The paternal mutation is a 25-bp deletion in exon 20 (2638de125) that leads to a frameshift and a premature termination codon 133 bp downstream from the site of deletion. This combination of mutations allowed expression of collagen VII mRNA. Immunofluorescence staining of the patient's skin and cultured keratinocytes with domain-specific collagen VII antibodies, however, demonstrated markedly reduced levels of alpha1(VII) polypeptides, and no stable collagen VII protein could be extracted from the patient's cells. Electron microscopy showed severely hypoplastic fibrils below the lamina densa, without evidence of normal anchoring fibrils. The clinically unaffected parents were heterozygous for the mutations, suggesting that both COL7A1 gene defects were recessively inherited disease-causing mutations that are "silent" in heterozygous carriers but in combination can severely interfere with the dermal-epidermal adhesion and lead to severe EBD.
Asunto(s)
Colágeno/genética , Mutación del Sistema de Lectura , Mutación Puntual , Especificidad de Anticuerpos , Preescolar , Colágeno/inmunología , ADN/análisis , Epidermólisis Ampollosa Distrófica/genética , Técnica del Anticuerpo Fluorescente , Genes Recesivos , Genotipo , Heterocigoto , Humanos , Queratinocitos/química , Microscopía Electrónica , Datos de Secuencia Molecular , Ácidos Nucleicos Heterodúplex/análisis , Fenotipo , Reacción en Cadena de la Polimerasa , Empalme del ARN , ARN Mensajero/metabolismo , Piel/química , Piel/ultraestructuraRESUMEN
Neutralizing antibodies are necessary and sufficient for protection against infection with vesicular stomatitis virus (VSV). The in vitro neutralization capacities and in vivo protective capacities of a panel of immunoglobulin G monoclonal antibodies to the glycoprotein of VSV were evaluated. In vitro, neutralizing activity correlated with avidity and with neutralization rate constant, a measure of on-rate. However, in vivo, protection was independent of immunoglobulin subclass, avidity, neutralization rate constant, and in vitro neutralizing activity; above a minimal avidity threshold, protection depended simply on a minimum serum concentration. These two biologically defined thresholds of antibody specificity offer hope for the development of adoptive therapy with neutralizing antibodies.
Asunto(s)
Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos , Inmunización Pasiva , Glicoproteínas de Membrana , Infecciones por Rhabdoviridae/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Animales , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos , Encéfalo/virología , Ensayo de Inmunoadsorción Enzimática , Glicoproteínas/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Pruebas de Neutralización , Infecciones por Rhabdoviridae/virología , Virus de la Estomatitis Vesicular Indiana/crecimiento & desarrollo , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Antibody responses against antibodies, such as rheumatoid factors, are found in several immunopathological diseases and may play a role in disease pathogenesis. Experience shows that they are usually difficult to induce experimentally. Antibodies specific for immunoglobulin constant regions (anti-allotypic) or for variable regions (anti-idiotypic) have been investigated in animal models; the latter have even been postulated to regulate antibody and T cell responses via network-like interactions. Why and how such anti-antibodies are induced during autoimmune diseases, has remained largely unclear. Because repetitively arranged epitopes in a paracrystalline structure of a viral envelope cross-link B cell receptors efficiently to induce a prompt T-independent IgM response, this study used immune complexes containing viruses or bacteria to evaluate the role of antigen pattern for induction of anti-antibody responses. We present evidence that antibodies bound to strictly ordered, but not to irregularly arranged, antigens dramatically enhance induction of anti-antibodies, already after a single immunization and without using adjuvants. The results indicate a novel link between anti-antibody responses and infectious agents, and suggest a similar role for repetitive self-antigens such as DNA or collagen involved in chronic immunopathological diseases.
Asunto(s)
Anticuerpos Antiidiotipos/biosíntesis , Autoanticuerpos/inmunología , Epítopos/inmunología , Inmunoglobulina G/biosíntesis , Glicoproteínas de Membrana , Animales , Anticuerpos Monoclonales , Formación de Anticuerpos , Línea Celular , Cricetinae , Alotipos de Inmunoglobulinas/inmunología , Regiones Constantes de Inmunoglobulina/inmunología , Idiotipos de Inmunoglobulinas/inmunología , Inmunoglobulina M/inmunología , Región Variable de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos BALB C , Antígenos O/inmunología , Pseudomonas aeruginosa/inmunología , Proteínas Recombinantes de Fusión/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
During most clinically relevant infections with cytopathic viruses, neutralizing antibodies are generated early, i.e., within the first week of infection. As early as 4 days after immunization of mice with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, hybridomas could be isolated that secreted virus-neutralizing IgGs. Such antibodies were devoid of somatic mutations, showed high binding avidities (approximately 10(9) M-1), and used V gene fragments predominantly belonging to the VHQ52 and VK19-28 families. In contrast, most secondary and hyperimmune response IgGs isolated 12 and 150 days after infection used several additional V gene combinations. These, which used the VHQ52/VK19-28 combination of early IgGs, were point mutated but showed only marginally enhanced binding avidities. Since all VHQ52/ VK19-28-positive IgGs bound to one subsite within the major antigenic site of VSV-G irrespective of the presence or absence of somatic point mutations, fine specificity diversification of secondary and hyperimmune responses was achieved by newly appearing V gene combinations.