RESUMEN
PURPOSE: Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene. Migalastat, a pharmacological chaperone, binds to specific mutant forms of α-galactosidase A to restore lysosomal activity. METHODS: A pharmacogenetic assay was used to identify the α-galactosidase A mutant forms amenable to migalastat. Six hundred Fabry disease-causing mutations were expressed in HEK-293 (HEK) cells; increases in α-galactosidase A activity were measured by a good laboratory practice (GLP)-validated assay (GLP HEK/Migalastat Amenability Assay). The predictive value of the assay was assessed based on pharmacodynamic responses to migalastat in phase II and III clinical studies. RESULTS: Comparison of the GLP HEK assay results in in vivo white blood cell α-galactosidase A responses to migalastat in male patients showed high sensitivity, specificity, and positive and negative predictive values (≥0.875). GLP HEK assay results were also predictive of decreases in kidney globotriaosylceramide in males and plasma globotriaosylsphingosine in males and females. The clinical study subset of amenable mutations (n = 51) was representative of all 268 amenable mutations identified by the GLP HEK assay. CONCLUSION: The GLP HEK assay is a clinically validated method of identifying male and female Fabry patients for treatment with migalastat.Genet Med 19 4, 430-438.
Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Enfermedad de Fabry/genética , Mutación , alfa-Galactosidasa/genética , 1-Desoxinojirimicina/administración & dosificación , 1-Desoxinojirimicina/farmacología , Bioensayo , Línea Celular , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Enfermedad de Fabry/tratamiento farmacológico , Femenino , Células HEK293 , Humanos , Leucocitos/efectos de los fármacos , Leucocitos/enzimología , Masculino , Valor Predictivo de las Pruebas , Estudios de Validación como AsuntoRESUMEN
Infection with HIV-1, SIV, or simian HIV is associated with abnormalities in the number, size, and structure of germinal centers (GCs). To determine whether these histopathologic abnormalities are associated with abnormalities in Ab development, we analyzed nucleotide sequences of Igs from splenic GCs of simian HIV-infected macaques. Virus-specific GCs were identified in frozen splenic tissue sections by inverse immunohistochemistry using rHIV-1 gp120 as a probe. B cells from envelope-specific GCs were isolated from these sections using laser capture microdissection. Their Igs were amplified from cDNA using nested PCR, then cloned and sequenced. Nucleotide sequences were recovered from nine multimember clonal lineages. Within each lineage, sequences had similar V-D-J or V-J junctions but differed by somatic mutations distributed throughout the variable domain. The clones were highly mutated, similar to that previously reported for HIV-1-specific human IgG Abs. The average clone had 37 mutations in the V region, for a frequency of 0.11 mutations/base. The mutational pattern was strikingly nonrandom, with somatic mutations occurring preferentially at RGYW/WRCY hotspots. Transition mutations were favored over transversions, with C-->T and G-->A replacements together accounting for almost one-third of all mutations. Analysis of replacement and silent mutations in the framework and CDRs suggests that the Igs were subjected to affinity selection. These data demonstrate that the process of Ab maturation is not seriously disrupted in GCs during the early stages of immunodeficiency virus infection, and that Env-specific Igs developing in GCs are subject to extensive somatic mutation and profound selection pressures.
Asunto(s)
Centro Germinal/inmunología , Centro Germinal/metabolismo , Infecciones por VIH/inmunología , VIH-1/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Bazo/inmunología , Bazo/metabolismo , Secuencia de Aminoácidos , Animales , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/patología , Clonación Molecular/métodos , Productos del Gen env/genética , Productos del Gen env/metabolismo , Genes de Inmunoglobulinas , Centro Germinal/patología , Infecciones por VIH/genética , Infecciones por VIH/patología , VIH-1/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Macaca mulatta , Microdisección , Datos de Secuencia Molecular , Unión Proteica/genética , Unión Proteica/inmunología , Análisis de Secuencia de ADN , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Virus de la Inmunodeficiencia de los Simios/genética , Hipermutación Somática de Inmunoglobulina , Bazo/patologíaRESUMEN
Perivascular macrophages are uniquely situated at the intersection between the nervous and immune systems. Although combined myeloid marker detection differentiates perivascular from resident brain macrophages (parenchymal microglia), no single marker distinguishes perivascular macrophages in humans and mice. Here, we present the macrophage scavenger receptor CD163 as a marker for perivascular macrophages in humans, monkeys, and mice. CD163 was primarily confined to perivascular macrophages and populations of meningeal and choroid plexus macrophages in normal brains and in brains of humans and monkeys with human immunodeficiency virus or simian immunodeficiency virus (SIV) encephalitis. Scattered microglia in SIV encephalitis lesions and multinucleated giant cells were also CD163 positive. Consistent with prior findings that perivascular macrophages are primary targets of human immunodeficiency virus and SIV, all SIV-infected cells in the brain were CD163 positive. Using fluorescent dyes that definitively and selectively label perivascular macrophages in vivo, we confirmed that dye-labeled simian perivascular macrophages were CD163 positive and able to repopulate the central nervous system within 24 hours. Flow cytometric studies demonstrated a subset of monocytes (CD163(+)CD14(+)CD16(+)) that were immunophenotypically similar to brain perivascular macrophages. These findings recognize CD163(+) blood monocytes/macrophages as a source of brain perivascular macrophages and underscore the utility of this molecule in studying the biology of perivascular macrophages and their precursors in humans, monkeys, and mice.
Asunto(s)
Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Encéfalo/virología , Encefalitis Viral/inmunología , Infecciones por VIH/inmunología , Macrófagos/inmunología , Receptores de Superficie Celular/análisis , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Animales , Biomarcadores/análisis , Encéfalo/irrigación sanguínea , Encéfalo/inmunología , Arterias Cerebrales/inmunología , VIH , Haplorrinos , Humanos , Receptores de Lipopolisacáridos/análisis , Macrófagos/virología , Ratones , Ratones Endogámicos C3H , Monocitos/inmunología , Receptores de IgG/análisis , Virus de la Inmunodeficiencia de los SimiosRESUMEN
The etiology of the lymphadenopathy and follicular hyperplasia associated with human immunodeficiency virus type 1 (HIV-1) infection has remained unclear. To determine whether the B-lymphocyte expansions characteristic of this syndrome represent polyclonal and virus-specific processes, the antigen specificity of B cells in lymphoid tissues of monkeys infected with simian-human immunodeficiency virus (SHIV) chimeras was assessed using an inverse immunohistochemical assay with biotinylated HIV-1 envelope gp120 (Env) as an antigen probe. Env-binding B cells were found aggregated in lymph node and splenic germinal centers (GCs). Most Env-binding GCs also contained an unstained population of B cells, suggesting the GCs were formed by a polyclonal (oligoclonal) process. By day 42 following infection, Env-binding B cells were present in 19% of all lymph node GCs. Env-binding cells were present in 25% of GCs even during chronic infection. This extraordinarily high frequency of Env-specific B lymphocytes suggests that the expansion of virus-specific B cells may largely account for the follicular hyperplasia in AIDS virus-infected individuals.