RESUMEN
Our laboratory develops protocols to prevent or reverse ongoing anti-hFIX IgG inhibitors in haemophilia B mice with a F9 gene deletion on BALB/c and C3H/HeJ backgrounds. C3H/HeJ F9(-/Y) mice develop high titre anti-hFIX IgG1 inhibitors and anaphylaxis, whereas most BALB/c F9(-/Y) mice have mild anti-hFIX IgG1 inhibitors and no anaphylaxis. Our aim was to determine if hFIX-specific B- and T-cell responses in BALB/c and C3H/HeJ F9(-/Y) mice trigger the difference in anti-hFIX immune responses. BALB/c and C3H/HeJ F9(-/Y) mice were challenged weekly with recombinant hFIX protein. Humoral immune responses were determined by IgG1 and IgG2a anti-hFIX ELISA, Bethesda assay for inhibitors and B-cell ELISpot on bone marrow and spleen cells. T-cell studies measured the TH 1 (IFN-γ) and TH 2 (IL-4) cytokine responses in splenocytes at the mRNA and protein level in response to hFIX protein. Antibody responses were also measured in C3H/HeJ/OuJ F9(-/Y) mice with restored toll-like receptor 4 (TLR4) function. BALB/c F9(-/Y) mice have a TH 2 skewed response and a reduction in anti-hFIX secreting plasma cells in the bone marrow. Independent antigen challenge revealed both strains generated equivalent IgG1 antibody titres to an intravenously delivered antigen. C3H/HeJ F9(-/Y) mice have a mixed TH 1 and TH 2 response (mainly TH 2). Importantly, TLR4 signalling has a modulatory role in the C3H background on the levels of anti-hFIX IgG1 and incidence of anaphylaxis. The background strain strongly impacts the immune response to hFIX, which can be significantly impacted by mutations in innate immune sensors.
Asunto(s)
Factor IX/inmunología , Hemofilia B/inmunología , Receptor Toll-Like 4/inmunología , Animales , Factor IX/genética , Humanos , Ratones , Receptor Toll-Like 4/genéticaRESUMEN
BACKGROUND: Formation of inhibitory antibodies is a frequent and serious complication of factor (F) VIII replacement therapy for the X-linked bleeding disorder hemophilia A. Similarly, hemophilia A mice develop high-titer inhibitors to recombinant human FVIII after a few intravenous injections. OBJECTIVE: Using the murine model, the study sought to develop a short regimen capable of inducing tolerance to FVIII. METHODS: A 1-month immunomodulatory protocol, consisting of FVIII administration combined with oral delivery of rapamycin, was developed. RESULTS: The protocol effectively prevented formation of inhibitors to FVIII upon subsequent intravenous treatment (weekly for 3.5 months). Control mice formed high-titer inhibitors and had CD4(+) T effector cell responses characterized by expression of IL-2, IL-4 and IL-6. Tolerized mice instead had a CD4(+)CD25(+)FoxP3(+) T cell response to FVIII that suppressed antibody formation upon adoptive transfer, indicating a shift from Th2 to Treg if FVIII antigen was introduced to T cells during inhibition with rapamycin. CD4(+) T cells from tolerized mice also expressed TGF-ß1 and CTLA4, but not IL-10. The presence of FVIII antigen during the time of rapamycin administration was required for specific tolerance induction. CONCLUSIONS: The study shows that a prophylactic immune tolerance protocol for FVIII can be developed using rapamycin, a drug that is already widely in clinical application. Immune suppression with rapamycin was mild and highly transient, as the mice regained immune competence within a few weeks.
Asunto(s)
Anticuerpos/sangre , Linfocitos T CD4-Positivos/efectos de los fármacos , Coagulantes/administración & dosificación , Factor VIII/administración & dosificación , Hemofilia A/tratamiento farmacológico , Tolerancia Inmunológica/efectos de los fármacos , Inmunosupresores/administración & dosificación , Sirolimus/administración & dosificación , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/trasplante , Antígeno CTLA-4/metabolismo , Células Cultivadas , Coagulantes/inmunología , Modelos Animales de Enfermedad , Esquema de Medicación , Factor VIII/inmunología , Factores de Transcripción Forkhead/metabolismo , Hemofilia A/sangre , Hemofilia A/inmunología , Humanos , Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-4/metabolismo , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Recombinantes/administración & dosificación , Factores de Tiempo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
We report a family with a heterogeneous group of neurologic disorders associated with the mitochondrial DNA G8363A transfer ribonucleic acid (RNA)Lys mutation. The phenotype of one child in the family was consistent with autism. During his second year of life, he lost previously acquired language skills and developed marked hyperactivity with toe-walking, abnormal reciprocal social interaction, stereotyped mannerisms, restricted interests, self-injurious behavior, and seizures. Brain magnetic resonance imaging (MRI) and repeated serum lactate studies were normal. His older sister developed signs of Leigh syndrome with progressive ataxia, myoclonus, seizures, and cognitive regression. Her laboratory studies revealed increased MRI T2-weighted signal in the putamen and posterior medulla, elevated lactate in serum and cerebrospinal fluid, and absence of cytochrome c oxidase staining in muscle histochemistry. Molecular analysis in her revealed the G8363A mutation of the mitochondrial transfer RNA(Lys) gene in blood (82% mutant mitochondrial DNA) and muscle (86%). The proportions of mutant mitochondrial DNA from her brother with autism were lower (blood 60%, muscle 61%). It is likely that the origin of his autism phenotype is the pathogenic G8363A mitochondrial DNA mutation. This observation suggests that certain mitochondrial point mutations could be the basis for autism in some individuals.
Asunto(s)
Trastorno Autístico/genética , ADN Mitocondrial/genética , Enfermedad de Leigh/genética , Mutación Puntual , ARN de Transferencia de Lisina/genética , Adulto , Trastorno Autístico/fisiopatología , Trastorno Autístico/psicología , Niño , Preescolar , Análisis Mutacional de ADN , Epilepsia/genética , Femenino , Genotipo , Humanos , Discapacidades para el Aprendizaje/genética , Masculino , Músculo Esquelético/patología , Linaje , Fenotipo , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: The mitochondrial DNA polymerase gamma is the principal polymerase required for mitochondrial DNA replication. Primary or secondary deficiencies in the activity of DNA polymerase gamma may lead to mitochondrial DNA depletion. We describe a sensitive and robust clinical assay for this enzyme. METHODS: The assay was performed on mitochondria isolated from skeletal muscle biopsies. High-molecular weight polynucleotide reaction products were captured on ion-exchange paper, examined qualitatively by autoradiography, and quantified by scintillation counting. RESULTS: Kinetic analysis of DNA polymerase gamma by this method showed a K(m) for dTTP of 1.43 micromol/L and a K(i) for azidothymidine triphosphate of 0.861 micromol/L. The assay was linear from 0.1 to 2 microgram of mitochondrial protein. The detection limit was 30 units (30 fmol dTMP incorporated in 30 min). The linear dynamic range was three orders of magnitude; 30-30 000 units. Imprecision (CV) was 6.4% within day and 12% between days. Application of this assay to a mixed population of 38 patients referred for evaluation of mitochondrial disease revealed a distribution with a range of 0-2506 U/microgram, reflecting extensive biologic variation among patients with neuromuscular disease. CONCLUSION: This assay provides a useful adjunct to current laboratory methods for the evaluation of patients with suspected mitochondrial DNA depletion syndromes.
Asunto(s)
ADN Mitocondrial/análisis , ADN Polimerasa Dirigida por ADN/análisis , Animales , Autorradiografía , Bovinos , Cromatografía por Intercambio Iónico , Cromatografía en Papel , ADN Polimerasa gamma , Humanos , Mitocondrias Cardíacas/química , Músculo Esquelético/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Deficiency of mitochondrial DNA polymerase gamma activity was found in a patient with mtDNA depletion and Alpers' syndrome. Metabolic evaluation revealed fasting hypoglycemia, dicarboxylic aciduria, and reduced activity of the electron transport chain in skeletal muscle. The patient died in early childhood of fulminant hepatic failure, refractory epilepsy, lactic acidemia, and coma. mtDNA content was 30% of normal in skeletal muscle and 25% in the liver. The activity of mtDNA polymerase gamma was undetectable.