RESUMO
Hepatitis B is considered to be a worldwide public health problem. An immunosuppressor microenvironment has been proposed to contribute to viral persistence during chronic disease. Understanding the intracellular signaling cascade in T-cells from HBV-infected patients, will contribute to unravel the mechanisms that control the development of immune response during hepatitis B. We analyze lipid rafts formation and early activation signals in chronic HBV infected patients, compared to naturally immune subjects (NIS). Patients show: (1) diminished GM1 clustering, (2) A deficient lipid rafts recruitment of CD3ζ/ZAP-70/Grb2, and (3) these proteins do not merge with GM1 within the lipid rafts. Finally, immunoprecipitation assays proved that ZAP-70 does not associate to CD3ζ. These results show for the first time, defects regarding early key events in T-cell activation, in chronically infected HBV patients, which may contribute not only to understand HBV immune tolerance, but to reveal new potential therapeutic targets to control the infection.
Assuntos
Complexo CD3/imunologia , Proteína Adaptadora GRB2/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Microdomínios da Membrana/imunologia , Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/imunologia , Imunidade Adaptativa , Complexo CD3/metabolismo , Citometria de Fluxo , Proteína Adaptadora GRB2/metabolismo , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ativadoras de Esfingolipídeos/imunologia , Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
A 2-year-old child had a metachromatic leukodystrophy-variant phenotype mainly involving the peripheral nervous system (PNS) that was caused by saposin-B deficiency. Bone marrow transplantation resulted in transient deterioration then continuous improvement of PNS functions. These findings were supported by nerve conduction velocity measurements, but the symptoms ultimately worsened. Magnetic resonance imaging showed persistent white matter lesions and progressive pontocerebellar atrophy.
Assuntos
Transplante de Medula Óssea , Glicoproteínas/deficiência , Leucodistrofia Metacromática/terapia , Encéfalo/patologia , Pré-Escolar , Humanos , Leucodistrofia Metacromática/patologia , Leucodistrofia Metacromática/fisiopatologia , Imageamento por Ressonância Magnética , Masculino , Condução Nervosa , Sistema Nervoso Periférico/fisiologia , Saposinas , Proteínas Ativadoras de EsfingolipídeosRESUMO
The lysosomal removal of the sulfate moiety from sulfatide requires the action of two proteins, arylsulfatase A and sphingolipid activator protein-1 (SAP-1). Recently, patients have been identified who have a variant form of metachromatic leukodystrophy which is characterized by mutations in the gene coding for SAP-1, which is also called "prosaposin." All of the mutations characterized in these patients result in (a) deficient mature SAP-1, as determined by immunoblotting after SDS-PAGE of tissue and cell extracts, and (b) decreased ability of cultured skin fibroblasts to metabolize endocytosed [14C]-sulfatide. We now report the insertion of the full-length prosaposin cDNA into the Moloney murine leukemia virus-derived retroviral vector, pLJ, and the infection of cultured skin fibroblasts from a newly diagnosed and molecularly characterized patient with SAP-1 deficiency. The cultured cells infected with the prosaposin cDNA construct now show both production of normal levels of mature SAP-1 and completely normal metabolism of endocytosed [14C]-sulfatide. These studies demonstrate that the virally transferred prosaposin cDNA is processed normally and is localized within lysosomes, where it is needed for interaction between sulfatide and arylsulfatase A. In addition, normal as well as mutant sequences can now be found by allele-specific oligonucleotide hybridization of PCR-amplified genomic DNA by using exonic sequences as primers.