RESUMEN
B lymphocyte stimulator (BLyS; also known as TNFSF20, BAFF, TALL-1, zTNF4, and THANK), a tumor necrosis factor ligand family member, has recently been identified as a factor that promotes expansion and differentiation of the B cell population, leading to increases in serum immunoglobulin levels. Here, pharmacokinetic parameters for BLyS administered i.v. and s.c. to mice are described, and the effects of different dosing regimens on serum and salivary immunoglobulin levels as well as splenic cell populations are reported. The pharmacokinetics of BLyS following i.v. injection are monophasic with a half-life of 160 min, a clearance of 0.22 ml/min-kg, and a volume of distribution of 53 ml/kg. Systemic administration of BLyS to mice resulted in increased serum IgG, IgA, IgM, and IgE and salivary IgA as well as splenic B cell population expansion and differentiation. The i.v. and s.c. routes of administration were pharmacologically equivalent, even though s.c. bioavailability of BLyS is only 25%. BLyS (s.c.) dramatically elevated serum IgG and IgA levels, and the duration of the responses after cessation of treatment (t(1/2) = 4.4 and 1.3 days, respectively) are similar to the half-lives of endogenous IgG and IgA in mice. The IgM response is more modest than that of IgG and IgA but lasts longer (t(1/2) = 7.0 days) than the half-life of endogenous IgM. A linear pharmacodynamic response was identified between days of dosing x log(dose), and increases in serum IgG, IgA, and IgM indicating that the response is more sensitive to the duration of dosing than to the cumulative dose. The implications of these findings for therapeutic administration of BLyS are discussed.
Asunto(s)
Inmunidad Celular/efectos de los fármacos , Proteínas de la Membrana/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Factor Activador de Células B , Semivida , Humanos , Inmunoglobulinas/metabolismo , Inyecciones Intravenosas , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos BALB C , Tamaño de los Órganos/efectos de los fármacos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Saliva/inmunología , Bazo/citología , Bazo/efectos de los fármacosRESUMEN
B-lymphocyte stimulator (BLyS) is a recently identified novel member of the tumor necrosis factor ligand superfamily shown to exist in a membrane-bound and soluble form. BLyS was found to be specifically expressed on cells of myeloid lineage and to selectively stimulate B-lymphocyte proliferation and immunoglobulin production. The expression of a cytokine involved in potentiation of humoral immune responses, such as BLyS, is expected to be strictly controlled. The goal of the present study was to examine regulation of BLyS levels in monocytic cells in response to cytokines and during their differentiation to macrophages and dendritic cells. The presence of BLyS on the cell surface and in the culture medium of both normal blood monocytes and on tumor cells of myelomonocytic origin was demonstrated. BLyS gene expression and levels of membrane-associated and soluble BLyS were found to be regulated by cytokines, in particular interferon (IFN)-gamma and to a lesser extent interleukin-10 (IL-10). The expression of BLyS on monocyte membranes was retained following differentiation into macrophages, but detection on the surface of monocyte-derived dendritic cells required stimulation with IFN-gamma. Both IFN-gamma and IL-10 enhanced the release of soluble BLyS that was active in B-cell proliferation assays. Cells transfected with BLyS complementary DNA mutated in a predicted cleavage site failed to release BLyS into the culture medium, thereby suggesting that soluble BLyS was derived from the membrane form. These results provide further support for an important role for BLyS expressed in myeloid cells in B-cell expansion and antibody responses.
Asunto(s)
Proteínas de la Membrana/genética , Células Mieloides/metabolismo , Factor de Necrosis Tumoral alfa/genética , Anticuerpos/metabolismo , Factor Activador de Células B , Linfocitos B/citología , División Celular/efectos de los fármacos , Citocinas/farmacología , Células Dendríticas/química , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Macrófagos/química , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Monocitos/metabolismo , Péptido Hidrolasas/metabolismo , ARN Mensajero/metabolismo , Solubilidad , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Expression of the region of the feline calicivirus (FCV) ORF1 encoded by nucleotides 3233 to 4054 in an in vitro rabbit reticulocyte system resulted in synthesis of an active proteinase that specifically processes the viral nonstructural polyprotein. Site-directed mutagenesis of the cysteine (Cys1193) residue in the putative active site of the proteinase abolished autocatalytic cleavage as well as cleavage of the viral capsid precursor, suggesting that this "3C-like" proteinase plays an important role in proteolytic processing during viral replication. Expression of the region encoding the C-terminal portion of the FCV ORF1 (amino acids 942 to 1761) in bacteria allowed direct N-terminal sequence analysis of the virus-specific polypeptides produced in this system. The results of these analyses indicate that the proteinase cleaves at amino acid residues E960-A961, E1071-S1072, E1345-T1346, and E1419-G1420; however, the cleavage efficiency is varied. The E1071-S1072 cleavage site defined the N terminus of a 692-amino-acid protein that contains sequences with similarity to the picornavirus 3C proteinase and 3D polymerase domains. Immunoprecipitation of radiolabeled proteins from FCV-infected feline kidney cells with serum raised against the FCV ORF1 C-terminal region showed that this "3CD-like" proteinase-polymerase precursor protein is apparently stable and accumulates in cells during infection.
Asunto(s)
Calicivirus Felino/enzimología , Cisteína Endopeptidasas/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/genética , Proteasas Virales 3C , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calicivirus Felino/genética , Catálisis , Gatos , Línea Celular , Mapeo Cromosómico , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Humanos , Datos de Secuencia Molecular , Mutagénesis , Sistemas de Lectura Abierta , Proteínas/metabolismo , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Proteínas Virales/metabolismoRESUMEN
The tumor necrosis factor (TNF) superfamily of cytokines includes both soluble and membrane-bound proteins that regulate immune responses. A member of the human TNF family, BLyS (B lymphocyte stimulator), was identified that induced B cell proliferation and immunoglobulin secretion. BLyS expression on human monocytes could be up-regulated by interferon-gamma. Soluble BLyS functioned as a potent B cell growth factor in costimulation assays. Administration of soluble recombinant BLyS to mice disrupted splenic B and T cell zones and resulted in elevated serum immunoglobulin concentrations. The B cell tropism of BLyS is consistent with its receptor expression on B-lineage cells. The biological profile of BLyS suggests it is involved in monocyte-driven B cell activation.
Asunto(s)
Linfocitos B/inmunología , Activación de Linfocitos , Proteínas de la Membrana/fisiología , Monocitos/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Secuencia de Aminoácidos , Animales , Factor Activador de Células B , Receptor del Factor Activador de Células B , Subgrupos de Linfocitos B/inmunología , Línea Celular , Células Cultivadas , Humanos , Inmunoglobulinas/sangre , Interferón gamma/farmacología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Receptores de Citocinas/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Especificidad de la Especie , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia ArribaRESUMEN
The capsid protein of the Hawaii strain of human calicivirus was expressed in the transient MVA/bacteriophage T7 polymerase hybrid expression system in order to examine its processing in mammalian cells. Selected amino acid modifications (an insertion, deletion, and substitution) at the predicted amino terminus of the capsid protein as well as the presence or absence of the ORF3 gene were examined for their effect on capsid expression. The protein was expressed efficiently in cell lines derived from three different species, with most of the expressed protein remaining localized within the cells. There was no evidence for N-linked glycosylation or myristylation of the 57 kDa capsid protein. Hawaii virus-like particles (HV VLPs), efficiently produced in the baculovirus expression system, were not observed in this expression system under the conditions in this study.
Asunto(s)
Caliciviridae/metabolismo , Cápside/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Células CACO-2 , Caliciviridae/genética , Cápside/genética , Línea Celular , Línea Celular Transformada , Chlorocebus aethiops , Clonación Molecular , Expresión Génica , Genes Virales , Humanos , Mamíferos , Plásmidos , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Feline calicivirus (FCV), a member of the Caliciviridae, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3'-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates for trans cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1' position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.
Asunto(s)
Calicivirus Felino/enzimología , Cápside/metabolismo , Endopeptidasas/metabolismo , Animales , Secuencia de Bases , Calicivirus Felino/genética , Gatos , Línea Celular , Mapeo Cromosómico , ADN Viral , Endopeptidasas/genética , Expresión Génica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Biosíntesis de Proteínas , Precursores de Proteínas/metabolismo , Transcripción GenéticaRESUMEN
The hepatitis E virus (HEV) capsid antigen has been proposed as a candidate subunit vaccine for the prevention of hepatitis E. The full-length HEV ORF2 protein product is predicted to contain 660 amino acids and to weigh 72,000 daltons. Expression of the HEV ORF2 capsid gene from recombinant baculoviruses in insect cells produced multiple immunoreactive proteins ranging in size from 30 to 100 kDa. The most abundant HEV proteins had molecular weights of 72, 63, 56, and 53 kDa. Temporal expression kinetics of these viral polypeptides indicated that the 72- and 63-kDa polypeptides were produced abundantly within the initial 36 h. postinfection but were replaced by 56- and 53-kDa polypeptides in the cell and medium, respectively, by 48 h postinfection. The 53-kDa protein was secreted as early as 24 h. postinfection, and accumulation in the medium peaked by 72 h postinfection. Purification of the 53-, 56-, and 63-kDa viral polypeptides was accomplished by anion-exchange and subsequent gel filtration chromatography. Sequence analysis of the 53-, 56-, and 63-kDa HEV polypeptides indicated that the amino terminus was amino acid residue 112 of the predicted full-length protein product. The results of carboxy terminal amino acid sequencing indicated that the carboxy terminus of the 53-, 56-, and 63-kDa HEV proteins was located at amino acid residues 578, 607, and 660, respectively. The molecular masses of the 53- and 56-kDa HEV polypeptides were 53,872 and 56,144 as determined by mass spectroscopy.