Screening for MECP2 mutations in Spanish patients with an unexplained mental retardation.

Rett syndrome (RTT) is an X-linked progressive encephalopathy. Mutations in the MECP2 (methyl-CpG-binding protein) gene have been found to cause RTT. In the past few years, the role of MECP2 mutations in patients with mental disorders other than RTT has been studied, finding that mutations in MECP2 also contribute to non-syndromic entities. More recently, it has been demonstrated that RTT shares clinical features with those of Angelman syndrome, another neurodevelopmental disorder. These observations must be confirmed in a large series, to better understand the criteria needed for justifying a molecular test. Consequently, we have searched for MECP2 mutations in 294 patients (43 Angelman and Prader-Willi like included) with mental retardation (MR) of unknown aetiology. We found six polymorphisms (three novel, three previously reported) in 10 patients, one novel unclassified silent change (p.V222V) in a man, and one causative mutation in a girl with MR. Once this case was clinically reviewed, the girl presented symptoms of atypical RTT. The mutation (p.Y141C) lies within the methyl-binding domain, and has only been reported once in another atypical RTT. Our results show that the MECP2 mutations account for a low frequency (1/416 chromosomes = 0.24%) among mentally retarded individuals, which imply that it is necessary to perform an exhaustive clinical examination of patients before determining whether analysis of MECP2 is required or not.

Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus.

Porcine T-cell recognition of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP) was tested using in vitro lymphoproliferative responses. Lymphocytes were obtained from outbred pigs experimentally infected with FMDV. Of the different NSP, polypeptides 3A, 3B, and 3C gave the highest stimulations in the in vitro assays. The use of overlapping synthetic peptides allowed the identification of amino acid regions within these proteins that were efficiently recognized by the lymphocytes. The sequences of some of these antigenic peptides were highly conserved among different FMDV serotypes. They elicited major histocompatibility complex-restricted responses with lymphocytes from pigs infected with either a type C virus or reinfected with a heterologous FMDV. A tandem peptide containing the T-cell peptide 3A[21-35] and the B-cell antigenic site VP1[137-156] also efficiently stimulated lymphocytes from infected animals in vitro. Furthermore, this tandem peptide elicited significant levels of serotype-specific antiviral activity, a result consistent with the induction of anti-FMDV antibodies. Thus, inclusion in the peptide formulation of a T-cell epitope derived from the NSP 3A possessing the capacity to induce T helper activity can allow cooperative induction of anti-FMDV antibodies by B cells.

Recombinant viruses expressing the foot-and-mouth disease virus capsid precursor polypeptide (P1) induce cellular but not humoral antiviral immunity and partial protection in pigs.

The importance of the induction of virus neutralizing antibodies to provide protection against foot-and-mouth disease virus (FMDV) infection is well established. However, recent studies with recombinant adenovirus expressing the precursor polypeptide of the viral capsid (P1) indicate that cattle inoculated with this recombinant vector developed partial protection against FMDV infection, in the absence of a detectable specific humoral response. Other viral vectors have been widely used to induce protective immunity against many pathogens, and it has been reported that the use of different vectors for priming and boosting injections can provide a synergistic effect on this response. In this work, we determined the immunogenicity of two recombinant viruses (adenovirus and vaccinia) expressing P1-FMDV, administered either individually or sequentially, and the protection that they induced against FMDV challenge in pigs. A double immunization with the adeno-P1 virus was the most effective strategy at inducing protective immunity. In contrast to previous reports, the use of two different vectors for priming and boosting did not show a synergistic effect on the protection induced against FMD. Interestingly, immunized pigs developed FMDV-specific T cell responses but not detectable antibodies. Thus, the protection observed was likely to be mediated by a cellular immune response.

Evidence of partial protection against foot-and-mouth disease in cattle immunized with a recombinant adenovirus vector expressing the precursor polypeptide (P1) of foot-and-mouth disease virus capsid proteins.

A recombinant live vector vaccine was produced by insertion of cDNA encoding the structural proteins (P1) of foot-and-mouth disease virus (FMDV) into a replication-competent human adenovirus type 5 vaccine strain (Ad5 wt). Groups of cattle (n = 3) were immunized twice, by the subcutaneous and/or intranasal routes, with either the Ad5 wt vaccine or with the recombinant FMDV Ad5-P1 vaccine. All animals were challenged by intranasal instillation of FMDV 4 weeks after the second immunizations. In the absence of a detectable antibody response to FMDV, significant protection against viral challenge was seen in all of the animals immunized twice by the subcutaneous route with the recombinant vaccine. The observed partial protection against clinical disease was not associated with a reduction in titre of persistent FMDV infections in the oropharynx of challenged cattle.

Analysis of the B and T cell response in guinea pigs induced with recombinant vaccinia expressing foot-and-mouth disease virus structural proteins.

Recombinant vaccinia viruses expressing foot-and-mouth disease virus (FMDV) P1 and VP1 genes have been used to study the immune response induced by these viral polypeptides in guinea pigs. Anti-FMDV antibodies, but not neutralizing activity, were detected in the sera from immunized animals. The results indicate that both CD4+ and CD8+ FMDV-specific T cells were induced by the vaccinia recombinants. Consistently with the activation of CD4+ T cells, lymphocytes from immunized animals specifically proliferated in vitro in response to whole virus. The induction of virus-specific CD8+ T cells was determined by CTL assay of immune splenocytes restimulated in vitro with FMDV infected cells. Altogether, the results obtained indicate that both B and T cell immune responses to FMDV are elicited upon immunization of guinea pigs with vaccinia recombinants expressing FMDV structural polypeptides.

Infection with foot-and-mouth disease virus results in a rapid reduction of MHC class I surface expression.

The modulation of MHC class I molecule expression on the surface of cells as a consequence of foot-and-mouth disease virus (FMDV) infection has been examined. On cells infected with FMDV, class I expression was reduced to approximately 70% of the initial value 3 h after the infection and to 53% after 6 h. On cells depleted of surface class I complexes by acid treatment, the appearance of newly assembled class I-peptide complexes on the cell surface of non-infected cells increased immediately upon neutralization and original class I levels were recovered in about 20 h. In contrast, the appearance of new peptide-bound class I molecules on the cell surface was inhibited as early as 30 min after FMDV infection. Since the shut-down of FMDV-mediated host protein synthesis occurs approximately 2-3 h post-infection, this result suggests that an earlier event, which prevents the surface expression of newly synthesized complexes, is induced following FMDV infection. Thus, FMDV-infected cells rapidly become unable to present viral peptides in association with MHC class I molecules to T lymphocytes. Such a mechanism would assist virus evasion of the cytotoxic immune response of the host.