Truncation of the mu heavy chain alters BCR signalling and allows recruitment of CD5+ B cells.

Ig are multifunctional molecules with distinct properties assigned to individual domains. To assess the importance of IgM domain assembly in B cell development we generated two transgenic mouse lines with truncated muH chains by homologous integration of the neomycin resistance gene (neo(r)) into exons C(mu)1 and C(mu)2. Upon DNA rearrangement shortened muH chain transcripts, V(H)-D-J(H)-C(mu)3-C(mu)4, are produced independent of the transcriptional orientation and termination signals provided by neo(r). The truncated muH chain of approximately 52 kDa associates non-covalently with the L chain to form a monovalent HL heterodimer. Surface IgM is assembled into a defective BCR complex which has lost important signalling capacity. In immunizations with T-dependent and T-independent antigens, specific IgM antibodies cannot be detected, whilst IgG responses remain normal. B cell development in the bone marrow is characterized by an increase in early B cells, but a decrease of B220(+) cells from the stage when muH chain rearrangement is completed. The peritoneal lymphocyte population has elevated levels of CD5(+) B cells and their expansion may be the result of a negative feedback mechanism. The results show that antigenic stimulation is compromised by truncated monovalent IgM and that this deficit in stimulation leads to reduced levels of conventional B-2 lymphocytes, but dramatically increased levels of B-1 cells.

Cationic lipids and polymers are able to enhance adenoviral infection of cultured mouse myotubes.

Adenovirus-mediated gene transfer has been used to promote efficient expression of various reporter and therapeutic transgenes such as minidystrophin in skeletal muscle tissue. However, down-regulation of the adenovirus internalisation receptors, alpha(v)/beta3 and alpha(v)beta5, in adult myofibres and in mature cultured myotubes makes them less susceptible to infection than neonatal muscle or cultured myoblasts. It has been reported elsewhere that adenoviral transduction of cells that are normally refractory to infection can be enhanced by complexing virus particles with cationic lipids or cationic polymers. In this study we describe increased levels of adenovirus-mediated transduction of cultured C2C12 myotubes, when the vector is complexed with either of the cationic lipids Lipofectamine or 1,3-dioleoyloxy-2-(6-carboxyspermyl)propylamide (DOSPER) or the cationic polymer polyethylenimine. The presence of polycations allowed a smaller dose of adenovirus vector to be used to attain the same level of infection seen with adenovirus alone, which has important relevance to future in vivo studies. Electron microscopic analysis of adenovirus/polycation complexes showed large aggregates as opposed to single adenovirus particles in the absence of polycations. Finally, by complexing adenovirus particles with polycations, partial protection against the neutralising effect of adenovirus antiserum was observed.

Dystrophin expression in the hair cells of the cochlea.

Dystrophin is normally expressed in a number of tissues including muscle, brain and the outer plexiform layer of the retina. In Duchenne and Becker muscular dystrophy abnormal or deficient dystrophin expression leads to muscle degeneration and has been implicated in mental retardation and a form of night blindness. We have examined the expression of dystrophin immunoreactivity in cochlear tissues of normal guinea-pig and mouse, and whether expression is perturbed in the cochlea of the dystrophic MDX mouse. A single band of approximately 427 kDa, corresponding to a full-length isoform of dystrophin was detected in guinea-pig and normal mouse but was absent from the MDX mouse. Cochleae from guinea-pig, normal and MDX mouse also showed a second dystrophin isoform of 116 kDa molecular weight with the C-terminal specific antibody. Immunostained guinea pig cochlear half turns were examined by laser scanning confocal microscopy. Dystrophin was localized in both inner and outer hair cells with staining patterns which were qualitatively similar with both antibodies. In the outer hair cells labelling of the lateral wall was especially distinctive. The synaptic region of both hair cell types was also strongly labelled.

Adenovirus-mediated gene transfer to murine retinal cells in vitro and in vivo.

Adenovirus-mediated gene transfer to retinal cells was evaluated using the replication-defective recombinant adenovirus vector Ad2/CMVlacZ-1 (coding for beta-galactosidase) both in an in vitro murine culture model and in vivo in adult mice. In vitro, no difference in infectability of neuronal and glial cells was observed, and 50% of neurons expressed the exogenous gene at low viral concentration (10 pfu/cell). In vivo, intraocular injection of 3 x 10(6) pfu Ad2/CMVlacZ-1 resulted in expression of the transferred beta-galactosidase gene in retinal pigment epithelium and ganglion cells. These results demonstrate that Ad2/CMVlacZ-1 is an effective vector for gene transfer into retinal cells.

Independent localization of dystrophin N- and C-terminal regions to the sarcolemma of mdx mouse myofibres in vivo.

Dystrophin has been proposed to associate with the skeletal muscle membrane by way of a glycoprotein complex that interacts with its C-terminal domains. Transfection of mdx mouse myotubes in culture or myofibres in vivo with recombinant genes encoding human dystrophin deletion mutants shows, however, that not only the C terminus of dystrophin but also its N-terminal actin-binding domain can locate independently to the muscle sarcolemma. This observation suggests that lack of sarcolemma-associated dystrophin in Duchenne muscular dystrophy (DMD) muscle may result from enhanced degradation of truncated mutation products rather than their inability per se to associate with the sarcolemma.

Human dystrophin gene transfer: production and expression of a functional recombinant DNA-based gene.

The identification and cloning of the gene responsible for Duchenne muscular dystrophy (DMD) and characterization of the protein product of the gene, dystrophin, has led to major advances in diagnostic and genetic counselling procedures for this inherited disorder. Due to its high mutation rate, however, individuals affected by DMD will continue to arise in large proportion by de novo mutations, and the search for direct therapies remains a high priority. In this respect direct genetic correction of dystrophin deficiency via grafting of healthy myoblast stem cells or direct introduction of functional DNA into diseased muscle tissue have both been proposed as potential therapeutic approaches. We describe here, the first example of the engineering and cloning of a synthetic gene encoding recombinant human dystrophin and its stable transfer to and expression in mammalian cells. This DMD gene construction represents a primary step towards evaluating direct DNA-mediated gene transfer as a potential treatment for this debilitating disorder.