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The ubiquitotis Sm polypeptides B/B' (28 and 29 kD) and the highly homologous tissue-specific Sm N polypeptide (29 kD) share several autoepitopes recognized by systemic lupus erythematosus (SLE) sera. Previous studies on the antigenicity of nuclear antigens recognized by human autoantibodies have not discriminated between ubiquitous and tissue-specific forms. We set out to examine whether a tissue-specific nuclear antigen, Sm N, is autoantigenic in SLE by comparing the immunoreactivity of the most unique sequences in this polypeptide. Synthetic peptides from the two regions of least sequence homology that occur between Sm N and Sm B/B', a dodecamer (amino acid residues 179-190 containing five substitutions) and an undecamer (residues 203-213 containing four substitutions) were coupled to a carrier protein. These conjugates were used to quantify IgG anti-peptide antibodies in sera from patients with SLE, Of 43 sera with anti-Sm specificity, six bound to the B/B' 179-190 peptide but not to the N version. None of 17 anti-Sm-negative SLE sera bound these peptides. The second region of least sequence homology between N and B/B' (203-213) was not antigenic. Our data suggest that a subset of SLE patients with anti-Sm reactivity have IgG autoantibodies capable of discriminating between Sm N and SmB/B' polypeptides by binding a previously unreported SmB/B' specific autoepitope. The data also indicate that brain and heart-specific anti-Sm antibodies do not exist in SLE sera, suggesting that these tissues do not participate in the induction or maintenance of the autoimmune anti-Sm response. [ABSTRACT FROM AUTHOR]

The design of recombinant HSV-1 vectors for delivery of transgenes to the central nervous system is undergoing constant development. Problems associated with the construction and use of such vectors include the requirement for detection of recombinant versus nonrecombinant virus in vitro and also the identification of transduced cells in vivo. This could be overcome by the insertion of reporter genes such as lacZ or green fluorescent protein (GFP) under a separate promoter to the transgene to be expressed. In this case, however, reporter gene expression does not necessarily confirm transgene expression as a separate RNA must be produced. This study reports the use of an encephalomyocarditis virus internal ribosome entry site (IRES) to enable the translation of two reporter genes from a single mRNA transcript driven by the same promoter within a disabled HSV vector, and discusses the potential advantages of this approach. [ABSTRACT FROM AUTHOR]

The safe and efficient use of herpes simplex virus (HSV)-based vectors to deliver genes of potentially therapeutic benefit to the central nervous system will require their effective disablement by the inactivation of viral genes required for lytic growth. Here we report that viruses lacking functional genes for ICP27 (which is required for growth in all cell types) and ICP34.5 (which is required for growth in nondividing cell types) can deliver a marker gene to both the rodent and primate CNS with high efficiency whilst producing relatively minimal damage and having no effect on sodium currents in dorsal root ganglion neurons. Such viruses paradoxically deliver genes at much higher efficiency than the less disabled single mutant lacking ICP34.5 alone and also, as expected, produce less damage in vivo. Moreover, unlike the single mutant lacking ICP27 the double mutant viruses cannot revert to wild-type by acquistion of complimenting gene sequences during growth of virus stocks in vitro on dividing cells expressing ICP27 since artificial expression of ICP34.5 in these cells is not required. Such ICP27-; ICP34.5- viruses thus offer a platform for the development of vectors which are sufficiently safe for ultimate use in human gene therapy. [ABSTRACT FROM AUTHOR]

Herpes simplex virus (HSV) has often been suggested as a vector for gene delivery to the nervous system although it is also capable of infecting many other cell types. HSV also has the ability to package large genetic insertions so the expression of multiple genes from a single virus is possible. Here we show that a green fluorescent protein (GFP) expressing HSV1 vector can transduce two primary human cell types – quiescent human CD34+ hematopoietic progenitor cells and dendritic cells – which are both hard to transduce by other means. We also show that GFP is an effective marker when expressed from an HSV vector in vivo in the mouse brain. When GFP is expressed together with a second gene (in this case lacZ) from a sin-gle virus, transduced GFP-positive CD34+ hematopoietic progenitor cells or dendritic cells can both be generated at an effective efficiency of 100% for the second gene. Here transduction with the vector is combined with flow cytometry allowing GFP-positive cells to be sorted from the untransduced population. Such completely transduced populations of quiescent CD34+ hematopoietic progenitor and dendritic cells cannot easily be achieved by other means, and might thus allow experimental or therapeutic protocols to be carried out requiring high-level transduction which would not otherwise be possible. Such an approach using HSV vectors might also be applicable to other cell types for which transduction is as yet unreliable or of low efficiency. [ABSTRACT FROM AUTHOR]

Direct injection of viral vectors into the central nervous system has become a valuable technique for exploring the function of neurological systems and is a potential therapy for neural disease. To this end a number of herpes simplex virus (HSV)-derived vectors are currently being developed for the introduction of foreign DNA into the brain. In this study a non-neurovirulent HSV 17+ mutant, variant 1716, deleted in the γ34.5 gene and expressing the marker gene lacZ under the control of the latency-associated transcripts promoter was injected stereotactically into the central nervous system of two strains of rat (AO and PVG). We show (1) that transgene expression was low at the site of injection, in the striatum, at all times studied (12 h to 30 days after injection); (2) dramatically more transgene expression was observed at distant sites which contain neurons projecting directly to the site of injection, with maximal expression at these sites being at 1–2 days; (3) immunostaining with a polyclonal anti-HSV antibody and with an antibody which detects a 65 kDa HSV DNA binding protein (the product of the UL42 gene of the virus) demonstrated that viral gene products could be detected at the injection site as early as 12 h and up to 1 week after injection. Moreover these could also be detected at several secondary sites not all of which have direct connections with the injection site. These findings suggest that γ34.5 negative vectors have potential for gene transfer but may require further attenuation to limit viral antigen expression before they can be used successfully for gene therapy in the brain. [ABSTRACT FROM AUTHOR]

The myocardium is a potential target for the expression of exogenous genes to treat inherited and acquired diseases. Although adenovirus-mediated gene transfer has resulted in high-level gene transfer in vivo via direct intramyocardial injection and via a percutaneous intra-arterial route, the time-course of gene expression is limited by host immune responses. It was the aim of this study to test whether cationic liposome-mediated gene transfer, which does not suffer from the aforementioned problems, was feasible in the adult rabbit myocardium via a percutaneous transluminal approach. Doses of plasmid DNA encoding lacZ from 200–800 μg complexed to cationic liposomes resulted in X-gal conversion at day 3 with associated myocardial damage. We hypothesised that the damage was associated with macro-aggregates of cationic liposomes–DNA occluding the microcirculation. When such aggregates were excluded no X-gal conversion was seen in vivo. In order to show that X-gal conversion occurs in areas of infarction in the myocardium we caused closed chest infarction by deploying a platinum micro-embolisation coil in the circumflex coronary artery. At day 3 X-gal conversion was observed in the territory supplied by the occluded artery. Thus, microinfarction causes the false positive appearance of gene transfer when using a lacZ reporter gene. [ABSTRACT FROM AUTHOR]

There is increasing evidence to suggest that abnormalities in apoptosis may play a part in the pathogenesis of systemic lupus erythematosus (SLE). For example, there is now considerable evidence that bel-2 expression is enhanced in a proportion of peripheral T cells, but not in B cells, in SLE patients and correlates with overall disease activity regardless of the activity index employed. Further work is required to establish whether enhanced bel-2 expression by some T cells in SLE patients is related to their activation or intrinsically enhanced by genetic predisposition. Mutations in Fas result in a lymphoproliferative syndrome and may play a role in accelerating autoimmune disease. A report of three children with mutations in Fas has once again focused attention on this regulator of apoptosis. The relationships between inducers and inhibitors of apoptosis may differ in different cell types, and must be elucidated before the implications of observations made in lymphocytes from SLE patients can be fully understood. [ABSTRACT FROM PUBLISHER]