The human IgG3 hinge mediates the formation of antigen dimers that enhance humoral immune responses to DNA immunisation.

A series of plasmid DNA constructs containing the 45W antigen gene from Taenia ovis were used to investigate the impact of antigen dimerisation on the humoral immune response to genetic immunisation. Genes encoding dimeric 45W were generated via fusion to the hinge region of human IgG3 (hIg). This region was selected because it is compact and contains 11 inter-chain disulphide-bridges. The DNA encoding the IgG3 hinge contains four exons, with the last three exons being repeats and possibly superfluous. Plasmids containing the 45W gene linked to exons 1-2, 1-3 or 1-4 of the hIgG3 hinge, were compared to a control plasmid containing a form of the 45W gene which encodes secreted, monomeric 45W protein. Western blot analysis was used to investigate the formation of the fusion-proteins in transfected Cos-7 cells. The full-length fusion construct expressed predominantly dimeric forms of the fusion-protein, while truncation of the hinge region decreased the abundance of dimeric fusion-protein and increased the proportion monomeric fusion antigen. In immunised BALB/c mice, 45W-specific antibody titres were increased 3 to 4-fold via fusion to the full-length hinge region, whereas the truncated constructs were similar to the control. IgG subclass analysis indicated that all mice generated predominantly IgG1, IgG2a and IgG2b antibodies. Therefore, these results suggest that the efficient formation of dimeric antigen, via fusion to the full-length hinge of human IgG3, can increase the immunogenicity of expressed antigens without altering the form of the immune response elicited by DNA immunisation.     

Nonrandom tissue distribution of mutant mtDNA

Heteroplasmic mitochondrial DNA (mtDNA) defects are an important cause of inherited human disease. On a cellular level, the percentage of mutant mtDNA is the principal factor behind the expression of the genetic defect. Marked variation in the level of mutant mtDNA among tissues is thought to be responsible for the diverse clinical phenotypes associated with the same pathogenic mtDNA mutation. This study was designed to determine whether the percentage level of a pathogenic mtDNA molecule is determined by a purely random process. The tissue distribution of the A3243G MELAS point mutation was analyzed in five individuals who were members of a family with maternally inherited diabetes and deafness. The level of mutant mtDNA was measured in four tissues in three individuals and three tissues in two individuals. The highest level of mutant mtDNA occurred in skeletal muscle, followed by hair follicles, and then buccal mucosa, with the lowest levels in blood (leucocyte/platelet fraction). The probability of observing any strict hierarchy in family is 4.82 × 10⁻⁵. These results indicate that the distribution of the A3243G mutation is not solely determined by random processes. Am. J. Med. Genet. 85:498–501, 1999. © 1999 Wiley-Liss, Inc.     

Molecular analysis of cytochromec Oxidase deficiency in Leigh's syndrome

Cytochrome c oxidase deficiency is the most common biochemical defect associated with Leigh's syndrome. The genetic defect responsible for this deficiency has not been identified in any patient with Leigh's syndrome. Given that this disorder appears to be inherited as an autosomal recessive trait, this would suggest prima facie that one of the nuclear DNA—encoded cytochrome c oxidase subunits is affected. We report the first detailed sequence analysis of all 10 cytochrome c oxidase nuclear complementary DNAs and the cytochrome c oxidase mitochondrial genes in a Leigh's syndrome patient with cytochrome c oxidase deficiency. No pathological mutations were identified in any of the cytochrome c oxidase structural genes.     

Role of mitochondrial DNA mutations in human aging: Implications for the central nervous system and muscle

It has been proposed that one mechanism for nerve and muscle dysfunction with age involves the mitochondria. Mitochondria contain the only DNA outside the nucleus in mammalian cells. Mitochondrial DNA (mtDNA) has a high mutation rate, and low levels of pathogenic mutations have been found in tissues from elderly subjects. However, the role of these mutations in the aging process is uncertain unless a mechanism can be identified that would lead to a biochemical defect. In muscle tissue from normal elderly subjects we show that there are muscle fibers with very low activity of cytochrome c oxidase, suggestive of a mtDNA defect. In these cytochrome c oxidase-deficient fibers we have found very high levels of mutant mtDNA. In addition, different mtDNA mutations are present in different fibers, which explains why there is a low overall incidence of an individual mutation in tissues from elderly subjects. These studies show a direct age-related correlation between a biochemical and genetic defect in normal human tissues and that mtDNA abnormalities are involved in the aging process in human muscle.     

Synthetic peptide antigens induce antibodies to Taenia ovis oncospheres

Sheep immunised with the Taenia ovis recombinant 45W antigen are protected from infection with the parasite. Two peptides were synthesised corresponding to putative host-protective regions at the N- and C-termini of 45W. Sera from sheep immunised with 45W or related recombinant proteins reacted strongly with the N-terminal peptide. Approximately 40% of the antibody directed against 45WB/X, a truncated form of 45W, was found to be directed against the N-terminal peptide sequence. Sheep were immunised with the N- and C-terminal peptides alone or conjugated to a carrier protein. The N-terminal peptide was found to be highly immunogenic whereas the C-terminal peptide required conjugation to a carrier protein to be immunogenic. Antibodies raised against each of these immunogens crossreacted with the parent protein, 45WB/X, however, only antibodies specific for the N-terminal peptide were found to bind to antigens from the T. ovis oncosphere.     

Excellent Local Control of Paraganglioma in the Head and Neck with Fractionated Radiotherapy

AimsRadiotherapy is an important treatment option for paraganglioma in the head and neck region. It seems to be highly effective and avoids important surgical morbidity, which can impair quality of life. The aim of this study was to evaluate the outcomes of radiotherapy for paraganglioma of the head and neck region in order to inform our future practice.Materials and methodsThe cohort of patients for the present study comprised 21 patients who received radiotherapy between 1998 and 2008. Follow-up ranged from 6 to 132 months, median 55 months. The mean age was 48.7 years, range 20–78 years. The female:male ratio was 2 : 1. Two patients had confirmed familial tumour syndromes. The gross tumour volume in 20 cases ranged from 1.3 to 74 cm³, mean 23.2 cm³, median 14.7 cm³. Five patients were treated with intensity-modulated radiotherapy. The median dose was 50 Gy in 30 fractions.ResultsThe crude 5-year local control rate was 95% (20/21), although the 5-year actuarial local control rate was 87%. The one patient who relapsed, at 45 months after radiotherapy, had a comparatively small tumour of 10.8 cm³. A relationship between tumour volume and local control seems unlikely. It was possible to obtain details of side-effects from electronic records for 11 patients. Grade 3 headache, which resolved, was the most serious acute side-effect. One patient had three teeth extracted due to exacerbation of dental caries, and one had deterioration of hearing thought to be due to a combination of tumour and radiotherapy. There were two serious complications in patients who had embolisation, which we no longer use.ConclusionsOur results show a high level of efficacy for fractionated external beam radiotherapy, with minimal toxicity, in keeping with other series. This should encourage the use of radiotherapy as primary treatment for paragangliomas of the head and neck region.     

Induction of protection against porcine cysticercosis by vaccination with recombinant oncosphere antigens

Two recombinant Taenia solium oncosphere antigens, designated TSOL18 and TSOL45-1A, were investigated as vaccines to prevent transmission of the zoonotic disease cysticercosis through pigs. Both antigens were effective in inducing very high levels of protection (up to 100%) in three independent vaccine trials in pigs against experimental challenge infection with T. solium eggs, which were undertaken in Mexico and Cameroon. This is the highest level of protection that has been achieved against T. solium infection in pigs by vaccination with a defined antigen. TSOL18 and TSOL45-1A provide the basis for development of a highly effective practical vaccine that could assist in the control and, potentially, the eradication of human neurocysticercosis.