A truncating PET100 variant causing fatal infantile lactic acidosis and isolated cytochrome c oxidase deficiency

Isolated mitochondrial complex IV (cytochrome c oxidase) deficiency is an important cause of mitochondrial disease in children and adults. It is genetically heterogeneous, given that both mtDNA-encoded and nuclear-encoded gene products contribute to structural components and assembly factors. Pathogenic variants within these proteins are associated with clinical variability ranging from isolated organ involvement to multisystem disease presentations. Defects in more than 10 complex IV assembly factors have been described including a recent Lebanese founder mutation in PET100 in patients presenting with Leigh syndrome. We report the clinical and molecular investigation of a patient with a fatal, neonatal-onset isolated complex IV deficiency associated with multiorgan involvement born to consanguineous, first-cousin British Asian parents. Exome sequencing revealed a homozygous truncating variant (c.142C>T, p.(Gln48*)) in the PET100 gene that results in a complete loss of enzyme activity and assembly of the holocomplex. Our report confirms PET100 mutation as an important cause of isolated complex IV deficiency outside of the Lebanese population, extending the phenotypic spectrum associated with abnormalities within this gene.     

Serum antibody response following parenteral immunization with hydatid cyst fluid in sheep infected with Echinococcus granulosus

Production of specific serum antibodies following immunization with hydatid cyst fluid antigens was investigated in sheep with Echinococcus granulosus infection and in noninfected controls. Six of 10 infected animals responded to intramuscular injection of antigen by rapid production of antibodies detected in indirect hemagglutination assays. Similar responses did not occur in any of 10 noninfected controls. It is suggested that differences in the rate of response to immunization with cyst fluid antigens between groups of sheep could be exploited in serodiagnosis of E. granulosus infection in sheep. The results also suggest that low levels of antibody found in the serum of sheep infected with E. granulosus are not the result of immunosuppression or immunological tolerance, but are due to sequestration of antigen from the immune system of the host.     

Epitope specificities and antibody responses to the EG95 hydatid vaccine

Antibody isotype and epitope specificities were examined in sheep immunized with EG95, a protective recombinant vaccine against hydatid disease. All sheep immunized with EG95 as a fusion protein with glutathione S-transferase (GST) produced prominent IgG antibodies against the EG95 portion of the protein. Linear, antibody-binding epitope specificities of EG95 were mapped using a series of 25 overlapping synthetic peptides. Three immunodominant regions were identified which generated specific IgG₁ and IgG₂ antibodies in the majority of vaccinated sheep. These regions corresponded to the EG95-derived sequences SLKAVNPSDPLVYKRQTAKF, DIETPRAGKKESTVMTSGSA and SALTSAIAGFVFSC. An additional immunogenic region was identified which induced almost exclusively IgG₂ antibody. This epitope was located within the sequence TETPLRKHFNLTPV. The anti-parasitic, protective effects of the EG95 vaccine correlated with the detection of specific antibody to two or more of the four linear immunogenic regions. The identification of these immunogenic peptides of EG95 maybe useful in the development of a synthetic peptide vaccine as a derivative of the EG95 recombinant.     

Protection against hydatid disease induced with the EG95 vaccine is associated with conformational epitopes

This paper describes attempts to map the location of host-protective epitopes of a recombinant vaccine antigen by assessing the ability of truncated regions of the antigen to elicit protective immune responses in sheep. Sheep were immunised with three truncated regions (EG95-1, EG95-2 and EG95-3) of the hydatid vaccine antigen, EG95. These regions overlapped each other and corresponded to amino acids 1-70 (EG95-1), 51-106 (EG95-2) and 89-153 (EG95-3) of the full length recombinant protein. Each region elicited antibody which reacted with the parent antigen, although these reactivities were a small proportion of the level of reactivity generated by immunisation with the full length antigen. Antisera raised against each of the truncated proteins reacted with the native parasite antigen. In vaccination and parasite challenge trials in sheep, none of the truncated regions elicited significant protection against challenge infection or antibody which was lethal to the parasite in vitro. Antibodies from sheep immunised with the combination of all three overlapping truncations elicited a comparatively low but significant level of lysis of the parasite in vitro. These antigens did not inhibit anti-EG95 antibody reactivity with EG95 nor did they inhibit in vitro oncosphere killing induced by anti-EG95 antibodies. These results indicate that the major part of the immune response induced by EG95 vaccination is directed against conformational epitopes and that the host-protective epitope(s) is/are conformational.     

Accumulation of Nav1 mRNAs at differentiating postsynaptic sites in rat soleus muscles

Acetylcholine receptors (AChRs) and voltage-gated sodium channels (Na(V)1s) accumulate at different times in the development of the murine neuromuscular junction (NMJ). We used in situ hybridization to study the relationship of Na(V)1 mRNA accumulation to this difference. mRNAs encoding both muscle Na(V)1 isoforms, Na(v)1.4 and Na(v)1.5, were first concentrated at NMJs at birth, when the proteins start to accumulate. Within 4 weeks, Na(v)1.4 mRNA increased 5-fold at the NMJ while Na(v)1.5 mRNA became undetectable. Na(V)1 mRNA accumulation occurred even if the nerve was cut at birth. Like AChR mRNA, Na(V)1 mRNA accumulated at denervated synaptic sites on regenerating muscles and in response to ectopically expressed neural agrin. Clustering of Na(V)1 at the NMJ follows that of its mRNA while AChR clustering precedes its mRNA clustering by several days. This suggests that factors other than local mRNA upregulation determine the timing of clustering of these two important postsynaptic ion channels.     

Cysticercosis/taeniasis in Asia and the Pacific

Three taeniid tapeworms infect humans in Asia and the Pacific: Taenia solim, Taenia saginata, and Taenia asiatica. Although there is continuing debate about the definition of a new species, phylogenetic analyses of these parasites have provided multiple lines of evidence that T. asiatica is an independent species and the sister species of T. saginata. Here we review briefly the morphology, pathology, molecular biology, distribution and control options of taeniasis/cysticercosis in Asia and the Pacific and comment on the potential role which dogs may play in the transmission of T. solium. Special attention is focused on Indonesia: taeniasis caused by T. asiatica in North Sumatra, taeniasis/cysticercosis of T. solium and taeniasis of T. saginata in Bali, and taeniasis/cysticercosis of T. solium in Papua (formerly Irian Jaya). Issues relating to the spread of taeniasis/cysticercosis caused by T. solium in Papua New Guinea are highlighted, since serological evidence suggests that cysticercosis occurs among the local residents. The use of modern techniques for detection of taeniasis in humans and cysticercosis in humans, pigs and dogs, with the possible adoption of new control measures will provide a better understanding of the epidemiology of taeniasis/cysticercosis in Asia and the Pacific and lead to improved control of zoonotic and simultaneously meat-borne disease transmission.     

Molecular cloning of a vaccine antigen against infection with the larval stage of Echinococcus multilocularis

Alveolar and cystic hydatidosis are caused by infection with the larval stages of Echinococcus multilocularis and Echinococcus granulosus, respectively. A host-protective antigen has been identified in E. granulosus. Here we identify the presence of a closely related protein in E. multilocularis, characterize and express a cDNA encoding the antigen (designated EM95), determine the structure of the em95 gene, and demonstrate that the EM95 recombinant protein can be used to induce significant levels of protection against challenge infection with E. multilocularis eggs in mice.     

The comparative efficacy of CTLA-4 and L-selectin targeted DNA vaccines in mice and sheep

The access of antigens to antigen presenting cells (APCs) appears to be a rate-limiting step in the generation of immune responses to DNA vaccines. The cytotoxic T lymphocyte antigen 4 (CTLA-4) and L-selectin represent attractive ligands for use in the targeting of antigen to APCs and lymph nodes. CTLA-4 binds with high affinity to the B7 membrane antigen on APCs, while L-selectin functions as a lymphocyte homing marker and binds to CD34 on the surface of high endothelial venule cells. DNA vaccines encoding human immunoglobulin (HIg), fused to either CTLA-4 or L-selectin, have been shown to generate up to 10,000-fold higher anti-HIg antibody responses than DNA vaccines encoding HIg alone. In this study, the ability of CTLA-4 or L-selectin mediated targeting to enhance the humoral immune response to an alternate vaccine antigen was investigated. DNA vaccines encoding CTLA-4-HIg and L-selectin-HIg fused to the host-protective 45W antigen from Taenia ovis were constructed. In BALB/c mice, the L-selectin targeted vaccine did not improve either the magnitude or speed of antibody responses of vaccinated mice. In contrast, the CTLA-4 targeted DNA vaccine generated 45W-specific antibody responses which were up to 30-fold higher than those achieved with non-targeted DNA vaccination. The kinetic of the antibody response generated following CTLA-4 targeted DNA vaccination was also significantly faster than that achieved with non-targeted DNA vaccination, or with adjuvanted protein vaccination. Vaccination of outbred sheep with DNA vaccines expressing either murine or ovine CTLA-4 targeted antigen failed to enhance immune responses. These findings indicate that CTLA-4 targeting may find application in the improvement of DNA vaccines, but requires further development for applications in large animal species.     

Molecular basis for treatment of mitochondrial myopathies

Mitochondrial DNA (mtDNA) is the only autonomously replicating source of DNA outside the nucleus. The mitochondrial genome encodes thirteen essential polypeptides of the mitochondrial respiratory chain. Defects of the mitochondrial genome can cause severe neurological and multi-systemic disorders. In many patients there is a mixture of mutated and wild-type mtDNA in the same cell (a situation termed heteroplasmy). In these patients the ratio of mutated to wild-type mtDNA is crucial and a biochemical defect only occurs with relatively high levels of mutated mtDNA within an individual cell. This threshold also seems to be critical in the development of mtDNA disease. Since the gentic defect causes a dysfunction in the terminal stage of oxidative metabolism, there is little potential for pharmacological intervention. Molecular techniques must be developed to reverse the ratio of mutated and wild-type mtDNA. In this paper we summarise our approach using both antigenomic peptide nucleic acis and cell necrosis.