Animal Models, Pathogenesis, and Potential Treatment of Thoracic Aortic Aneurysm.

Thoracic aortic aneurysm (TAA) has a prevalence of 0.16-0.34% and an incidence of 7.6 per 100,000 person-years, accounting for 1-2% of all deaths in Western countries. Currently, no effective pharmacological therapies have been identified to slow TAA development and prevent TAA rupture. Large TAAs are treated with open surgical repair and less invasive thoracic endovascular aortic repair, both of which have high perioperative mortality risk. Therefore, there is an urgent medical need to identify the cellular and molecular mechanisms underlying TAA development and rupture to develop new therapies. In this review, we summarize animal TAA models including recent developments in porcine and zebrafish models: porcine models can assess new therapeutic devices or intervention strategies in a large mammal and zebrafish models can employ large-scale small-molecule suppressor screening in microwells. The second part of the review covers current views of TAA pathogenesis, derived from recent studies using these animal models, with a focus on the roles of the transforming growth factor-beta (TGFß) pathway and the vascular smooth muscle cell (VSMC)-elastin-contractile unit. The last part discusses TAA treatment options as they emerge from recent preclinical studies.

Toxocara seroprevalence in Canada-Climate, environment and culture.

Human infection with larvae of canine and feline roundworms belonging to the genus Toxocara can lead to devastating visceral, neural or ocular larvae migrans disease. However, such overt disease represents a fraction of cases. Far more common is covert toxocariasis, a less severe, but clinically symptomatic form of disease, and those who are exposed to infective larvae and seroconvert, but appear to be asymptomatic. Canada represents a unique epidemiological environment for Toxocara infection and exposure. Although the freezing conditions of the vast Arctic Tundra region of the North are thought unlikely to support the lifecycle of Toxocara spp., exposure and seroconversion does occur in people belonging to Inuit communities of this region. Further south, in the sub-Arctic of northern Quebec and Saskatchewan, there is a higher seroprevalence in many Canadian First Nations communities. The epidemiology of these infections is different to that seen in the non-Indigenous communities of the Humid Continental region. Poverty and climate play a major part in the risk of Toxocara seropositive status in Canada, but other factors such as unique cultural practices, population density of humans and reservoir hosts, and contact with wildlife are also factors in exposure and subsequent seroconversion in Canadian communities. This review discusses previous Toxocara seroprevalence studies performed in Canada, summarizes the data for domestic and wild animal reservoir hosts of Toxocara canis, Toxocara cati, Toxocara vitulorum and the closely related helminth, Toxascaris leonina. It also discusses how the unique and varied aspects of climate, culture and environment impacts human Toxocara exposure in Canada.

Effect of differing +2 amino acids on export of a heterologous PhoA lipoprotein in Mycoplasma gallisepticum.

The significance of the amino acid adjacent to the amino terminal cysteine of lipoproteins, the +2 amino acid, has been well documented in E. coli and there have also been limited studies on Gram-positive bacteria. In this study we investigated whether there was any preference for specific residues and any targeting role attributable to different residues following the cysteine at the amino terminus in lipoproteins of Mycoplasma gallisepticum. There were found to be distinct preferences in this position that vary considerably from the preferences seen in Gram-positive and Gram-negative bacteria. The effect of different amino acids at the +2 position was studied using the pTAP vector, which has been shown to express PhoA as a lipoprotein. Replacement of the threonine at the +2 position in the PhoA lipoprotein with hydrophobic amino acids resulted in higher levels of expression of alkaline phosphatase, while replacement with hydrophilic amino acids resulted in lower levels of expression of alkaline phosphatase. Changes in the +2 amino acid did not appear to alter export of the PhoA lipoprotein to the membrane fraction, but a difference was seen in susceptibility to proteolysis in PhoA lipoproteins with differing +2 amino acids. This is the first study to examine the role of the +2 amino acid in mycoplasma lipoproteins and establish a difference between M. gallisepticum and Gram-positive and Gram-negative bacteria and will assist in optimization of the design of recombinant lipoprotein genes in mycoplasmas for maximal levels of expression and stability on the cell surface.

The Effect of an Alternate Start Codon on Heterologous Expression of a PhoA Fusion Protein in Mycoplasma gallisepticum.

While the genomes of many Mycoplasma species have been sequenced, there are no collated data on translational start codon usage, and the effects of alternate start codons on gene expression have not been studied. Analysis of the annotated genomes found that ATG was the most prevalent translational start codon among Mycoplasma spp. However in Mycoplasma gallisepticum a GTG start codon is commonly used in the vlhA multigene family, which encodes a highly abundant, phase variable lipoprotein adhesin. Therefore, the effect of this alternate start codon on expression of a reporter PhoA lipoprotein was examined in M. gallisepticum. Mutation of the start codon from ATG to GTG resulted in a 2.5 fold reduction in the level of transcription of the phoA reporter, but the level of PhoA activity in the transformants containing phoA with a GTG start codon was only 63% of that of the transformants with a phoA with an ATG start codon, suggesting that GTG was a more efficient translational initiation codon. The effect of swapping the translational start codon in phoA reporter gene expression was less in M. gallisepticum than has been seen previously in Escherichia coli or Bacillus subtilis, suggesting the process of translational initiation in mycoplasmas may have some significant differences from those used in other bacteria. This is the first study of translational start codon usage in mycoplasmas and the impact of the use of an alternate start codon on expression in these bacteria.

A novel transposon construct expressing PhoA with potential for studying protein expression and translocation in Mycoplasma gallisepticum.

BACKGROUND: Mycoplasma gallisepticum is a major poultry pathogen and causes severe economic loss to the poultry industry. In mycoplasmas lipoproteins are abundant on the membrane surface and play a critical role in interactions with the host, but tools for exploring their molecular biology are limited. RESULTS: In this study we examined whether the alkaline phosphatase gene (phoA ) from Escherichia coli could be used as a reporter in mycoplasmas. The promoter region from the gene for elongation factor Tu (ltuf) and the signal and acylation sequences from the vlhA 1.1 gene, both from Mycoplasma gallisepticum , together with the coding region of phoA , were assembled in the transposon-containing plasmid pISM2062.2 (pTAP) to enable expression of alkaline phosphatase (AP) as a recombinant lipoprotein. The transposon was used to transform M. gallisepticum strain S6. As a control, a plasmid containing a similar construct, but lacking the signal and acylation sequences, was also produced (pTP) and also introduced into M. gallisepticum . Using a colorimetric substrate for detection of alkaline phosphatase activity, it was possible to detect transformed M. gallisepticum . The level of transcription of phoA in organisms transformed with pTP was lower than in those transformed with pTAP, and alkaline phosphatase was not detected by immunoblotting or enzymatic assays in pTP transformants, eventhough alkaline phosphatase expression could be readily detected by both assays in pTAP transformants. Alkaline phosphatase was shown to be located in the hydrophobic fraction of transformed mycoplasmas following Triton X-114 partitioning and in the membrane fraction after differential fractionation. Trypsin proteolysis confirmed its surface exposure. The inclusion of the VlhA lipoprotein signal sequence in pTAP enabled translocation of PhoA and acylation of the amino terminal cysteine moiety, as confirmed by the effect of treatment with globomycin and radiolabelling studies with [14C]palmitate. PhoA could be identified by mass-spectrometry after separation by two-dimensional electrophoresis. CONCLUSION: This is the first study to express PhoA as a lipoprotein in mycoplasmas. The pTAP plasmid will facilitate investigations of lipoproteins and protein translocation across the cell membrane in mycoplasmas, and the ease of detection of these transformants makes this vector system suitable for the simultaneous screening and detection of cloned genes expressed as membrane proteins in mycoplasmas.

Development and immunogenicity of recombinant Mycoplasma gallisepticum vaccine strain ts-11 expressing chicken IFN-gamma.

Mycoplasma gallisepticum (MG) is a poultry pathogen that causes respiratory disease and loss of egg production worldwide. A live attenuated vaccine, ts-11, has been used for the control of MG in several countries. To improve the functionality of the vaccine and investigate its potential as a delivery vector for host immune molecules and foreign antigens, we have developed ts-11 as a vector to express and secrete chicken IFN-gamma (ts-11 C3) using a transposon-based delivery vector. Following administration of ts-11 C3 in chickens by eye drop, up to 2 weeks post-vaccination, neither significant systemic IFN-gamma expression nor an antibody response as determined by the rapid serum agglutination (RSA) could be detected, while moderate RSA scores were detected in birds vaccinated with ts-11. However, the MG-specific IFN-gamma response in spleen cultures was significantly enhanced in ts-11 C3 vaccinated chickens and, more interestingly, significant heterophil infiltration was detected in the tracheal epithelium in ts-11 C3 vaccinated birds, but not in ts-11 vaccinated birds. These results indicate that the IFN-gamma expressed by ts-11 C3 enhanced host cellular immunity rather than humoral immunity and may also have stimulated mucosal heterophil infiltration. These results also suggest that ts-11 is a promising vector for protective antigens of other chicken respiratory pathogens.