Circulating biomarkers are not associated wtih endoleaks after endovascular repair of abdominal aortic aneurysms

Objective: Endoleak is a common complication of endovascular repair (EVAR) for abdominal aortic aneurysm (AAA), but can only be detected through prolonged follow-up with repeated aortic imaging. This study examined the potential for circulating matrix metalloproteinase-9 (MMP9), osteoprotegerin (OPG), D-dimer, homocysteine (HCY) and C-reactive protein (CRP) to act as diagnostic markers for endoleak in AAA patients undergoing elective EVAR. Methods: Linear mixed effects models were constructed to assess differences in AAA diameter after EVAR, between groups of patients who did, and did not develop endoleak during follow-up, adjusting for potential confounders. Circulating MMP9, OPG, D-dimer, HCY and CRP concentrations were measured in pre- and post-operative plasma samples. The association of these markers with endoleak diagnosis was assessed using linear mixed effects adjusted as above. The potential for each marker to diagnose endoleak was assessed using receiver operator characteristic (ROC) curves. Results: Seventy-five patients were included in the current study, 24 of whom developed an endoleak during follow-up. Patients with an endoleak had significantly large AAA sac diameters than those that did not have an endoleak. None of the assessed markers showed a significant association with endoleak. This was confirmed through ROC curve analyses indicating poor diagnostic ability for all markers. Conclusions: Circulating concentrations of MMP9, OPG, D-dimer, HCY and CRP were not associated with endoleak in patients undergoing EVAR in this study

Immune responses in Burkholderia pseudomallei infection, relapse, reactivation and protection

Burkholderia pseudomallei is the bacterium that causes the potentially fatal disease\ud melioidosis. Endemic regions include South-east Asia and northern Australia. The\ud immunopathogenesis of melioidosis is not fully understood as the bacterium can evade\ud the host immune responses surviving within cells becoming apparent when the host is\ud immunocompromised. Since B. pseudomallei is an intracellular organism, cell-mediated\ud immune (CMI) responses are important in clearance and protection. The processes\ud involved in the induction of a protective immune response is necessary to understand\ud how the disease would progress and for developing vaccine strategies against\ud B. pseudomallei. Therefore, the focus of the research in this thesis is to determine the\ud extent of exposure to B. pseudomallei in northern Queensland and to characterise the\ud CMI responses of seropositive healthy individuals from this endemic region. The\ud immune responses involved in relapse and latent B. pseudomallei infection will also be\ud determined by analysing the clonality of isolates from patients with recurrent melioidosis\ud and developing a murine model of latency. The protective effect of DNA constructs of\ud putative virulence factors will be studied in an experimental model of melioidosis.\ud \ud Using the indirect haemagglutination assay, 1500 blood donors from northern\ud Queensland were assessed for antibodies to B. pseudomallei. The results demonstrated\ud 2.47% (n=37) of the population had an antibody response to B. pseudomallei with a titre\ud of ≥1:40. Using these seropositive donors, the Melioidosis IgG Rapid Cassette test was\ud evaluated as a potential screening test for melioidosis. The results found only 32% were\ud positive using this rapid test. This study demonstrated that individuals living in endemic\ud regions may have been exposed to B. pseudomallei and consequently produced an\ud antibody response. By characterising the CMI response of these seropositive individuals\ud it will be possible to determine whether these individuals develop protective immunity\ud against the bacterium.\ud \ud Lymphocyte proliferation assays and flow cytometry were used to characterise the CMI\ud response in seropositive individuals (n=8). Lymphocytes were isolated and stimulated with B. pseudomallei antigen (BpLy1) the proliferative response was assessed, as well as\ud activation of T cell subsets and cytokine production. Results demonstrated no significant\ud differences in the proliferative response of lymphocytes in seropositive individuals when\ud compared to seronegative individuals. There were no significant differences observed in\ud CD4+ and CD8+ populations, interferon (IFN)-γ or interleukin (IL)-4 production. These\ud results did not demonstrate an induction of a CMI response following stimulation with\ud BpLy1. Although these seropositive individuals may have produced an antibody\ud response to B. pseudomallei, the exposure was not sufficient to produce a CMI response.\ud \ud Clonality of B. pseudomallei isolates from patients who had relapsed with melioidosis\ud was determined using pulsed-field gel electrophoresis (PFGE). Fragment patterns\ud produced following PFGE demonstrated clonal isolates were responsible for each relapse\ud episode in four patients. One patient who had relapsed with melioidosis nine times over a\ud period of eight years was due to the same B. pseudomallei isolate recrudescing rather\ud than infection with a different isolate. These results suggest that the bacterium is able to\ud evade the immune responses and cause continual episodes of melioidosis, therefore,\ud treatment of the disease may need to be reassessed.\ud \ud The development of a latent model of melioidosis involved C57BL/6 mice being infected\ud with a low dose of a low virulence strain of B. pseudomallei (NCTC13179). The mice\ud were allowed to clear the infection over a period of 30 days with bacterial load, delayed type\ud hypersensitivity (DTH) and lymphocyte proliferation responses assessed at days 10,\ud 20 and 30. These results demonstrated that a CMI response had been produced with\ud significant differences seen in the DTH response and proliferative response of\ud lymphocytes following stimulation with BpLy1. There was also an apparent clearance of\ud B. pseudomallei, with negative culture from spleen and liver of infected animals. Mice\ud were then immunosuppressesed with dexamethasone and monitored for survival, DTH\ud and lymphocyte proliferation responses and bacterial load. Following\ud immunosuppression, there was a significantly reduced immune response demonstrated by\ud the DTH response. The proliferative response of lymphocytes in infected,\ud immunosuppressed mice was not reflective of observations seen in vivo with no differences observed in proliferation of lymphocytes, following stimulation with BpLy1,\ud in infected, immunosuppressed mice compared to infected, non-immunosuppressed mice.\ud Interestingly, results from bacterial loads demonstrated that by bacterial culture,\ud B. pseudomallei was not present, however using real-time PCR (qPCR) B. pseudomallei\ud DNA was detected in the spleens of both groups of mice. There was apparent\ud reactivation of B. pseudomallei infection with a significant decrease in survival of\ud infected mice that had been immunosuppressed and infected mice that had not been\ud immunosuppressed. This aspect of the investigation demonstrates the initial development\ud of an animal model of latent melioidosis.\ud \ud Potential DNA vaccine constructs were evaluated using a BALB/c mouse model of\ud experimental B. pseudomallei infection to determine their protective potential. The DNA\ud vaccine constructs were developed against selective putative virulence factors of\ud B. pseudomallei. Mice were vaccinated at three time points using a gene gun and\ud subsequently challenged with a low virulence strain of B. pseudomallei (NCTC13179).\ud Mice were monitored for survival and bacterial load. There were no significant\ud differences seen in survival of mice vaccinated with different DNA constructs. When a\ud combination of DNA constructs or CpG, an immunostimulator, was added to the\ud vaccination regime, no significant differences were seen in survival of mice in these\ud groups when compared to control mice. Splenic bacterial loads were not reduced in\ud vaccinated groups when compared to control mice.\ud \ud The results of this present study have provided further knowledge into host-pathogen\ud interactions of B. pseudomallei. There is evidence that although there is exposure to\ud B. pseudomallei in northern Queensland, detected by assessing antibody production, this\ud exposure does not produce a CMI response which is essential in protection against the\ud bacterium. Initial studies in determining whether B. pseudomallei persists within the host\ud has been modelled in a murine model of latency. To produce a successful vaccine to\ud protect against B. pseudomallei infection, further understanding of the immune responses\ud and interaction of the bacterium is required

Development of DNA vaccines against Burkholderia pseudomallei

Burkholdena pseudomallei is the causative agent of melioidosis, a disease endemic in Southeast Asia and NorthemAustralia. The bacteria cause infection via subcutaneous or inhaled routes, resulting in either acute lethal sepsis or chronic and eventually fatal disease. Currently no licensed vaccine is available to provide protection against this pathogen. In this study, eleven DNA vaccines encoding the B. pseudomallei proteins were constructed and used to immunise mice and subsequent protective capability analysed following live challenge with B. pseudomallei.\ud \ud Mice immunised with DNA vaccines encoding Hagl; Hag2 and heat shock protein demonstrated the greatest level of protection against bacterial infection, however, the levels of protection seen were still quite limited. There was no increase in survival seen following immunisation with the remaining eight vaccines. This study highlights the challenges in designing vaccines against B. pseudomallei and suggests that a successful DNA, vaccine may need to be used in conjunction with adjuvant such as CpG oligodeoxynuc1eotide, or combined in a prime-boost regime with recombinant protein in order to increase protective capabilities

Assessment of a DNA vaccine encoding Burkholderia pseudomallei bacterioferritin

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in Southeast Asia and Northern Australia. The bacteria cause infection via subcutaneous or inhaled routes, resulting in either acute lethal sepsis or chronic and eventually fatal disease. Currently no licensed vaccine is available to provide protection against this pathogen. Intracellular enzymatic proteins of other bacterial species, such as the iron storage protein bacterioferritin, have been shown to be potent inducers of the immune response. In this study, a DNA vaccine encoding the B. pseudomallei bacterioferritin protein was constructed. The DNA vaccine was then used to immunise mice and analyse subsequent immune responses and protective capability following live challenge with B. pseudomallei. There was a substantial increase in anti-bacterioferritin IgG titers following immunisation, however the cellular response and survival following challenge was limited, suggesting that the vaccine may need to be used in conjunction with adjuvant such as CpG or in a multicomponent vaccine in order to increase protective capabilities

Seropositivity to Burkholderia pseudomallei does not\ud reflect the development of cell-mediated immunity

Cell-mediated immunity to Burkholderia pseudomallei, the causative agent of melioidosis, provides protection from disease progression. An indirect haemagglutination assay was used to detect antibodies to B. pseudomallei in 1500 healthy donors in an endemic region of Australia. Lymphocyte proliferation, activation and cytokine expression to B. pseudomallei antigen were determined in eight donors who were seropositive and in eight age- and sex-matched controls. In North Queensland, 2.5% of the population was seropositive for B. pseudomallei, which is less than half that which was previously described. Of clinical significance was the observation that while 75% of the seropositive individuals had increased lymphocyte proliferation to B. pseudomallei antigens, there were no significant differences observed in lymphocyte activation or production of cytokines

A Randomised Controlled Trial Assessing the Effects of Peri-operative Fenofibrate Administration on Abdominal Aortic Aneurysm Pathology: Outcomes From the FAME Trial

Objective: Experimental studies suggest that fenofibrate prevents abdominal aortic aneurysm (AAA) development by lowering aortic osteopontin (OPN) concentration and reducing the number of macrophages infiltrating the aortic wall. The current study examined the effects of a short course of fenofibrate on AAA pathology in people with large AAAs awaiting aortic repair. Methods: This randomised double blind parallel trial included male and female participants aged ≥ 60 years who had an asymptomatic AAA measuring ≥ 50 mm and were scheduled to undergo open AAA repair. Participants were allocated to fenofibrate (145 mg/day) or matching placebo for at least two weeks before elective AAA repair. Blood samples were collected at recruitment and immediately prior to surgery. AAA biopsies were obtained during aortic surgery. The primary outcomes were (1) AAA OPN concentration; (2) serum OPN concentration; and (3) number of AAA macrophages. Exploratory outcomes included circulating and aortic concentrations of other proteins previously associated with AAA. Outcomes assessed at a single time point were compared using logistic regression. Longitudinal outcomes were compared using linear mixed effects models. Results: Forty-three participants were randomised. After three withdrawals, 40 were followed until the time of surgery (21 allocated fenofibrate and 19 allocated placebo). As expected, serum triglycerides reduced significantly from recruitment to the time of surgery in participants allocated fenofibrate. No differences in any of the primary and exploratory outcomes were observed between groups. Conclusion: A short course of 145 mg of fenofibrate/day did not lower concentrations of OPN or aortic macrophage density in people with large AAAs

Depletion of CD11c+ dendritic cells in apolipoprotein E-deficient mice limits angiotensin II-induced abdominal aortic aneurysm formation and growth

Objective: The role of chronic inflammation in abdominal aortic aneurysm (AAA) is controversial. CD11c+ antigen-presenting cells (APCs) (dendritic cells (DCs)) have been reported in human AAA samples but their role is unclear. The effect of conditional depletion of CD11c+ cells on experimental AAA was investigated in the angiotensin II (AngII)-infused apolipoprotein E-deficient (ApoE–/–) mouse model. Approach: CD11c-diphtheria toxin (DT or D.tox) receptor (DTR), ovalbumin (OVA) fragment aa 140–386, and enhanced green fluorescent protein (eGFP)-ApoE–/– (CD11c.DOG.ApoE–/–) mice were generated and CD11c+ cell depletion achieved with D.tox injections (8 ng/g body weight, i.p., every-other-day). AAA formation and growth were assessed by measurement of supra-renal aortic (SRA) diameter in vivo by serial ultrasound and by morphometry assessment of harvested aortas at the end of the study. Results: Depletion of CD11c+ cells by administration of D.tox on alternative days was shown to reduce the maximum diameter of AAAs induced by 28 days AngII infusion compared with controls (D.tox, 1.58 ± 0.03 mm vs Vehicle control, 1.81 ± 0.06 mm, P<0.001). CD11c+ depletion commencing after AAA establishment by 14 days of AngII infusion, was also shown to lead to smaller AAAs than controls after a further 14 days (D.tox, 1.54 ± 0.04 mm vs Vehicle control, 1.80 ± 0.03 mm, P<0.001). Flow cytometry revealed significantly lower numbers of circulating CD44hi CD62Llo effector CD4 T cells, CD44hi CD62Llo effector CD8 T cells and B220+ B cells in CD11c+ cell-depleted mice versus controls. CD11c+ depletion attenuated SRA matrix degradation indicated by decreased neutrophil elastase activity (P=0.014), lower elastin degradation score (P=0.012) and higher collagen content (P=0.002). Conclusion: CD11c+ cell-depletion inhibited experimental AAA development and growth associated with down-regulation of circulating effector T cells and attenuated matrix degradation. The findings suggest involvement of autoreactive immune cells in AAA pathogenesis