Melbourne Medical School Collected Works - Research Publications
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ItemDeep Sequencing of RNA from Three Different Extracellular Vesicle (EV) Subtypes Released from the Human LIM1863 Colon Cancer Cell Line Uncovers Distinct Mirna-Enrichment Signatures(PUBLIC LIBRARY SCIENCE, 2014-10-17)Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863--shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs--miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.
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ItemDendritic cell immunotherapy: clinical outcomes(WILEY, 2014-07)The use of tumour-associated antigens for cancer immunotherapy studies is exacerbated by tolerance to these self-antigens. Tolerance may be broken by using ex vivo monocyte-derived dendritic cells (DCs) pulsed with self-antigens. Targeting tumour-associated antigens directly to DCs in vivo is an alternative and simpler strategy. The identification of cell surface receptors on DCs, and targeting antigens to DC receptors, has become a popular approach for inducing effective immune responses against cancer antigens. Many years ago, we demonstrated that targeting the mannose receptor on macrophages using the carbohydrate mannan to DCs led to appropriate immune responses and tumour protection in animal models. We conducted Phase I, I/II and II, clinical trials demonstrating the effectiveness of oxidised mannan-MUC1 in patients with adenocarcinomas. Here we summarise DC targeting approaches and their efficacy in human clinical trials.
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ItemPredictors of Mean Arterial Pressure Morning Rate of Rise and Power Function in Subjects Undergoing Ambulatory Blood Pressure Recording(PUBLIC LIBRARY SCIENCE, 2014-03-25)BACKGROUND: We determined clinical predictors of the rate of rise (RoR) in blood pressure in the morning as well as a novel measure of the power of the BP surge (BP(power)) derived from ambulatory blood pressure recordings. METHODS: BP(power) and RoR were calculated from 409 ambulatory blood pressure (ABP) recordings from subjects attending a cardiovascular risk clinic. Anthropometric data, blood biochemistry, and history were recorded. The 409 subjects were 20-82 years old (average 57, SD = 13), 46% male, 9% with hypertension but not on medication and 34% on antihypertensive medication. RESULTS: Average RoR was 11.1 mmHg/hour (SD = 8) and BP(power) was 273 mmHg(2)/hour (SD = 235). Only cholesterol, low density lipoprotein and body mass index (BMI) were associated with higher BP(power) and RoR (P<0.05) from 25 variables assessed. BP(power) was lower in those taking beta-blockers or diuretics. Multivariate analysis identified that only BMI was associated with RoR (4.2% increase/unit BMI, P = 0.020) while cholesterol was the only remaining associated variable with BP(power) (17.5% increase/mmol/L cholesterol, P = 0.047). A follow up of 213 subjects with repeated ABP after an average 1.8 years identified that baseline cholesterol was the only predictor for an increasing RoR and BP(power) (P<0.05). 37 patients who commenced statin subsequently had lower BP(power) whereas 90 age and weight matched controls had similar BP(power) on follow-up. CONCLUSIONS: Cholesterol is an independent predictor of a greater and more rapid rise in morning BP as well as of further increases over several years. Reduction of cholesterol with statin therapy is very effective in reducing the morning blood pressure surge.
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ItemCirculating antibodies against Plasmodium falciparum histidine-rich proteins 2 interfere with antigen detection by rapid diagnostic tests(BMC, 2014-12-06)BACKGROUND: Rapid diagnostic tests (RDTs) for detection of Plasmodium falciparum infection that target P. falciparum histidine-rich protein 2 (PfHRP2), a protein that circulates in the blood of patients infected with this species of malaria, are widely used to guide case management. Understanding determinants of PfHRP2 availability in circulation is therefore essential to understanding the performance of PfHRP2-detecting RDTs. METHODS: The possibility that pre-formed host anti-PfHRP2 antibodies may block target antigen detection, thereby causing false negative test results was investigated in this study. RESULTS: Anti-PfHRP2 antibodies were detected in 19/75 (25%) of plasma samples collected from patients with acute malaria from Cambodia, Nigeria and the Philippines, as well as in 3/28 (10.7%) asymptomatic Solomon Islands residents. Pre-incubation of plasma samples from subjects with high-titre anti-PfHRP2 antibodies with soluble PfHRP2 blocked the detection of the target antigen on two of the three brands of RDTs tested, leading to false negative results. Pre-incubation of the plasma with intact parasitized erythrocytes resulted in a reduction of band intensity at the highest parasite density, and a reduction of lower detection threshold by ten-fold on all three brands of RDTs tested. CONCLUSIONS: These observations indicate possible reduced sensitivity for diagnosis of P. falciparum malaria using PfHRP2-detecting RDTs among people with high levels of specific antibodies and low density infection, as well as possible interference with tests configured to detect soluble PfHRP2 in saliva or urine samples. Further investigations are required to assess the impact of pre-formed anti-PfHRP2 antibodies on RDT performance in different transmission settings.
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ItemAssessment of Anthelmintic Efficacy of Mebendazole in School Children in Six Countries Where Soil-Transmitted Helminths Are Endemic(PUBLIC LIBRARY SCIENCE, 2014-10)BACKGROUND: Robust reference values for fecal egg count reduction (FECR) rates of the most widely used anthelmintic drugs in preventive chemotherapy (PC) programs for controlling soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, and hookworm) are still lacking. However, they are urgently needed to ensure detection of reduced efficacies that are predicted to occur due to growing drug pressure. Here, using a standardized methodology, we assessed the FECR rate of a single oral dose of mebendazole (MEB; 500 mg) against STHs in six trials in school children in different locations around the world. Our results are compared with those previously obtained for similarly conducted trials of a single oral dose of albendazole (ALB; 400 mg). METHODOLOGY: The efficacy of MEB, as assessed by FECR, was determined in six trials involving 5,830 school children in Brazil, Cambodia, Cameroon, Ethiopia, United Republic of Tanzania, and Vietnam. The efficacy of MEB was compared to that of ALB as previously assessed in 8,841 school children in India and all the above-mentioned study sites, using identical methodologies. PRINCIPAL FINDINGS: The estimated FECR rate [95% confidence interval] of MEB was highest for A. lumbricoides (97.6% [95.8; 99.5]), followed by hookworm (79.6% [71.0; 88.3]). For T. trichiura, the estimated FECR rate was 63.1% [51.6; 74.6]. Compared to MEB, ALB was significantly more efficacious against hookworm (96.2% [91.1; 100], p<0.001) and only marginally, although significantly, better against A. lumbricoides infections (99.9% [99.0; 100], p = 0.012), but equally efficacious for T. trichiura infections (64.5% [44.4; 84.7], p = 0.906). CONCLUSIONS/SIGNIFICANCE: A minimum FECR rate of 95% for A. lumbricoides, 70% for hookworm, and 50% for T. trichiura is expected in MEB-dependent PC programs. Lower FECR results may indicate the development of potential drug resistance.
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ItemProduction and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2(BMC, 2014-07-18)BACKGROUND: Early and accurate diagnosis of Plasmodium falciparum infection is important for providing appropriate treatment to patients with malaria. However, technical limitations of currently available diagnostic tests limit their use in control programs. One possible explanation for the vulnerability of current antibodies used in RDTs is their propensity to degrade at high ambient temperatures. Isolation of new antibodies with better thermal stability represents an appealing approach to improve the performance of RDTs. METHODS: In this study, phage display technology was deployed to isolate novel binders by screening a human naïve scFv antibody library against recombinant Plasmodium falciparum histidine rich protein 2 (rPfHRP2). The isolated scFv clones were reformatted to whole IgG and the recombinant mAbs were produced in a mammalian CHO cell expression system. To verify the biological activity of these purified recombinant mAbs, range of functional assays were characterized. RESULTS: Two unique clones (D2 and F9) were isolated after five rounds of biopanning. The reformatted and expressed antibodies demonstrated high binding specificity to malaria recombinant PfHRP2 and native proteins. When 5 μg/mL of mAbs applied, mAb C1-13 had the highest sensitivity, with an OD value of 1, the detection achieved 5 ng/mL of rPfHRP2, followed by mAbs D2 and F9 at 10 ng/mL and 100 ng/mL of rPfHRP2, respectively. Although the sensitivity of mAbs D2 and F9 was lower than the control, these recombinant human mAbs have shown better stability compared to mouse mAb C1-13 at various temperatures in DSC and blot assays. In view of epitope mapping, the predominant motif of rPfHRP2 recognized by mAb D2 was AHHAADAHHA, whereas mAb F9 was one amino acid shorter, resulting in AHHAADAHH. mAb F9 had the strongest binding affinity to rPfHRP2 protein, with a KD value of 4.27 × 10(-11) M, followed by control mAb C1-13 at 1.03 × 10(-10) M and mAb D2 at 3.05 × 10(-10) M. CONCLUSIONS: Overall, the performance of these mAbs showed comparability to currently available PfHRP2-specific mouse mAb C1-13. The stability of these novel binders indicate that they merit further work to evaluate their utility in the development of new generation point of care diagnosis of malaria.
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ItemPlasmodium falciparum parasites lacking histidine-rich protein 2 and 3: a review and recommendations for accurate reporting(BMC, 2014-07-22)Malaria rapid diagnostic tests (RDTs) play a critical role in malaria case management, surveillance and case investigations. Test performance is largely determined by design and quality characteristics, such as detection sensitivity, specificity, and thermal stability. However, parasite characteristics such as variable or absent expression of antigens targeted by RDTs can also affect RDT performance. Plasmodium falciparum parasites lacking the PfHRP2 protein, the most common target antigen for detection of P. falciparum, have been reported in some regions. Therefore, accurately mapping the presence and prevalence of P. falciparum parasites lacking pfhrp2 would be an important step so that RDTs targeting alternative antigens, or microscopy, can be preferentially selected for use in such regions. Herein the available evidence and molecular basis for identifying malaria parasites lacking PfHRP2 is reviewed, and a set of recommended procedures to apply for future investigations for parasites lacking PfHRP2, is proposed.
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ItemNo Preview AvailablePlasma Dihydroceramide Species Associate with Waist Circumference in Mexican American Families(WILEY, 2014-03)OBJECTIVE: Waist circumference (WC), the clinical marker of central obesity, is gaining popularity as a screening tool for type 2 diabetes (T2D). While there is epidemiologic evidence favoring the WC-T2D association, its biological substantiation is generally weak. Our objective was to determine the independent association of plasma lipid repertoire with WC. METHODS: Samples and data from the San Antonio Family Heart Study of 1208 Mexican Americans from 42 extended families were used. Association of plasma lipidomic profiles with the cross-sectionally assessed WC was determined. Plasma lipidomic profiling entailed liquid chromatography with mass spectrometry. Statistical analyses included multivariable polygenic regression models and bivariate trait analyses using the SOLAR software. RESULTS: After adjusting for age and sex interactions, body mass index, homeostasis model of assessment-insulin resistance, total cholesterol, triglycerides, high density lipoproteins and use of lipid lowering drugs, dihydroceramides as a class were associated with WC. Dihydroceramide species 18:0, 20:0, 22:0, and 24:1 were significantly associated and genetically correlated with WC. Two sphingomyelin species (31:1 and 41:1) were also associated with WC. CONCLUSIONS: Plasma dihydroceramide levels independently associate with WC. Thus, high resolution plasma lipidomic studies can provide further credence to the biological underpinnings of the association of WC with T2D.
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ItemHip Structural Parameters over 96 Weeks in HIV-Infected Adults Switching Treatment to Tenofovir-Emtricitabine or Abacavir-Lamivudine(PUBLIC LIBRARY SCIENCE, 2014-04-10)BACKGROUND: Therapy with tenofovir is associated with lower bone mineral density (BMD), higher markers of bone turnover and increased fracture risk in HIV-infected adults. Bone structural parameters generated by hip structural analysis may represent a separate measure of bone strength, but have not been assessed in HIV. METHODS: Dual-energy X-ray absorptiometry (DXA) scans from 254 HIV-infected adults randomised to simplify their existing dual nucleoside analogue reverse transcriptase inhibitor therapy to coformulated tenofovir-emtricitabine or abacavir-lamivudine were analysed using DXA-derived hip structural analysis software. Hip structural parameters included femoral strength index, section modulus, cross-sectional area, and cross-sectional moment of inertia. We used one-way ANOVA to test the relationship between nucleoside analogue type at baseline and structural parameters, multivariable analysis to assess baseline covariates associated with femoral strength index, and t-tests to compare mean change in structural parameters over 96 weeks between randomised groups. RESULTS: Participants taking tenofovir at baseline had lower section modulus (-107.3 mm2, p = 0.001), lower cross-sectional area (-15.01 mm3, p = 0.001), and lower cross-sectional moment of inertia (-2,036.8 mm4, p = 0.007) than those receiving other nucleoside analogues. After adjustment for baseline risk factors, the association remained significant for section modulus (p = 0.008) and cross-sectional area (p = 0.002). Baseline covariates significantly associated with higher femoral strength index were higher spine T-score (p = 0.001), lower body fat mass (p<0.001), lower bone alkaline phosphatase (p = 0.025), and higher osteoprotegerin (p = 0.024). Hip structural parameters did not change significantly over 96 weeks and none was significantly affected by treatment simplification to tenofovir-emtricitabine or abacavir-lamivudine. CONCLUSION: In this population, tenofovir use was associated with reduced composite indices of bone strength as measured by hip structural analysis, but none of the structural parameters improved significantly over 96 weeks with tenofovir cessation. TRIAL REGISTRATION: ClinicalTrials.gov NCT00192634.
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ItemA comparative analysis of risk stratification tools for emergency department patients with chest pain(BMC, 2014)BACKGROUND: Appropriate disposition of emergency department (ED) patients with chest pain is dependent on clinical evaluation of risk. A number of chest pain risk stratification tools have been proposed. The aim of this study was to compare the predictive performance for major adverse cardiac events (MACE) using risk assessment tools from the National Heart Foundation of Australia (HFA), the Goldman risk score and the Thrombolysis in Myocardial Infarction risk score (TIMI RS). METHODS: This prospective observational study evaluated ED patients aged ≥30 years with non-traumatic chest pain for which no definitive non-ischemic cause was found. Data collected included demographic and clinical information, investigation findings and occurrence of MACE by 30 days. The outcome of interest was the comparative predictive performance of the risk tools for MACE at 30 days, as analyzed by receiver operator curves (ROC). RESULTS: Two hundred eighty-one patients were studied; the rate of MACE was 14.1%. Area under the curve (AUC) of the HFA, TIMI RS and Goldman tools for the endpoint of MACE was 0.54, 0.71 and 0.67, respectively, with the difference between the tools in predictive ability for MACE being highly significant [chi2 (3) = 67.21, N = 276, p < 0.0001]. CONCLUSION: The TIMI RS and Goldman tools performed better than the HFA in this undifferentiated ED chest pain population, but selection of cutoffs balancing sensitivity and specificity was problematic. There is an urgent need for validated risk stratification tools specific for the ED chest pain population.