Validation of Immunoassay-Based Tools for the Comprehensive Quantification of Aß40 and Aß42 Peptides in Plasma

Recent advances in neuroimaging and cerebrospinal fluid (CSF) biomarker assays have provided evidence of a long preclinical stage of Alzheimer''s disease (AD). This period is being increasingly targeted for secondary prevention trials of new therapies. In this context, the interest of a noninvasive, cost-effective amyloid-ß (Aß) blood-based test does not need to be overstated. Nevertheless, a thorough validation of these bioanalytical methods should be performed as a prerequisite for confident interpretation of clinical results. The aim of this study was to validate ELISA sandwich colorimetric ABtest40 and ABtest42 for the quantification of Aß40 and Aß42 in human plasma. The validation parameters assessed included precision, accuracy, sensitivity, specificity, recovery, and dilution linearity. ABtest40 and ABtest42 proved to be specific for their target peptide using Aß peptides with sequence similar to the target. Mean relative error in the quantification was found to be below 7.5 for both assays, with high intra-assay, inter-assay, and inter-batch precision (CV <9.0 on average). Sensitivity was assessed by determination of the limit of quantification fulfilling precision and accuracy criteria; it was established at 7.60 pg/ml and 3.60 pg/ml for ABtest40 and ABtest42, respectively. Plasma dilution linearity was demonstrated in PBS; however, dilution in a proprietary formulated buffer significantly increased the recovery of both Aß40 and Aß42 masked by matrix interactions, allowing a more comprehensive assessment of the free and total peptide levels in the plasma. In conclusion, both assays were successfully validated as tools for the quantification Aß40 and Aß42 in plasma

Library of Seleno-Compounds as Novel Agents against Leishmania Species

The in vitro leishmanicidal activities of a series of 48 recently synthesized selenium derivatives against Leishmania infantum and Leishmania braziliensis parasites were tested using promastigotes and intracellular amastigote forms. The cytotoxicity of the tested compounds for J774.2 macrophage cells was also measured in order to establish their selectivity. Six of the tested compounds (compounds 8, 10, 11, 15, 45, and 48) showed selectivity indexes higher than those of the reference drug, meglumine antimonate (Glucantime), for both Leishmania species; in the case of L. braziliensis, compound 20 was also remarkably selective. Moreover, data on infection rates and amastigote numbers per macrophage showed that compounds 8, 10, 11, 15, 45, and 48 were the most active against both Leishmania species studied. The observed changes in the excretion product profile of parasites treated with these six compounds were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds were potent inhibitors of Fe superoxide dismutase (Fe-SOD) in the two parasite species considered, whereas their impact on human CuZn-SOD was low. The high activity, low toxicity, stability, low cost of the starting materials, and straightforward synthesis make these compounds appropriate molecules for the development of affordable antileishmanicidal agents

Plasma Aβ42/40 ratio alone or combined with FDG-PET can accurately predict amyloid-PET positivity: a cross-sectional analysis from the AB255 Study

Background: To facilitate population screening and clinical trials of disease-modifying therapies for Alzheimer’s disease, supportive biomarker information is necessary. This study was aimed to investigate the association of plasma amyloid-beta (Aβ) levels with the presence of pathological accumulation of Aβ in the brain measured by amyloid-PET. Both plasma Aβ42/40 ratio alone or combined with an FDG-PET-based biomarker of neurodegeneration were assessed as potential AD biomarkers. Methods: We included 39 cognitively normal subjects and 20 patients with mild cognitive impairment from the AB255 Study who had undergone PiB-PET scans. Total Aβ40 and Aβ42 levels in plasma (TP42/40) were quantified using ABtest kits. Subjects were dichotomized as Aβ-PET positive or negative, and the ability of TP42/40 to detect Aβ-PET positivity was assessed by logistic regression and receiver operating characteristic analyses. Combination of plasma Aβ biomarkers and FDG-PET was further assessed as an improvement for brain amyloidosis detection and diagnosis classification. Results: Eighteen (30.5%) subjects were Aβ-PET positive. TP42/40 ratio alone identified Aβ-PET status with an area under the curve (AUC) of 0.881 (95% confidence interval [CI] = 0.779–0.982). Discriminating performance of TP42/40 to detect Aβ-PET-positive subjects yielded sensitivity and specificity values at Youden’s cutoff of 77.8% and 87.5%, respectively, with a positive predictive value of 0.732 and negative predictive value of 0.900. All these parameters improved after adjusting the model for significant covariates. Applying TP42/40 as the first screening tool in a sequential diagnostic work-up would reduce the number of Aβ-PET scans by 64%. Combination of both FDG-PET scores and plasma Aβ biomarkers was found to be the most accurate Aβ-PET predictor, with an AUC of 0.965 (95% CI = 0.913–0.100). Conclusions: Plasma TP42/40 ratio showed a relevant and significant potential as a screening tool to identify brain Aβ positivity in preclinical and prodromal stages of Alzheimer’s disease

Acute and reproductive effects of Align, an insecticide containing azadirachtin, on grape berry moth, Lobesia botrana.

Azadirachtin, derived from the neem tree, Azadirachta indica A. Juss (Sapindales: Meliaceae), seems promising for use in integrated pest management programs to control a variety of pest species. A commercial formulation of azadirachtin, Align, has been evaluated against different developmental stages of the European grape berry moth, Lobesia botrana Denis and Schiffermüller (Lepidoptera: Tortricidae). When administered orally, Align reduced the fecundity and fertility of adults treated with 1, 5, and 10 mg litre(-1). At the highest doses, fecundity and fertility were zero, but longevity was not affected. An LC(50) of 231.5 mg litre(-1) was obtained when Align was sprayed on eggs less than 1 day old. Hatching of all egg classes was significantly reduced, and this reduction was more pronounced for eggs less than 24 h old. LC(50) values of 2.1 mg litre(-1) for first instars and 18.7 mg litre(-1) for third instars were obtained when Align was present in the diet. Larvae reared on a diet containing different concentrations of Align did not molt into adults at the highest concentrations (0.3, 0.6, 1.2), and 50% molted at the lowest concentration (0.15). Phenotypic effects included inability to molt properly and deformities. The combination of acute toxicity and low, effective concentrations of Align observed in this study could lead to the inclusion of insecticides containing azadirachtin in integrated management programs against this pest

Effects of azadirachtin on the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae).

Effects of sublethal azadirachtin exposure to the biological performance of Tetranychus urticae Koch was studied under laboratory conditions. Bioassay was used to asses the effect of different concentrations of azadirachtin on longevity, fecundity, fertility, and offspring development. Azadirachtin (64 and 128 ppm) affected fecundity and mortality but had no effect on fertility and offspring development. A subsequent life-table study with 80 ppm of azadirachtin found that the compound caused a reduction of 50% in survival to adult stage. The peak of reproduction was reached at 5 days causing a decrease in the mean fecundity to almost eight times than of untreated females. The net reproductive rate (R 0), the intrinsic rate of increase (r m), and the finite rate of increase () of treated females were lower. Treatment showed a negative value of r m, resulting in a declining population. These results suggest that azadirachtin could be incorporated in Integrated Pest Management (IPM) programmes of T. urticae. © Springer 2005

Plasma amyloid β 42/40 ratios as biomarkers for amyloid β cerebral deposition in cognitively normal individuals.

INTRODUCTION: Plasma amyloid β (Aβ) peptides have been previously studied as candidate biomarkers to increase recruitment efficiency in secondary prevention clinical trials for Alzheimer's disease. METHODS: Free and total Aβ42/40 plasma ratios (FP42/40 and TP42/40, respectively) were determined using ABtest assays in cognitively normal subjects from the Australian Imaging, Biomarker and Lifestyle Flagship Study. This population was followed-up for 72 months and their cortical Aβ burden was assessed with positron emission tomography. RESULTS: Cross-sectional and longitudinal analyses showed an inverse association of Aβ42/40 plasma ratios and cortical Aβ burden. Optimized as a screening tool, TP42/40 reached 81% positive predictive value of high cortical Aβ burden, which represents 110% increase over the population prevalence of cortical Aβ positivity. DISCUSSION: These findings support the use of plasma Aβ42/40 ratios as surrogate biomarkers of cortical Aβ deposition and enrichment tools, reducing the number of subjects submitted to invasive tests and, consequently, recruitment costs in clinical trials targeting cognitively normal individuals

Venetoclax improves CD20 immunotherapy in a mouse model of MYC/BCL2 double-expressor diffuse large B-cell lymphoma

BackgroundApproximately one-third of diffuse large B cell lymphoma (DLBCL) patients exhibit co-expression of MYC and BCL2 (double-expressor lymphoma, DEL) and have a dismal prognosis. Targeted inhibition of the anti-apoptotic protein BCL2 with venetoclax (ABT-199) has been approved in multiple B-cell malignancies and is currently being investigated in clinical trials for DLBCL. Whether BCL2 anti-apoptotic function represents a multifaceted vulnerability for DEL-DLBCL, affecting both lymphoma B cells and T cells within the tumor microenvironment, remains to be elucidated.MethodsHere, we present novel genetically engineered mice that preclinically recapitulate DEL-DLBCL lymphomagenesis, and evaluate their sensitivity ex vivo and in vivo to the promising combination of venetoclax with anti-CD20-based standard immunotherapy.ResultsVenetoclax treatment demonstrated specific killing of MYC+/BCL2(+) lymphoma cells by licensing their intrinsically primed apoptosis, and showed previously unrecognized immunomodulatory activity by specifically enriching antigen-activated effector CD8 T cells infiltrating the tumors. Whereas DEL-DLBCL mice were refractory to venetoclax alone, inhibition of BCL2 significantly extended overall survival of mice that were simultaneously treated with a murine surrogate for anti-CD20 rituximab.ConclusionsThese results suggest that the combination of anti-CD20-based immunotherapy and BCL2 inhibition leads to cooperative immunomodulatory effects and improved preclinical responses, which may offer promising therapeutic opportunities for DEL-DLBCL patients