Ana M. Lacosta

Validation of Immunoassay-Based Tools for the Comprehensive Quantification of Aβ40 and Aβ42 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-β (Aβ) 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 Aβ40 and Aβ42 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 Aβ 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 Aβ40 and Aβ42 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 Aβ40 and Aβ42 in plasma.

Immunization of dogs with C-terminal sequences of beta-amyloid protein is safe and depletes the protein from the blood

Background: Immunization with Ab has been proposed as a promising therapeutic way in the fight against Alzheimer disease. Methods: We have carried out a pilot immunization assay against Ab peptide in four groups of dogs (n 1⁄4 3) that were vaccinated with 7 biweekly injections containing 400 mg of Ab x-42 conjugated to either hemocyanin (groups A and B) or albumin (groups C and D) and mixed with Th1-type adjuvant (groups B and D), or Th2-type adjuvant (groups A and C). Antibody titers were determined by direct ELISA assay. Levels of Ab 1-42 and Ab 1-40 in plasma were measured with high sensitive proprietary ELISA assay kits. All animals received clinical examination, including blood cell and chemistry analyses, at the beginning and the end of the experiment and were diagnosed to be healthy. Results: After the third immunization, dogs of groups C and D reached high antibody titers, which remained practically unchanged up to the time of the seventh immunization. At this time point, plasma levels of both Ab 1-42 and Ab 1-40 had dropped 100 % and 62 %, respectively, with regard to pre-immune levels. Four months after the last immunization the antibody titers in these dogs had fallen down to the preimmunization levels again. At this time point, the plasma levels of Ab 1-42 and Ab 1-40 showed a tendency to increase, but they were still significantly lower than in the pre-immune samples.Dogs in groups A and B did not respond to immunization and did not show significant changes in the levels of plasma Ab 1-42 and Ab 1-40 across the experiment. Conclusions: Vaccines designed from the C-terminal sequence of Ab are safe for the animals and efficiently eliminate plasmatic Ab in the dog, which represents a natural model for testing Ab modulating therapies against Alzheimer disease.

ABVAC40 TREATMENT IN DOGS INDUCES SPECIFIC ANTIBODY RESPONSE AND INCREMENT OF Aβ40 LEVELS IN PLASMA

colocalized with LRP1 in SH-SY5Y cells. The MAPKs (P38, JNK, ERK) were activated (P<0.05), the expression level of LRP1 (P<0.01) were also significantly increased, compared to the saline group, while the SH-SY5Y cells were treated with Ab1-42. By using the p38 MAPK inhibitor SB202190, the expression of LRP1 level significantly decreased (P<0.05). ERK1/2 MAPK inhibitor decreases the level of LRP1 (P<0.01) expression. However, in JNK MAPK inhibitor treated group, the level of LRP1 (P<0.05) significantly increased. In the study of Ab induced toxicity, Ab (5 mM, 18 h) up-regulates the expression of Bcl2/Bax (P<0.05), and increases the Cyt c (P<0.01) release. The p38, ERK1/2 MAPK inhibitor could up-regulate the Bcl2/Bax (P<0.05) level, decrease the level of Cyt c (P<0.05) release. JNK MAPK inhibitor has no significant effects on the level of Bcl2/Bax and Cyt c. Conclusions: Exogenous Ab could be internalized by SH-SY5Y cells; MAPK signaling pathways were activated by Ab; The MAPK signaling pathways could regulate the Ab level through the LRP1; Ab was internalized and could induce toxic effect on SH-SY5Y cells.

AN ACTIVE ANTI-AB40 VACCINE (ABVAC40) PROVED TO BE SAFE AND IMMUNOGENIC IN THE PHASE I CLINICAL TRIAL

cebo were studied. Comprehensive neuropathological assessments were performed ranging from 4 months to 14 years after the trial. Large coronal paraffin sections of cerebral hemisphere were immunostained for Ab, scanned, then (i) the entire neocortical ribbon was scored for plaques in a CERAD-like manner (frequent1⁄43, moderate1⁄42, sparse1⁄41, none1⁄40) and (ii) false coloured to permit visualisation of staining patterns at low power. Results: 16/21 had a distribution of tangles and at least some residual Ab pathology indicating the cause of dementia had been AD, whereas 5/21 had alternative causes for dementia (PSP1⁄41, DLB1⁄41, VaD1⁄41and FTDTDP431⁄42). 18/21 had received the active agent and 3/21 the placebo. Scanning and false colouration of large Ab immunostained sections generated images suitable for comparison with amyloid PET scans as used in current immunotherapy trials. 13/15 AD subjects receiving the active agent had evidence of plaque removal (almost complete removal1⁄45, extensive patches of removal1⁄44, small patches of removal1⁄44, no plaque removal1⁄42). In the immunised AD group the mean plaque score was 1.46 (range1⁄40.05-2.9) vs. 2.4 for the single placebo AD case. There was a significant inverse correlation between peripheral blood anti-Ab titres and plaque scores. Conclusions: AD patients actively immunised against Ab can remain virtually plaque–free for up to 14 years. Nearly a quarter of the patients examined in this study did not have neuropathologically verified AD. Neuropathology follow up of patients in therapeutic trials for AD provides valuable information on the cause of dementia and effects of treatment.

The Expression of Procalcitonin in the Central Nervous System of the RatEmbryo

Procalcitonin mRNA is abundantly synthesized in the thyroid gland in mammals. In recent years, procalcitonin or its posttranslational product calcitonin, has been identified in numerous non-thyroidal adult tissues. Nevertheless, little is known about the implications of procalcitonin in development. We have previously discovered that chick embryo expresses procalcitonin in the developing floor plate and the dorsal rhombencephalon from the early stages of embryogenesis. This finding focused our attention on investigating whether procalcitonin is present in the nervous system of mammalian embryos. In a previous study by our laboratory, the central expression of calcitonin was not found in rat embryos when isotopic deoxyoligonucleotides were used as probes for in situ hybridization analyses. In this study, the expression of procalcitonin mRNA was detected in rat embryos from 11 to 18 days post-coitum when analyzed using techniques such as RT-PCR. In situ hybridization histochemistry with riboprobes revealed the expression of procalcitonin mRNA in the roof of the diencephalon from 12 to 16 days post-coitum and this expression was quantified by Ribonuclease Protection Assay technique. The immunohistochemical analysis confirmed the presence of the protein in this region and in the roof (velum medullare) of the rhombencephalon. Notably, numerous microglial cells were present in the developing diencephalon only during the period during which the expression of procalcitonin was detected. These findings suggest roles for procalcitonin not only in diencephalon development but also in the process of microglial colonization of the central nervous system.

Early diagnosis and preventive treatment of Alzheimer's with isoform-specific Aβ antibodies

Background: Until now attempts to measure Ab peptides in blood have given inconclusive and discouraging results; mostly for a variety of technical reasons that certainly compromise, but in our opinion do not invalidate the working hypothesis. Due to their biochemical nature, Ab1-40 and Ab1-42 peptides will be found free in the plasma (FP), bound to the plasma components and bound to the blood cells (comprehensively described here as the bamyloid pool in blood (bAPB)). Consequently, a complete quantification of Ab in blood should consider the levels of Ab1-40 and Ab1-42 total in plasma (TP) and bound to cells (CB), which obviously require the use of Ab C-terminal specific antibodies. In addition, contrasting to the preferred N-terminal targeted strategy, immunization against the C-terminus of Ab, which has been suggested to be mainly hidden in the fibrillar aggregates within senile plaques and vascular deposits, might be advantageous to remove Ab soluble monomers and oligomers without undesirable side effects. Methods: We carried out a pilot study including 16 healthy controls, 8 mild cognitive impairment patients and 16 Alzheimer disease patients to explore if quantification of the bAPB by ELISA with Ab C-terminus specific antibodies could provide useful and reliable biomarkers for early stages of Alzheimer disease. Simultaneously, the same C-terminus fragments used to generate these antibodies were used for the active immunization of aged dogs, considered as a very useful animal model for AD research. Results: Main results in the diagnostic study were that several directly markers, including TP and CB Ab140, FP and CB Ab1-42 and other calculated markers as the total bAPB reached a sensitivity and specificity over 80 % to discriminate between MCI patients and healthy controls. For the other side the active immunization of dogs with C-terminus fragment of Ab40 and Ab42 was completely safe, immunogenic and produced a complete wash up of the Ab peptides from the blood. Conclusions: Thus, these results suggest that C-terminal fragments of Ab peptides can be used to generate specific antibodies for the diagnosis of early stages of AD and for preventive therapeutic strategies. Validation of these results in a large sample population is in progress.