The role of adjuvant in mediating antigen structure and stability

The purpose of this study was to probe the fate of a model antigen, a cysteine-free mutant of bacteriophage T4 lysozyme, to the level of fine structural detail, as a consequence of its interaction with an aluminum (Al)-containing adjuvant. Fluorescence spectroscopy and differential scanning calorimetry were used to compare the thermal stability of the protein in solution versus adsorbed onto an Al-containing adjuvant. Differences in accessible hydrophobic surface areas were investigated using an extrinsic fluorescence probe, 8-Anilino-1-naphthalenesulfonic acid (ANS). As has been observed with other model antigens, the apparent thermal stability of the protein decreased following adsorption onto the adjuvant. ANS spectra suggested that adsorption onto the adjuvant caused an increase in exposure of hydrophobic regions of the protein. Electrostatic interactions drove the adsorption, and disruption of these interactions with high ionic strength buffers facilitated the collection of two-dimensional 15N heteronuclear single quantum coherence nuclear magnetic resonance data of protein released from the adjuvant. Although the altered stability of the adsorbed protein suggested changes to the protein\u27s structure, the fine structure of the desorbed protein was nearly identical to the protein\u27s structure in the adjuvant-free formulation. Thus, the adjuvant-induced changes to the protein that were responsible for the reduced thermal stability were not observed upon desorption. © 2011 Wiley Periodicals, Inc

Monitoring Temporal Changes in the Specificity of an Oral HIV Test: A Novel Application for Use in Postmarketing Surveillance

BACKGROUND: Postmarketing surveillance is routinely conducted to monitor performance of pharmaceuticals and testing devices in the marketplace. However, these surveillance methods are often done retrospectively and, as a result, are not designed to detect issues with performance in real-time. METHODS AND FINDINGS: Using HIV antibody screening test data from New York City STD clinics, we developed a formal, statistical method of prospectively detecting temporal clusters of poor performance of a screening test. From 2005 to 2008, New York City, as well as other states, observed unexpectedly high false-positive (FP) rates in an oral fluid-based rapid test used for screening HIV. We attempted to formally assess whether the performance of this HIV screening test statistically deviated from both local expectation and the manufacturer's claim for the test. Results indicate that there were two significant temporal clusters in the FP rate of the oral HIV test, both of which exceeded the manufacturer's upper limit of the 95% CI for the product. Furthermore, the FP rate of the test varied significantly by both STD clinic and test lot, though not by test operator. CONCLUSIONS: Continuous monitoring of surveillance data has the benefit of providing information regarding test performance, and if conducted in real-time, it can enable programs to examine reasons for poor test performance in close proximity to the occurrence. Techniques used in this study could be a valuable addition for postmarketing surveillance of test performance and may become particularly important with the increase in rapid testing methods

Running wheel exercise reduces α-synuclein aggregation and improves motor and cognitive function in a transgenic mouse model of Parkinson's disease.

Exercise has been recommended to improve motor function in Parkinson patients, but its value in altering progression of disease is unknown. In this study, we examined the neuroprotective effects of running wheel exercise in mice. In adult wild-type mice, one week of running wheel activity led to significantly increased DJ-1 protein concentrations in muscle and plasma. In DJ-1 knockout mice, running wheel performance was much slower and Rotarod performance was reduced, suggesting that DJ-1 protein is required for normal motor activity. To see if exercise can prevent abnormal protein deposition and behavioral decline in transgenic animals expressing a mutant human form of α-synuclein in all neurons, we set up running wheels in the cages of pre-symptomatic animals at 12 months old. Activity was monitored for a 3-month period. After 3 months, motor and cognitive performance on the Rotarod and Morris Water Maze were significantly better in running animals compared to control transgenic animals with locked running wheels. Biochemical analysis revealed that running mice had significantly higher DJ-1, Hsp70 and BDNF concentrations and had significantly less α-synuclein aggregation in brain compared to control mice. By contrast, plasma concentrations of α-synuclein were significantly higher in exercising mice compared to control mice. Our results suggest that exercise may slow the progression of Parkinson's disease by preventing abnormal protein aggregation in brain

Exercise increased α-synuclein concentration in blood plasma in 15-month old Y39C transgenic mouse.

<p>Mouse blood plasma samples were processed by Western blot analysis using antibodies to LB509 (<b>A</b>) and Syn-1 (<b>B</b>). Both monomer and dimer α-synucleins were found in mouse plasma. Sample images are shown for each Western blot. (<b>C, D</b>) Quantitative α-synuclein plasma levels are shown for each group after being normalized to mouse serum albumin. With both LB509 (<b>A</b>) and Syn-1 (<b>B</b>) antibodies, there were significant increases in α-synuclein monomer, dimer and total (monomer plus dimer) fractions in Exercise versus Non-Exercise mouse plasma (n = 7, t-test, *<i>p</i><0.05).</p

Exercise reduced α-synuclein oligomer formation in 15 month-old Y39C transgenic mouse brain.

<p>Brain tissues (cortex) from Exercise and Non-Exercise transgenic mice were analyzed for α-synuclein aggregation using LB509 (human only) and Syn-1 (mouse-plus-human) antibodies. (<b>A</b>) Western blots with LB509 show that exercise dramatically reduced α-synuclein oligomer formation in brain compared to mice of the same age not performing exercise. α-Synuclein monomer levels were not changed. The sample blots show Exercise (<b>Ex</b>) and Non-Exercise (<b>nEx</b>) animals. (<b>B</b>) Western blots with Syn-1 antibody show that Exercise mice had reduced α-synuclein oligomer and dimer fractions, as compared to Non-Exercise mice. (<b>C, D</b>) Western blot images were quantified. The relative levels of α-synuclein oligomer, monomer, and total (oligomer plus monomer) to β-actin are shown for Exercise and Non-Exercise transgenic mice. There were significant reductions in brain oligomer (n = 7, t-test, **<i>p</i><0.01) and total (n = 7, t-test, *<i>p</i><0.05) in the Exercise group compared to the Non-Exercise group.</p

Y39C human mutant α-synuclein transgenic mice performed daily running wheel exercise for three months which led to improved motor and cognitive function.

<p>12-month-old Y39C transgenic mice were divided into Exercise and Non-Exercise groups (n = 7 for each group) following pre-testing of all 14 animals in individual cages with running wheels. Animals were assigned to Exercise and Non-Exercise groups by alternating rank order following their week-long pre-test. Exercise mice had free access to individual cage-mounted running wheels and Non-Exercise mice had a locked, non-functioning running wheel in individual cages. Daily running distances of the Exercise animals were recorded and averaged for each week. (<b>A</b>) Data show that all animals continued running for 12 weeks with some reduction in running speed. Average distance in the first week was 3.76 ± 0.87 miles per day. Average distance in the 12^th week was 2.71 ± 0.53 miles per day (no statistical difference between 1^st and 12^th week, n = 7, multi-variance ANOVA, <i>p</i> = 0.33). After 12-weeks of running wheel activity, all mice were tested for high intensity motor activity on the Rotarod (<b>B</b>) and cognitive function using a Morris water maze (<b>C</b>). (<b>B</b>) In the Rotarod test, the Exercise group could remain on the rod significantly longer at 26 rpm than the Non-Exercise group (n = 7, multi-variance ANOVA, **<i>p =</i> 0.001). (<b>C</b>) In the Morris water maze, the Exercise mice took significantly less time to find the hidden platform at Day 5 than Non-Exercise transgenic mice (n = 7, multi-variance ANOVA, *<i>p =</i> 0.02).</p

Running wheel exercise in wild-type mice increased DJ-1 expression in muscle, plasma, and brain.

<p>Adult FVB/N wild-type mice (4–6 months old) were assigned to either an exercise group or a control group (n = 5 each). After one week of running wheel exercise, animals were sacrificed and tissues collected for DJ-1 protein analysis by Western blot (muscle and brain) and ELISA (plasma). (<b>A</b>) Quantitative data normalized to β-actin show that muscle DJ-1 levels were significantly higher in the exercise group than controls (t-test, **<i>p</i><0.01). (<b>B</b>) DJ-1 levels in plasma were significantly increased in exercise mice compared to control mice (t-test, **<i>p</i><0.01). (<b>C</b>) Brain DJ-1 levels were higher in exercise mice than control mice, but not significantly (t-test, <i>p</i> = 0.06). (<b>D</b>) Correlations between muscle and plasma DJ-1 (dashed line) and brain DJ-1 (solid line) in exercising mice. Both plasma and brain DJ-1 levels were significantly correlated with the exercise-induced change in muscle DJ-1 (plasma vs. muscle, <i>p</i> = 0.04 and brain vs. muscle, <i>p</i> = 0.03). All DJ-1 values are relative to non-exercise controls.</p

Exercise increased DJ-1, Hsp70 and BDNF levels in 15-month old Y39C transgenic mouse brain and also increased DJ-1 levels in muscle and plasma.

<p>(<b>A</b>) Brain tissues (cortex) were processed for Western blot analysis using antibodies to DJ-1, Hsp70, BDNF and β-actin after 3 months of running wheel Exercise (<b>Ex</b>) or no Exercise (<b>nEx</b>) in Y39C transgenic animals. Representative images are shown for all Western blots. (<b>B-D</b>) Quantitative protein levels in brain are shown for each group after being normalized to β-actin. There were significant increases in DJ-1, Hsp70 and BDNF proteins in Exercise mouse brain compared to Non-Exercise mice (n = 7, t-test, *<i>p</i><0.05, **<i>p</i><0.01). (<b>E)</b> Muscle samples were processed for Western blot analysis using DJ-1 and β-actin antibodies after 3 months of running wheel Exercise (<b>Ex</b>) or no Exercise (<b>nEx</b>) in control animals. (<b>F</b>) Muscle DJ-1 Westerns were quantified and are shown for each group after being normalized to β-actin. Muscle DJ-1 was significantly increased in Exercise mice compared to Non-Exercise mice (n = 7, t-test, **<i>p</i><0.01). (<b>G</b>) Plasma DJ-1 levels were measured by ELISA. Results show that the Exercise group had significantly higher plasma DJ-1 concentrations than the Non-Exercise mice (n = 7, t-test, **<i>p</i><0.01).</p

DJ-1 knockout mice had impaired performance on running wheels and on the Rotarod but normal cognitive and exploratory activity.

<p>Ten months old homozygous DJ-1 knockout (DJ-1 KO) mice and C57BL/6 wild-type (WT) littermates were trained to perform running wheel exercise in their individual cages. Daily running distances were recorded for two weeks. (<b>A</b>) Wild-type mice ran 5.57 ± 0.21 miles per day, while DJ-1 knockout mice were significantly slower, running 0.89 ± 0.06 miles per day (n = 6, multi-variance ANOVA test, **<i>p</i><0.01). Ten months old DJ-1 knockout mice and C57BL/6 wild-type littermates were also tested on the Rotarod (<b>B</b>), Morris water maze (<b>C</b>), and open field (<b>D</b>). (<b>B</b>) DJ-1 knockout mice fell from the Rotarod after a shorter period of time than WT mice when tested at 20 and 26 rpm (n = 6, multi-variance ANOVA, F_{(4, 60)} = 16.93, <i>p</i> = 0.0089, *<i>p</i><0.05, **<i>p</i><0.01). (<b>C</b>) DJ-1 knockout mice and WT littermates had similar learning curves in the Morris water maze (n = 6, multi-variance ANOVA, <i>p</i>>0.1). (<b>D</b>) DJ-1 knockout mice and WT mice had similar exploratory activity in open field testing (n = 6, multi-variance ANOVA, <i>p</i>>0.1).</p