Faculty Bibliography

Amyloid plaques are a key pathological hallmark of Alzheimer's disease (AD). The detection of amyloid plaques in the brain is important for the diagnosis of AD, as well as for following potential amyloid targeting therapeutic interventions. Our group has developed several contrast agents to detect amyloid plaques using magnetic resonance microimaging (microMRI) in AD transgenic mice, where we used mannitol to enhance blood brain barrier (BBB) permeability. In the present study, we used bifunctional ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, chemically coupled with Abeta1-42 peptide to image amyloid plaque deposition in the mouse brain. We coupled the nanoparticles to polyethylene glycol (PEG) in order to improve BBB permeability. These USPIO-PEG-Abeta1-42 nanoparticles were injected intravenously in AD model transgenic mice followed by initial and subsequent muMRI. A 3D gradient multi-echo sequence was used for imaging with a 100 microm isotropic resolution. The amyloid plaques detected by T2*-weighted muMRI were confirmed with matched histological sections. The region of interest-based quantitative measurement of T2* values obtained from the muMRI showed contrast injected AD Tg mice had significantly reduced T2* values compared to wild-type mice. In addition, the scans were examined with voxel-based analysis (VBA) using statistical parametric mapping (SPM) for comparison of USPIO-PEG-Abeta1-42 injected AD transgenic and USPIO alone injected AD transgenic mice. The regional differences seen by VBA in the USPIO-PEG-Abeta1-42 injected AD transgenic correlated with the amyloid plaque distribution histologically. Our results indicate that USPIO-PEG-Abeta1-42 can be used for amyloid plaque detection by intravenous injection without the need to co-inject an agent which increases permeability of the BBB. This technique could aid the development of novel amyloid targeting drugs by allowing therapeutic effects to be followed longitudinally in model AD mice.

The CD40 receptor-CD40 ligand (CD40-CD40L) interaction has been shown to affect both immune and non-immune cells and is implicated in diverse activities including immunoglobulin class switching (IgM to IgG), atherosclerosis, chronic inflammation and Alzheimer's disease pathogenesis. A number of groups have studied the role of CD40 in prion disease, however, the results are conflicting presumably due to the use of different scrapie agent-host strain combinations and routes of infection. In the current study, we clarify the effect of CD40 on: (i) replication, progression to clinical disease, PrP(Sc) profile, and neuropathology associated with infection of a single host genotype with three distinct mouse-adapted scrapie strains, and (ii) the immune response of double knockout (PrP, CD40) transgenic mice to recombinant PrP as assessed by the generation of anti-PrP antibodies. Our results suggest that CD40: (i) results in slower disease progression and scrapie strain-specific differences in incubation periods, (ii) does not affect the level of scrapie strain-specific PrP(Sc), (iii) does not influence disease-associated neuropathology, but (iv), as expected, is required to mount an immune response generating anti-PrP IgG antibodies.

Desmosterol is a C27 sterol intermediate in cholesterol synthesis generated during the metabolic pathway that transforms lanosterol into cholesterol. It has become of particular interest in the pathogenesis of Alzheimer's disease (AD) because of the report that the activity of the gene coding for the enzyme DHCR24, which metabolizes desmosterol to cholesterol, is selectively reduced in the affected areas of the brain. Any change in the pattern of C27 sterol intermediates in cholesterol synthesis merits investigation with respect to the pathogenesis of AD, since neurosteroids such as progesterone can modulate the tissue levels. We therefore analyzed the C27 sterol composition using a metabolomics approach that preserves the proportion of the different sterol intermediates. In AD, the proportion of desmosterol was found to be less than that of age-matched controls. The findings do not directly support the focus on Seladin-1, although they could reflect different stages of a slowly progressive disease.

Cyclic diguanylate (c-di-GMP), a bacterial signaling molecule, possesses protective immunostimulatory activity in bacterial challenge models. This study explored the potential of c-di-GMP as a vaccine adjuvant comparing it with LPS, CpG oligonucleotides, and a conventional aluminum salt based adjuvant. In this evaluation, c-di-GMP was a more potent activator of both humoral and Th1-like immune responses as evidenced by the robust IgG2a antibody response it induced in mice and the strong IFN-gamma, TNF-alpha and IP-10 responses, it elicited in mice and in vitro in non-human primate peripheral blood mononuclear cells. Further, compared to LPS or CpG, c-di-GMP demonstrated a more pronounced ability to induce germinal center formation, a hallmark of long-term memory, in immunized mice. Together, these data add to the growing body of evidence supporting the utility of c-di-GMP as an adjuvant in vaccination for sustained and robust immune responses and provide a rationale for further evaluation in appropriate models of immunization.

Background: Alzheimer's disease (AD) is the most common cause of dementia and has a major societal and economic impact. Immunomodulation has shown great promise as an AD therapy, even though the initial clinical trial was associated with severe adverse effects in a minority of patients. Our research group postulated stimulation of the innate immune system, via the Toll-like receptor 9 (TLR9), as a possible alternative method for ameliorating AD pathology, without associated toxicity. A significant concern with immunotherapy is clearance of vascular amyloid and associated microhemorrhages. This is an important issue, since cerebral amyloid angiopathy (CAA) is a common feature in AD and cognitively normal elderly individuals. We tested the efficacy of TLR9 signaling stimulation for reducing parenchymal and vascular amyloid, as well as tau related pathology. Methods: We utilized TLR9 agonist type B CpG oligonucleotides (ODNs) to stimulate innate immunity in 3xTg-AD mice, which develop both plaque and tangle pathology, and in Tg-SwDI mice with extensive CAA. Animals were divided into 2 study groups treated prior to or after the onset of AD pathology. Results: After treatment the mice were behaviorally tested. No statistical differences were observed between the groups in any of the locomotor parameters measured. CpG ODN treatment improved working memory in 3xTg-AD mice as indicated by radial arm maze testing. TLR9 stimulation was also effective at improving short-term memory in Tg-SwDI mice as evidenced by novel-object recognition testing. In 3xTg-AD mice TLR9 signaling reduced both amyloid deposits and hyperphosphorylated tau pathology. Semiquantitative analysis of hippocampal CA1 neurons revealed reduction in AT8 and PHF1 immunoreactivity in CpG ODN-treated 3xTg-AD mice. The reduction of plaque and tangle pathology was paralleled by an overall reduction in the numbers of activated microglia. There were no group differences in the levels of CNS astrocytosis; hence there was no evidence of encephalitis in the brains of treated mice. Further histological, biochemical analyses and characterization of immune responses are ongoing. Conclusions: Overall, stimulation of the TLR9 and thus innate immunity with CpG ODN (currently used in clinical trials for a variety of other diseases) represents a novel immunotherapeutic approach for AD

Background: Due to well-known markedly increased susceptibility contrast at ultra-high-field MR, this work is using 7T MR susceptibiltyweighted imaging (SWI) to better identify the histopathologic correlate of amyloid plaques containing iron and otherwise invisible subhippocampal structures of human post-mortem brain in patients with Alzheimer's disease. Methods: Post-mortem brain specimens of the frontal lobe and hippocampus were obtained from 8 patients (mean age 71.266.2 years) with clinically diagnosed AD and 6 age-matched healthy controls (72.4 66.6 years) without AD. Coronal 1w3 cm thick brain slices were preserved and fixed in 2% agar or formalin for this study. Imaging was performed on a 7.0T MR. A 24- element phased array head coil was used. High resolution 3D SWI was obtained with isotropic voxel size 150w320I=m. For imaging optimization to better visualize amyloid plaques, we varied TR, TE, bandwidth and flip angle from 30-100ms, 12-36ms, 60-140Hz/pixel and 10-40-; respectively. Results: Compared to controls, 7T SWI revealed a largely increased number of hypointense foci inADbrain samples along the cortical mantle of the frontal and entorhinal cortex due to enhanced susceptibility effects and superb signal. Figure 1 shows amyloid plaques identified with histologic slice (A) and in post-processed SWI image of the frontal cortex of an AD patient (B) as compared to a healthy control (C). The average phase value in the cortex region of AD data (2296 6 72) is significantly higher than that of control data (2032 6 64), which indicates higher iron amount in AD samples. In addition, 7T SWI also provides high resolution images for subregional hippocampal structures including CA1 CA2 CA3 subiculum, and dentate gyrus with significant atrophy of these structures in AD patients. Conclusions: Our findings suggest that SWI with optimization at ultra-high-field strength MR (i.e. 7T) has exhibited the capability to detect diminutive susceptibility contrast associated with iron deposition and otherwise invisible fine hippocampal structures with near histopathologic resolution. Therefore, SWI has great potential for direct detection and quantification of amyloid plaques in live human brain, and may become feasible in vivo in the near future

Background: Alzheimer's Disease (AD) is the most common of the conformational neurodegenerative disorders (NDD), while prion diseases are the most transmissible NDD. No highly effective treatment is currently available for any NDD. Previously, we have shown that mutated Ab peptides soluble and devoid of T-cell epitopes, produced a response which correlates with amelioration of AD pathology in animal models. Our lab has demonstrated that active immunization with antigens mimicking abnormal conformations such as polymerized ABri (pABri), could elicit antibody responses to oligomerized Ab and conformational aberrant hyperphosphorylated tau (ptau).We have now tested a combined vaccination using pBri and polymerized mutated Ab (pAb) in 3xTg mice and human PrP-Tg animals. Methods: We produced pBri and pAb1-30K18K19, which by electron microscopy formed oligomer-like structures. The pBri alone was used as an immunogen in HuPrPTg animals and in conjunction with pAb1-30KK in AD 3xTg mice using alum as an adjuvant. At the age of 15-16 months the AD mice were subjected to behavioral testing followed by histological and biochemical analysis. The HuPrPTg were assessed for the presence of antibodies against PrPRes. Results: 3xTg mice immunized animals showed no difference in locomotor activity compared to controls but a significant behavioral rescue by radial arm maze testing. The animals produced significant titers of IgM and IgG against Ab42 oligomers. Histological analysis showed a significant reduction of both amyloid and tau pathology. Biochemically, Ab42 and soluble ptau were significantly reduced. In HuPrPTg animals a systemic IgM and IgG response against PrPRes was observed, similar to the systemic response obtained in our prior successful mucosal PrP vaccinations. Conclusions: pBri conformational vaccination can be used to elicit antibodies that recognize multiple pathological proteins, including PrPRes, oligomer Ab and ptau. In 3x Tg mice with both Ab and tau pathology which were vaccinated with the combined pABri+pAb1-30KK, each of these pathologies were reduced histologically and biochemically, in association with a behavioral rescue. Such a combined approach targeting both pathological Ab and tau is more likely to be efficacious in AD patients

Background: Prionoses are transmissible, neurodegenerative disorders. Bovine spongioform encephalopathy (BSE) has crossed the species barrier to infect humans. Chronic wasting disease (CWD) infects large numbers of deer and elk, with the potential to infect humans. Currently no prionoses has an effective treatment. Previously, we have demonstrated we could prevent transmission of prions in a proportion of susceptible mice with a mucosal vaccine. Currently, we have attempted to produce an immune response in animals naturally susceptible to CWD, white tail deer, which could inhibit transmission. Methods: White tail deer were orally inoculated with attenuated Salmonella carrying deer or mouse PrP. Once established the mucosal response, the animals were boosted orally and locally in the tonsils and rectum with polymerized recombinant PrP. Blood, saliva and feces were collected regularly to assess IgA, IgM and IgG titers to PrP. The vaccinated and control animals were then challenged orally with CWD infected brains. Tonsil and rectal biopsies plus blood, saliva, feces and urine were collected every three months post-infection until the 9 month; and every 45 days thereafter. Results: Two vaccinated animals produced low antibody titers, two intermediate titers and one high titers of IgA and IgG anti-PrP. Both groups produced high titers of IgA and IgG against Salmonella. Six months post-infection 5 out of 6 controls and 3 out of 5 vaccinated showed histologically prion structures in the tonsils. Ayear post-infection one of the vaccinated animals remained prion free, with all controls being infected. The negative animal has the highest titers of IgA in saliva and IgG systemic against PrP. Immunoglobulins purified from saliva, feces and serum of this vaccinated deer reacts to PrPRes. Conclusions: Oral immunization can be used to overcome tolerance to self-PrP protein and produce a mucosal IgA and systemic IgG response to normal and conformational modified PrP in large mammals. High antibody titers might be enough to prevent transmission or to retard progression of PrP infection. This approach may lead to an effective anti-prion vaccine

Background: Amyloid plaques are a key pathological hallmark of Alzheimer's disease (AD). Their visualization is important for the diagnosis of AD, monitoring disease progression and evaluation of the efficacy of therapeutic interventions. We were the first group to use ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles coupled to an amyloid targeting peptide to visualize amyloid plaques. USPIO's have been widely used in animal and human imaging, and have been found to be very safe. However, our approach, so far, has required intra-carotid or intra-femoral injection, with mannitol to break the blood brain barrier (BBB). In the current study we sought to develop and test non-toxic, bifunctional USPIO particles which could be introduced via the femoral vein, without mannitol. Methods: 13 to 18 month old APP/PS1 transgenic mice and age-matched wild-type (C57Bl/6J) control mice were used. The USPIO nanoparticles were linked to Ab1-42 (targeting to amyloid plaques) and polyethyleneglycol (PEG, to increase BBB permeability), using EDC/NHS coupling methods. The potential neurotoxicity of USPIO-PEG-Ab42 was assessed in N2a cells using a MTS Assay. MRI scans were performed on a 7T micro- MRI system consisting of a 7-Telsa 200-mm horizontal bore magnet. All mice were scanned 6-hrs after iv injection (0.2 mmol Fe/kg body weight) of the USPIO-PEG-Ab42. Ex vivo imaging of mouse brains was also performed. Serial coronal sections were subject to anti-Ab immunohistochemistry and Perl staining to identify the USPIO. Results: The USPIO particles were non-toxic. Figure 1A showsmMRI and matching histology in an APP/ PS1 Tg mouse and wild type mouse injected with USPIO-PEG-Ab. Numerous dark spots are evident in 1A. To the right of 1A is a higher magnification of an area of the MRI which is matched to double Perl and Ab stain in figure 1B. A coronal section matching to 1A is seen in 1C. In 1D the blue areas point to Perl stain positive dots corresponding to USPIO particles while the red arrow points to the amyloid plaque. 1E shows a wild-type mouse injected with USPIO-PEG-Ab. 1F documents the lack of plaques in the WT mouse. Conclusions: Our non-toxic, non-invasive m MRI method has great potential for the longitudinal assessment of amyloid burden

Platelets play a supportive role in tumor metastasis. Impairment of platelet function within the tumor microenvironment may provide a clinically useful approach to inhibit metastasis. We developed a novel humanized single-chain antibody (scFv Ab) against integrin GPIIIa49-66 (named A11) capable of lysing activated platelets. In this study, we investigate the effect of A11 on the development of pulmonary metastases. In the Lewis lung carcinoma (LLC) metastatic model, A11 decreases the mean number of surface nodules and mean volume of pulmonary nodules. It protects against lung metastases in a time window that extended 4 hours prior to and 4 hours after the i.v. injection of LLCs. Co-injection of GPIIIa49-66 albumin reverses the anti-metastatic activity of A11 in B16 melanoma model, consistent with the pathophysiological relevance of the platelet GPIIIa-49-66 epitope. Significantly A11 had no effect on angiogenesis using both in vitro and in vivo assays. The underlying molecular mechanisms are a combination of inhibition of each of the following interactions: between activated platelets and tumor cells, platelets and endothelial cells, and platelets and monocytes, as well as disaggregation of an existing platelet/tumor thrombus. Our observations may provide a novel anti-metastatic strategy through lysing activated platelets in the tumor microenvironment using humanized anti-GPIIIa49-66 scFv Ab.