A P-insertion Screen Identifying Novel X-linked Essential Genes in Drosophila

Mechanisms of Development. Jan, 2002  |  Pubmed ID: 11744370

The recent determination and annotation of the entire euchromatic sequence of the Drosophila melanogaster genome predicted the existence of about 13600 different genes (Science 287 (2000) 2185; http://www.fruitfly.org/annot/index.html). In parallel, the Berkeley Drosophila Genome Project (BDGP) has undertaken systematic P-insertion screens, to isolate new lethals and misexpressing lines. To date, however, the genes of the X chromosome have been under-represented in the screens performed. In order both to characterize several X-linked genes of prime interest to our laboratories and contribute to the collection of lethal P-insertions available to the community, we performed a P-insertion mutagenesis of the X chromosome. Using the PlacW and PGawB P-elements as mutagens, we generated two complementary sets of enhancer-trap lines, l(1)(T)PL and l(1)(T)PG, respectively, which both contain a reporter gene whose developmental expression can be monitored when driven by nearby enhancer sequences. We report here the characterization of 260 new insertions, mapping to 133 different genes or predicted CGs. Of these, 83 correspond to genes for which no lethal mutation had yet been reported. For 64 of those, we could confirm that lethality was solely due to the P-element insertion. The primary molecular data, reporter gene expression patterns (observed in embryos, third instar larvae and adult ovaries) and proposed CG assignment for each strain can be accessed and updated on our website at the following address: http://www-cbd.ups-tlse.fr:8080/screen.

The Drosophila Gene Taranis Encodes a Novel Trithorax Group Member Potentially Linked to the Cell Cycle Regulatory Apparatus

Genetics. Feb, 2002  |  Pubmed ID: 11861561

Genes of the Drosophila Polycomb and trithorax groups (PcG and trxG, respectively) influence gene expression by modulating chromatin structure. Segmental expression of homeotic loci (HOM) initiated in early embryogenesis is maintained by a balance of antagonistic PcG (repressor) and trxG (activator) activities. Here we identify a novel trxG family member, taranis (tara), on the basis of the following criteria: (i) tara loss-of-function mutations act as genetic antagonists of the PcG genes Polycomb and polyhomeotic and (ii) they enhance the phenotypic effects of mutations in the trxG genes trithorax (trx), brahma (brm), and osa. In addition, reduced tara activity can mimic homeotic loss-of-function phenotypes, as is often the case for trxG genes. tara encodes two closely related 96-kD protein isoforms (TARA-alpha/-beta) derived from broadly expressed alternative promoters. Genetic and phenotypic rescue experiments indicate that the TARA-alpha/-beta proteins are functionally redundant. The TARA proteins share evolutionarily conserved motifs with several recently characterized mammalian nuclear proteins, including the cyclin-dependent kinase regulator TRIP-Br1/p34(SEI-1), the related protein TRIP-Br2/Y127, and RBT1, a partner of replication protein A. These data raise the possibility that TARA-alpha/-beta play a role in integrating chromatin structure with cell cycle regulation.

Evidence for a Mediator of RNA Polymerase II Transcriptional Regulation Conserved from Yeast to Man

Cell. Jul, 2002  |  Pubmed ID: 12150923

Mediator complexes (MED) link transcriptional regulators to RNA polymerase II. Here, we summarize the latest advances on the functional organization of yeast Mediator. We argue for the existence of a "universal" Mediator structurally conserved from yeast to man, based on an extensive analysis of sequence databases. Finally, we examine the implications of these observations for the physiological roles of metazoan MED subunits.

Beta-amyloid Deposition and Neurofibrillary Tangle Association with Caspase Activation in Down Syndrome

Neuroscience Letters. Sep, 2002  |  Pubmed ID: 12213643

Individuals with Down syndrome (DS) and Alzheimer's disease (AD) develop senile plaques, neurofibrillary tangles (NFT), and neuron loss. Recent studies demonstrate the activation of apoptotic pathways in AD; less data is available in DS. The DS brain was examined using immunocytochemistry and antibodies against the active fragment of caspase-8 (AC, 8) and to caspase-3 cleavage products of fodrin (CCP), a neuronal cytoskeleton protein. The hippocampus demonstrated widespread accumulation of fodrin CCP and AC8 in NFTs and dystrophic neurites. Individual neurons contained intracellular beta-amyloid (Abeta) and fodrin CCP providing evidence that caspase activation can occur with both NFT and Abeta. Abeta within or around neurons in addition to contributing to NFT formation may also trigger apoptotic pathways. Caspase activation may lead to the cleavage of critical cellular proteins and neuronal cell death associated with DS.

Evidence for a Direct Functional Antagonism of the Selector Genes Proboscipedia and Eyeless in Drosophila Head Development

Development (Cambridge, England). Feb, 2003  |  Pubmed ID: 12490563

Diversification of Drosophila segmental and cellular identities both require the combinatorial function of homeodomain-containing transcription factors. Ectopic expression of the mouthparts selector proboscipedia (pb) directs a homeotic antenna-to-maxillary palp transformation. It also induces a dosage-sensitive eye loss that we used to screen for dominant Enhancer mutations. Four such Enhancer mutations were alleles of the eyeless (ey) gene that encode truncated EY proteins. Apart from eye loss, these new eyeless alleles lead to defects in the adult olfactory appendages: the maxillary palps and antennae. In support of these observations, both ey and pb are expressed in cell subsets of the prepupal maxillary primordium of the antennal imaginal disc, beginning early in pupal development. Transient co-expression is detected early after this onset, but is apparently resolved to yield exclusive groups of cells expressing either PB or EY proteins. A combination of in vivo and in vitro approaches indicates that PB suppresses EY transactivation activity via protein-protein contacts of the PB homeodomain and EY Paired domain. The direct functional antagonism between PB and EY proteins suggests a novel crosstalk mechanism integrating known selector functions in Drosophila head morphogenesis.

Importance of IgG2c Isotype in the Immune Response to Beta-amyloid in Amyloid Precursor Protein/transgenic Mice

Neuroscience Letters. Feb, 2003  |  Pubmed ID: 12565127

A careful analysis of the immune response to immunization of amyloid precursor protein/transgenic (APP/Tg) mice with beta-amyloid (Abeta) may provide insights into why a subset of the patients in a clinical trial receiving Abeta-immunotherapy developed encephalomyelitis. Characterization of isotypic immune responses have been reported in different APP/Tg models. In these studies the relative ratios of IgG1 to IgG2a anti-Abeta antibodies has been used as an indirect measure of T helper 1 (Th1) and Th2 types immune responses. However, it has previously been shown that certain strains of mice, C57Bl/6, C57Bl/10, SJL, and NOD, have an IgG2c rather than an IgG2a gene. Since a substantial number of Abeta-immunization studies rely on APP/Tg mice that have at least one parental C57Bl/6 strain, we have investigated whether antibodies specific for IgG2a can be used for characterization of antibody isotypes in APP/Tg2576 mice. Our results suggest that APP/Tg2576 and major histocompatibilty complex-matched parental strains are not expressing IgG2a, producing instead IgG2c anti-Abeta antibodies.

The Induction of the TNFalpha Death Domain Signaling Pathway in Alzheimer's Disease Brain

Neurochemical Research. Feb, 2003  |  Pubmed ID: 12608703

The tumor necrosis factor-alpha death domain pathway contributes to cellular degeneration in a variety of conditions. This study investigates the hypothesis that this death domain pathway is progressively induced in the brain during the progression of Alzheimer's disease (AD). AD cases had increased levels of proapoptotic markers including tumor necrosis factor-alpha (TNFalpha), TNF receptor type 1 (TNF-R1), TNF receptor-associated death domain (TRADD), and caspase-3, 2- to 10-fold higher (P < .01) than age-matched controls and 1 to 3 times higher than transitional cases. In striking contrast, potentially neuroprotective TNF receptor type 2 (TNF-R2), and Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE) inhibitor protein (FLIP) were decreased in AD as compared with age-matched control cases (P < .01). Overall, there was an elevation in proapoptotic elements, including a 5-fold increase in TNF-R1 and a 12-fold decrease in FLIP in AD brains. These changes may translate to increased degenerative potential because the downstream effector caspase-3 and product of the TNF pathway was also increased in parallel with enhanced TNF proapoptotic conditions. Our findings suggest that the TNF death receptor pathway and caspases are activated in the early stages of neuronal degeneration in AD.

Adjuvant-dependent Modulation of Th1 and Th2 Responses to Immunization with Beta-amyloid

International Immunology. Apr, 2003  |  Pubmed ID: 12663680

The role of adjuvant on the T(h)1 and T(h)2 immune responses to Abeta-immunotherapy (Abeta(42 )peptide) was examined in wild-type mice. Fine epitope analysis with overlapping oligomers of the Abeta(42) sequence identified the 1-15 region as a dominant B cell epitope. The 6-20 peptide was recognized only weakly by antisera from mice administrated with Abeta(42) peptide formulated in complete Freund's adjuvant (CFA), alum or TiterMax Gold (TMG). However, mice immunized with Abeta(42) mixed with QS21 induced a significant antibody response to the 6-20 peptide. The only T cell epitope found was within the 6-28 sequence of Abeta(42). QS21 and CFA induced the strongest humoral response to Abeta, alum was intermediate, and TMG the weakest adjuvant. Analysis of antibody isotypes specific for Abeta indicates that alum induces primarily T(h)2-type immune response, whereas TMG, CFA and QS21 shift the immune responses toward a T(h)1 phenotype. Stimulation of splenocytes from Abeta-immunized mice with Abeta(40) peptide induced strikingly different cytokine expression profiles. QS21 and CFA induced significant IFN-gamma, IL-4 and tumor necrosis factor-alpha expression, whereas alum induced primarily IL-4 production. As T(h)1-type immune responses have been implicated in many autoimmune disorders, whereas T(h)2-type responses have been shown to inhibit autoimmune disease, the choice of adjuvant may be critical for the design of a safe and effective immunotherapy for Alzheimer's disease.

Fas and Fas Ligand Are Associated with Neuritic Degeneration in the AD Brain and Participate in Beta-amyloid-induced Neuronal Death

Neurobiology of Disease. Apr, 2003  |  Pubmed ID: 12742739

It has recently been suggested that neuronal cell death in response to many brain insults may be mediated by the upregulation of tumor necrosis factor receptor (TNFR) family members and their ligands. In the present study, we investigated whether the expression of the TNFR family death domain receptor, Fas, and its ligand, FasL, is altered in association with neuropathology and activated caspase markers in Alzheimer disease (AD) brain, and Abeta-induced neuronal cell death in vitro. To evaluate this hypothesis, we examined Fas and FasL expression in AD and control brain, and Abeta-treated primary neurons, using immunocytochemistry and Western blots. Neurons in both AD brain and Abeta-treated cultures exhibited FasL upregulation and changes in immunoreactivity for Fas receptor. Further, FasL expression was remarkably elevated in senile plaques and neurofilament-positive dystrophic neurites, and in association with caspase activation and neuritic apoptosis in AD brain. Based on these and previous data regarding protection of primary neuronal cultures from Abeta(1-42)-induced apoptosis by blockade of Fas-associated death domain signaling, we also tested the hypothesis that dynamic regulation of Fas and FasL may contribute to Abeta-mediated neuronal cell death. Accordingly, neuronal cultures derived from mice carrying inactivating mutations in Fas (Faslpr) or FasL (Fasgld) exhibited protection from Abeta(1-42)-induced cell death. These findings suggest that Fas-FasL interactions may contribute to mechanisms of neuronal loss and neuritic degeneration in AD.

Immunization with a Vaccine That Combines the Expression of MUC1 and B7 Co-stimulatory Molecules Prolongs the Survival of Mice and Delays the Appearance of Mouse Mammary Tumors

Clinical & Experimental Metastasis. 2003  |  Pubmed ID: 14598882

Human epithelial mucin (MUC1) is expressed by many carcinomas, including breast cancer cells. This breast cancer-associated antigen has been widely used for immunotherapy, despite the fact that cellular immune responses to MUC1 are impaired in breast cancer patients and MUC1 transgenic animals. Previously, we found that immunogenicity to MUC1 was also impaired in BALB/c mice injected with a mammary tumor cell line (410.4) expressing human MUC1. We suggested that one reason for its weak immunogenicity was the lack of expression of B7 molecules by 410.4 cells. Recognition of antigenic epitopes in conjunction with MHCI/II by the T-cell receptor without co-stimulation by B7/CD28 association resulted in T-cell anergy. Therefore, we attempted to enhance protective anti-MUC1-specific immunity in mice using B7 co-stimulatory molecules as a component of the MUC1 vaccine. We also compared cell-based with DNA-based vaccination strategies. One group of mice was vaccinated with an irradiated, 410.4 syngeneic mammary tumor cell line co-expressing human MUC1 and CD80 or CD86 co-stimulatory molecules, and a second group of mice was vaccinated with plasmids encoding MUC1 and CD80 or CD86. These mice along with appropriate controls were challenged with mammary tumor cell line 4T1, which expresses MUC1. There were significant inhibition on rates of tumor growth and survival in mice vaccinated with irradiated 410.4/MUC1 cells co-expressing either CD80 or CD86 molecules, compared to non-vaccinated animals. In addition, there were also significant delays in the appearance of measurable tumors and their growth in mice vaccinated by gene-gun immunization with plasmids encoding MUC1 and CD80 or CD86.

Generation and Characterization of the Humoral Immune Response to DNA Immunization with a Chimeric Beta-amyloid-interleukin-4 Minigene

European Journal of Immunology. Dec, 2003  |  Pubmed ID: 14635031

Active immunization with fibrillar beta-amyloid peptide (Abeta(42)) as well as passive transfer of anti-Abeta antibodies significantly reduces Abeta plaque deposition, neuritic dystrophy, and astrogliosis in the brain of mutant amyloid precursor protein (APP)-transgenic mice. Although the mechanism(s) of clearance of Abeta from the brain following active or passive immunization remains to be determined, it is clear that anti-Abeta antibodies are critical for clearance. DNA immunization provides an attractive alternative to direct peptide and adjuvant approaches for inducing a humoral response to Abeta. We constructed a DNA minigene with Abeta fused to mouse interleukin-4 (pAbeta(42)-IL-4) as a molecular adjuvant to generate anti-Abeta antibodies and enhance the Th2-type of immune responses. Gene gun immunizations induced primarily IgG1 and IgG2b anti-Abeta antibodies. Fine epitope analysis with overlapping peptides of the Abeta(42) sequence identified the 1-15 region as a dominant B cell epitope. The DNA minigene-induced anti-Abeta antibodies bound to Abeta plaques in brain tissue from an Alzheimer's disease patient demonstrating functional activity of the antibodies and the potential for therapeutic efficacy.

Microfluidic Multicompartment Device for Neuroscience Research

Langmuir : the ACS Journal of Surfaces and Colloids. Mar, 2003  |  Pubmed ID: 20725530

This paper describes and characterizes a novel microfabricated neuronal culture device. This device combines microfabrication, microfluidic, and surface micropatterning techniques to create a multicompartment neuronal culturing device that can be used in a number of neuroscience research applications. The device is fabricated in poly(dimethylsiloxane), PDMS, using soft lithography techniques. The PDMS device is placed on a tissue culture dish (polystyrene) or glass substrate, forming two compartments with volumes of less than 2 μL each. These two compartments are separated by a physical barrier in which a number of micron-size grooves are embedded to allow growth of neurites across the compartments while maintaining fluidic isolation. Cells are plated into the somal (cell body) compartment, and after 3-4 days, neurites extend into the neuritic compartment via the grooves. Viability of the neurons in the devices is between 50 and 70% after 7 days in culture; this is slightly lower than but comparable to values for a control grown on tissue culture dishes. Healthy neuron morphology is evident in both the devices and controls. We demonstrate the ability to use hydrostatic pressure to isolate insults to one compartment and, thus, expose localized areas of neurons to insults applied in soluble form. Due to the high resistance of the microgrooves for fluid transport, insults are contained in the neuritic compartment without appreciable leakage into the somal compartment for over 15 h. Finally, we demonstrate the use of polylysine patterning in combination with the microfabricated device to facilitate identification and visualization of neurons. The ability to direct sites of neuronal attachment and orientation of neurite outgrowth by micropatterning techniques, combined with fluidically isolated compartments within the culture area, offers significant advantages over standard open culture methods and other conventional methods for manipulating distinct neuronal microenvironments.

Caspase-mediated Degeneration in Alzheimer's Disease

The American Journal of Pathology. Aug, 2004  |  Pubmed ID: 15277211

Abeta Immunotherapy Leads to Clearance of Early, but Not Late, Hyperphosphorylated Tau Aggregates Via the Proteasome

Neuron. Aug, 2004  |  Pubmed ID: 15294141

Amyloid-beta (Abeta) plaques and neurofibrillary tangles are the hallmark neuropathological lesions of Alzheimer's disease (AD). Using a triple transgenic model (3xTg-AD) that develops both lesions in AD-relevant brain regions, we determined the consequence of Abeta clearance on the development of tau pathology. Here we show that Abeta immunotherapy reduces not only extracellular Abeta plaques but also intracellular Abeta accumulation and most notably leads to the clearance of early tau pathology. We find that Abeta deposits are cleared first and subsequently reemerge prior to the tau pathology, indicative of a hierarchical and direct relationship between Abeta and tau. The clearance of the tau pathology is mediated by the proteasome and is dependent on the phosphorylation state of tau, as hyperphosphorylated tau aggregates are unaffected by the Abeta antibody treatment. These findings indicate that Abeta immunization may be useful for clearing both hallmark lesions of AD, provided that intervention occurs early in the disease course.

Mimotopes of Conformational Epitopes in Fibrillar Beta-amyloid

Journal of Neuroimmunology. Nov, 2004  |  Pubmed ID: 15465592

In Alzheimer's disease (AD) beta-amyloid peptide accumulates in the brain in different forms including fibrils. Amyloid fibrils could be recognized as foreign by the mature immune system since they are not present during its development. Thus, using mouse antisera raised against the fibrillar form of Abeta42, we have screened two phage peptide libraries for the presence of foreign conformational mimotopes of Abeta. Antisera from wild type animals recognized predominately peptides with the EFRH motif from Abeta42 sequence, whereas amyloid precursor protein (APP) transgenic mice recognized mainly phage clones that mimic epitopes (mimotopes) within the fibrillar Abeta42 but lack sequence homology with this peptide.

Patterned Cell Culture Inside Microfluidic Devices

Lab on a Chip. Jan, 2005  |  Pubmed ID: 15616747

This paper describes a simple plasma-based dry etching method that enables patterned cell culture inside microfluidic devices by allowing patterning, fluidic bonding and sterilization steps to be carried out in a single step. This plasma-based dry etching method was used to pattern cell-adhesive and non-adhesive areas on the glass and polystyrene substrates. The patterned substrate was used for selective attachment and growth of human umbilical vein endothelial cells, MDA-MB-231 human breast cancer cells, NIH 3T3 mouse fibroblasts, and primary rat cortical neurons. Finally, we have successfully combined the dry-patterned substrate with a microfluidic device. Patterned primary rat neurons were maintained for up to 6 days inside the microfluidic devices and the neurons' somas and processes were confined to the cell-adhesive region. The method developed in this work offers a convenient way of micropatterning biomaterials for selective attachment of cells on the substrates, and enables culturing of patterned cells inside microfluidic devices for a number of biological research applications where cells need to be exposed to well-controlled fluidic microenvironment.

Prototype Alzheimer's Disease Vaccine Using the Immunodominant B Cell Epitope from Beta-amyloid and Promiscuous T Cell Epitope Pan HLA DR-binding Peptide

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2005  |  Pubmed ID: 15661919

Immunization of amyloid precursor protein transgenic mice with fibrillar beta-amyloid (Abeta) prevents Alzheimer's disease (AD)-like neuropathology. The first immunotherapy clinical trial used fibrillar Abeta, containing the B and T cell self epitopes of Abeta, as the immunogen formulated with QS21 as the adjuvant in the vaccine. Unfortunately, the clinical trial was halted during the phase II stage when 6% of the participants developed meningoencephalitis. The cause of the meningoencephalitis in the patients that received the vaccine has not been definitively determined; however, analysis of two case reports from the AN-1792 vaccine trial suggest that the meningoencephalitis may have been caused by a T cell-mediated autoimmune response, whereas production of anti-Abeta Abs may have been therapeutic to the AD patients. Therefore, to reduce the risk of an adverse T cell-mediated immune response to Abeta immunotherapy we have designed a prototype epitope vaccine that contains the immunodominant B cell epitope of Abeta in tandem with the synthetic universal Th cell pan HLA DR epitope, pan HLA DR-binding peptide (PADRE). Importantly, the PADRE-Abeta(1-15) sequence lacks the T cell epitope of Abeta. Immunization of BALB/c mice with the PADRE-Abeta(1-15) epitope vaccine produced high titers of anti-Abeta Abs. Splenocytes from immunized mice showed robust T cell stimulation in response to peptides containing PADRE. However, splenocytes from immunized mice were not reactivated by the Abeta peptide. New preclinical trials in amyloid precursor protein transgenic mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse events that occurred in the first clinical trial.

Homeotic Proboscipedia Function Modulates Hedgehog-mediated Organizer Activity to Pattern Adult Drosophila Mouthparts

Developmental Biology. Feb, 2005  |  Pubmed ID: 15680366

Drosophila proboscipedia (pb; HoxA2/B2 homolog) mutants develop distal legs in place of their adult labial mouthparts. Here we examine how pb homeotic function distinguishes the developmental programs of labium and leg. We find that the labial-to-leg transformation in pb mutants occurs progressively over a 2-day period in mid-development, as viewed with identity markers such as dachshund (dac). This transformation requires hedgehog activity, and involves a morphogenetic reorganization of the labial imaginal disc. Our results implicate pb function in modulating global axial organization. Pb protein acts in at least two ways. First, Pb cell autonomously regulates the expression of target genes such as dac. Second, Pb acts in opposition to the organizing action of hedgehog. This latter action is cell-autonomous, but has a nonautonomous effect on labial structure, via the negative regulation of wingless/dWnt and decapentaplegic/TGF-beta. This opposition of Pb to hedgehog target expression appears to occur at the level of the conserved transcription factor cubitus interruptus/Gli that mediates hedgehog signaling activity. These results extend selector function to primary steps of tissue patterning, and lead us to suggest the notion of a homeotic organizer.

A Microfluidic Culture Platform for CNS Axonal Injury, Regeneration and Transport

Nature Methods. Aug, 2005  |  Pubmed ID: 16094385

Investigation of axonal biology in the central nervous system (CNS) is hindered by a lack of an appropriate in vitro method to probe axons independently from cell bodies. Here we describe a microfluidic culture platform that polarizes the growth of CNS axons into a fluidically isolated environment without the use of targeting neurotrophins. In addition to its compatibility with live cell imaging, the platform can be used to (i) isolate CNS axons without somata or dendrites, facilitating biochemical analyses of pure axonal fractions and (ii) localize physical and chemical treatments to axons or somata. We report the first evidence that presynaptic (Syp) but not postsynaptic (Camk2a) mRNA is localized to developing rat cortical and hippocampal axons. The platform also serves as a straightforward, reproducible method to model CNS axonal injury and regeneration. The results presented here demonstrate several experimental paradigms using the microfluidic platform, which can greatly facilitate future studies in axonal biology.

Novel Abeta Peptide Immunogens Modulate Plaque Pathology and Inflammation in a Murine Model of Alzheimer's Disease

Journal of Neuroinflammation. 2005  |  Pubmed ID: 16332263

Alzheimer's disease, a common dementia of the elder, is characterized by accumulation of protein amyloid deposits in the brain. Immunization to prevent this accumulation has been proposed as a therapeutic possibility, although adverse inflammatory reactions in human trials indicate the need for novel vaccination strategies.

Prototype Alzheimer's Disease Epitope Vaccine Induced Strong Th2-type Anti-Abeta Antibody Response with Alum to Quil A Adjuvant Switch

Vaccine. Mar, 2006  |  Pubmed ID: 16368167

Beta-amyloid (Abeta) peptide has been proposed to be a causal factor in Alzheimer's disease (AD). Currently being investigated, active and passive Abeta-immunotherapy significantly reduce Abeta plaque deposition, neuritic dystrophy, and astrogliosis in the brains of APP transgenic (APP/Tg) mice. Immunization with Abeta42 formulated in the Th1-type adjuvant QS21 was beneficial for AD patients with significant titers of anti-Abeta antibodies, however, 6% of participants developed meningoencephalitis, likely due to anti-Abeta-specific autoimmune Th1 cells. Thus, successful Abeta vaccination requires the development of strong antibody responses without Th1-type cellular immunity. In this study, we compared the induction of humoral immune responses with Th1-type (Quil A) and Th2-type (Alum) adjuvants singly and in combination, using our novel epitope vaccine composed of self B cell epitope Abeta(1-15) and foreign T cell epitope PADRE (PADRE-Abeta(1-15)-MAP). Formulated in Quil A, this vaccine resulted in significantly higher anti-Abeta antibody responses in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, compared with Alum. Anti-Abeta antibodies induced by Alum were predominantly IgG1 type accompanied by lower levels of IgG2a and IgG2b. Quil A induced robust and almost equal titers of anti-Abeta antibodies of IgG1 and IgG2a isotypes and slightly lower levels of IgG2b. Switching adjuvants from Alum to Quil A induced higher concentrations of antibodies than injections with Alum only, however slightly lower than Quil A only. Switching both adjuvants did not change the profile of antibody responses generated by the initial adjuvant injected. These results suggest that switching from Alum to Quil A would be beneficial for AD patients because anti-Abeta antibody production was enhanced without changing the initially generated and likely beneficial Th2-type humoral response.

Immunization with Fibrillar Abeta(1-42) in Young and Aged Canines: Antibody Generation and Characteristics, and Effects on CSF and Brain Abeta

Vaccine. Apr, 2006  |  Pubmed ID: 16460841

We describe a study testing fibrillar beta-amyloid(1-42) (Abeta42) vaccination in dogs. Three young beagles (4.6 years) were immunized twice with Abeta42 and a Th1 adjuvant (TiterMax Gold). Animals generated primarily IgG2 and IgM antibody responses, which were specific for the Abeta(11-30) region of Abeta(1-42). Next, 3 aged beagles (8.9-13.8 years) were immunized 4 times with Abeta(42) and a Th2 adjuvant (Alum). We observed an acute increase in IgG2, a slower increase in IgG1 and Abeta antibodies of broader specificity (Abeta(1-15>) Abeta(11-30>) Abeta(6-20)). A nonsignificant increase in CSF Abeta(1-40) and decrease in Abeta(1-40/1-42) in cortex was detected. Canines may be a useful system for testing an Abeta vaccine.

Antitumor Efficacy of DNA Vaccination to the Epigenetically Acting Tumor Promoting Transcription Factor BORIS and CD80 Molecular Adjuvant

Journal of Cellular Biochemistry. Aug, 2006  |  Pubmed ID: 16741971

Cancer testis (CT) antigens are promising candidates for tumor vaccines due to their immunogenicity and tissue-restricted expression. Recently, we identified a novel cancer testis gene, BORIS, whose expression is restricted to male testis after puberty and is strictly absent in non-malignant female tissue. BORIS encodes a DNA-binding protein that shares 11 zing finger (ZF) with transcription factor CTCF and differs at the N- and C-termini. CTCF has been implicated in epigenetic regulation of imprinting, X chromosome inactivation, repression, and activation of cancer testis antigens. BORIS expression has been documented in cancers of diverse histological origin, including, but not limited to breast, prostate, ovary, gastric, liver, endometrial, glia, colon, and esophagus. Interestingly, BORIS induces demethylation and subsequent expression of many cancer-testis genes, including MAGE-A1 and NY-ESO-1, indicating that it is expressed very early in malignancy and might be an attractive candidate for immunotherapy. In this study we tested BORIS as a vaccine in a very aggressive, highly metastatic, and poorly immunogenic murine model of mammary carcinoma. Immunizations with a DNA encoding the mutant form of murine BORIS antigen (pmBORIS lacking DNA-binding function) significantly prolonged survival, and inhibited tumor growth in BALB/c mice inoculated with 4T1 cells. Priming with pmBORIS mixed with molecular adjuvant and boosting with adenoviral vector expressing mBORIS was generally more effective, suggesting that the vaccination protocol could be further optimized. This is the first report demonstrating the feasibility of vaccination with a cancer associated epigenetic regulator for the induction of tumor inhibition.

Novel Approaches for Immunotherapeutic Intervention in Alzheimer's Disease

Neurochemistry International. Jul, 2006  |  Pubmed ID: 16765487

Immunotherapy can attenuate amyloid neuropathology and improve cognitive function in transgenic models of Alzheimer's disease. However, the first clinical trial was halted when 6% of the Alzheimer's patients developed aseptic meningoencephalitis. Postmortem analysis of two cases with meningoencephalitis showed robust glial activation, T-cell infiltration and sporadic clearance of Abeta. Interestingly, transgenic mouse models of Alzheimer's disease failed as predictors of these adverse inflammatory events. However there are now several studies with amyloid precursor protein transgenic mice that have reported an increased risk of microhemorrhages at sites of cerebrovascular amyloid deposits and because approximately 80% of Alzheimer's patient's have cerebrovascular pathology, there is concern regarding clinical trials using passive administration of humanized anti-Abeta antibodies. Although many studies have now been published on immunotherapy in mouse models, the mechanism(s) of antibody-mediated clearance of beta-amyloid from the brain, and the cause of the antibody-induced microhemorrhages remain unclear. In this review, we will discuss the most recent results from the first clinical trial, offer speculation on possible causes for the failure of the trial, review data on antibody-mediated clearance mechanisms, explore the role of complement and inflammation in the clearance of beta-amyloid, and suggest novel strategies for avoiding problems in future clinical trials. The central hypothesis being proposed in this review is that anti-Abeta antibodies delivered directly to the CNS at the sites of amyloid deposits will be far more effective at clearing Abeta and safer than active or passive immunization strategies where the majority of the antibodies are in the periphery.

The Specification of a Highly Derived Arthropod Appendage, the Drosophila Labial Palps, Requires the Joint Action of Selectors and Signaling Pathways

Development Genes and Evolution. Jul-Aug, 2006  |  Pubmed ID: 16773339

The remarkable diversity of form in arthropods reflects flexible genetic programs deploying many conserved genes. In the insect model Drosophila melanogaster, diversity of form can be observed between serially homologous appendages or when a single appendage is transformed by homeotic mutations, such as the adult labial mouthparts that can present alternative antennal, prothoracic, or maxillary identities. We have examined the roles of the Hox selector genes proboscipedia (pb) and Sex combs reduced (Scr), and the antennal selectors homothorax (hth) and spineless (ss) in labial specification, by tissue-directed mitotic recombination. Whereas loss of pb function transforms labium to prothoracic leg, loss of Scr, hth, or ss functions results in little or no change in labial specification. Results of analysis of single and multiple mutant combinations support a genetic hierarchy in which the homeotic pb gene possesses a primary role. It is surprising to note that while loss of ss activity alone had no detected effect, all mutant combinations lacking both pb and ss yielded the most severe phenotype observed: stunted, apparently tripartite legs that may correspond to a default state. The roles of the four selector genes are functionally linked to a cell nonautonomous mechanism involving the coupled activities of the decapentaplegic (dpp)/TGF-beta and wingless (wg)/Wnt signaling pathways. Accordingly, several mutant combinations impaired in dpp signaling were seen to reorient labial-to-leg transformations toward antennal aristae. A crucial aspect of selector function in development and evolution may be in regulating diffusible signals, including those emitted by dpp and wg.

Relapsing Polychondritis with Features of Dementia with Lewy Bodies

Acta Neuropathologica. Aug, 2006  |  Pubmed ID: 16832629

We describe a 72-year old man with clinical features suggestive of dementia with Lewy bodies (DLB) who proved neuropathologically to have degeneration induced by relapsing polychondritis (RP), an autoimmune inflammatory disorder of cartilaginous tissues. There was lymphocytic infiltration of the leptomeninges, perivascular cuffing, reactive astrocytosis, and activation of microglia in multiple brain areas all consistent with an immunologically mediated process. There was widespread neuronal loss within the hippocampus, entorhinal cortex, and amygdala as well as diffuse myelin pallor of cortical pathways. Elevated levels of complement proteins and endothelial markers of inflammation were observed, which are similar to previous reports in DLB. This study demonstrates that qualitatively similar inflammation-associated neurodegeneration is present in widespread regions of the brain in a RP case presenting clinically as DLB.

Reduction of Soluble Abeta and Tau, but Not Soluble Abeta Alone, Ameliorates Cognitive Decline in Transgenic Mice with Plaques and Tangles

The Journal of Biological Chemistry. Dec, 2006  |  Pubmed ID: 17056594

Increasing evidence points to soluble assemblies of aggregating proteins as a major mediator of neuronal and synaptic dysfunction. In Alzheimer disease (AD), soluble amyloid-beta (Abeta) appears to be a key factor in inducing synaptic and cognitive abnormalities. Here we report the novel finding that soluble tau also plays a role in the cognitive decline in the presence of concomitant Abeta pathology. We describe improved cognitive function following a reduction in both soluble Abeta and tau levels after active or passive immunization in advanced aged 3xTg-AD mice that contain both amyloid plaques and neurofibrillary tangles (NFTs). Notably, reducing soluble Abeta alone did not improve the cognitive phenotype in mice with plaques and NFTs. Our results show that Abeta immunotherapy reduces soluble tau and ameliorates behavioral deficit in old transgenic mice.

Abeta-immunotherapy for Alzheimer's Disease Using Mannan-amyloid-Beta Peptide Immunoconjugates

DNA and Cell Biology. Oct, 2006  |  Pubmed ID: 17132088

In Alzheimer's disease (AD) the accumulation of pathological forms of the beta-amyloid (Abeta) peptide are believed to be causal factors in the neurodegeneration that results in the loss of cognitive function in patients. Anti-Abeta antibodies have been shown to reduce Abeta levels in transgenic mouse models of AD and in AN-1792 clinical trial on AD patients; however, the clinical trial was halted when some patients developed meningoencephalitis. Theories on the cause of the adverse events include proinflammatory "primed patients," a Th1-inducing adjuvant, and Abeta autoreactive T cells. New immunotherapy approaches are being developed to eliminate these putative risk factors. Mannan, which is recognized by pattern recognition receptors of the innate immune system, can be utilized as a molecular adjuvant to promote a Th2-mediated immune response to conjugated B cell epitopes. The N-terminus of Abeta was conjugated to mannan, and used to immunize mice with low concentrations of immunoconjugate, without a conventional adjuvant. Mannan induced a significant and highly polarized toward Th2 phenotype anti-Abeta antibody response not only in BALB/c, but also in B6SJL F1 mice. New preclinical trials in AD mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse immune response that occurred in the first clinical trial.

Elicitation of T Cell Responses to Histologically Unrelated Tumors by Immunization with the Novel Cancer-testis Antigen, Brother of the Regulator of Imprinted Sites

Journal of Immunology (Baltimore, Md. : 1950). Jan, 2007  |  Pubmed ID: 17182597

Brother of the regulator of imprinted sites (BORIS) was previously described as a transcription factor for epigenetic reprogramming the expression of which is strictly confined to germ cells of adult testes but is aberrantly activated in the vast majority of neoplastic cells. Considering the critical role of BORIS in cancerogenesis and the fact that its expression pattern may preclude thymic tolerance, we generated DNA- and protein-based mouse BORIS antitumor vaccines using a non-DNA-binding version of the BORIS molecule. Clinical use of BORIS as a vaccine Ag would require that certain safety concerns be met. Specifically, administration of the functional BORIS protein would hypothetically pose a risk of BORIS accelerating the progression of cancer. To alleviate such safety concerns, we have developed vaccines based on the BORIS molecule lacking the DNA-binding zinc fingers domain. To enhance anti-BORIS cellular immune responses, we used a standard molecular adjuvant approach. It consisted of plasmids encoding murine IL-12 and IL-18 for a DNA-based vaccine and conventional Th1 type adjuvant, Quil A, for a protein-based vaccine. Both DNA- and protein-based vaccines induced Ag-specific CD4(+) T cell proliferation with Th1 and Th2 cytokine profiles, respectively. Protein-based, but not DNA-based, BORIS vaccine induced a significant level of Ab production in immunized animals. Importantly, potent anticancer CD8(+)-cytotoxic lymphocytes were generated after immunization with the DNA-based, but not protein-based, BORIS vaccine. These cytolytic responses were observed across a wide range of different mouse cancers including mammary adenocarcinoma, glioma, leukemia, and mastocytoma.

Distinct Roles for Mediator Cdk8 Module Subunits in Drosophila Development

The EMBO Journal. Feb, 2007  |  Pubmed ID: 17290221

Mediator (MED) is a conserved multisubunit complex bridging transcriptional activators and repressors to the general RNA polymerase II initiation machinery. In yeast, MED is organized in three core modules and a separable 'Cdk8 module' consisting of the cyclin-dependent kinase Cdk8, its partner CycC, Med12 and Med13. This regulatory module, specifically required for cellular adaptation to environmental cues, is thought to act through the Cdk8 kinase activity. Here we have investigated the functions of the four Cdk8 module subunits in the metazoan model Drosophila. Physical interactions detected among the four fly subunits provide support for a structurally conserved Cdk8 module. We analyzed the in vivo functions of this module using null mutants for Cdk8, CycC, Med12 and Med13. Each gene is required for the viability of the organism but not of the cell. Cdk8-CycC and Med12-Med13 act as pairs, which share some functions but also have distinct roles in developmental gene regulation. These data reveal functional attributes of the Cdk8 module, apart from its regulated kinase activity, that may contribute to the diversification of genetic programs.

Anti-A Beta 1-11 Antibody Binds to Different Beta-amyloid Species, Inhibits Fibril Formation, and Disaggregates Preformed Fibrils but Not the Most Toxic Oligomers

The Journal of Biological Chemistry. Aug, 2007  |  Pubmed ID: 17545160

Different strategies proposed as therapy for Alzheimer disease (AD) have aimed to reduce the level of toxic forms of A beta peptide in the brain. Here, we directly analyze the therapeutic utility of the polyclonal anti-A beta(1-11) antibody induced in 3xTg-AD mice vaccinated with the second generation prototype epitope vaccine. Substoichiometric concentrations of purified anti-A beta(1-11) antibody prevented aggregation of A beta(42) and induced disaggregation of preformed A beta(42) fibrils down to nonfilamentous and nontoxic species. Anti-A beta(1-11) antibody delayed A beta(42) oligomer formation but ultimately appeared to stabilize nonfibrillar conformations, including oligomer-like assemblies. The reduced oligomer-mediated cytotoxicity observed upon preincubation of A beta oligomers with the anti-A beta(1-11) antibody in the absence of oligomer disaggregation suggests a possible oligomer rearrangement in the presence of the antibody. These in vitro observations suggest that preventive vaccination may protect from AD or may delay the onset of the disease, whereas therapeutic vaccination cannot disrupt the toxic oligomers and may only minimally alleviate preexisting AD pathology.

Phagocytosis of Amyloid-beta and Inflammation: Two Faces of Innate Immunity in Alzheimer's Disease

Journal of Alzheimer's Disease : JAD. Jul, 2007  |  Pubmed ID: 17656824

Innate immunity provides the first line of defense by recognizing pathogen-associated microbial patterns and inducing key co-stimulatory molecules and cytokines, which activate the mechanisms of the adaptive immune response. Innate immune cells perform phagocytosis, which can clear pathogens and tissue waste products but may also contribute to tissue injury due to harmful side effects of inflammation. Genetic studies of APOE4, cytokine polymorphisms and overexpression of inflammatory genes suggest that chronic inflammation may have adverse effects in patients with sporadic Alzheimer's disease. However, a vaccine against amyloid-beta induced beneficial clearance of amyloid-beta deposits, possibly through Fc receptor-mediated stimulation of microglial and macrophage uptake. A reconciliation of these two pathogenetic mechanisms is crucial to future progress in immune diagnosis and effective therapy. This may be possible by extending our recent observations suggesting that phagocytosis of amyloid-beta by macrophages is excellent in normal subjects but is deficient in most AD patients. Thus increased proinflammatory cytokine levels and activated microglia and macrophages in patients may be compensatory for defective clearance of amyloid-beta. Consequently, therapeutic interventions that increase phagocytosis of amyloid-beta might decrease brain inflammation as well as reduce inflammation-induced neurodegeneration. Finally, peripheral blood leukocytes are a superior model system for investigation of innate immune dysfunction in Alzheimer's disease.

External Force-assisted Cell Positioning Inside Microfluidic Devices

Biomedical Microdevices. Feb, 2007  |  Pubmed ID: 17091393

This paper describes straightforward approaches to positioning cells within microfluidic devices that can be implemented without special equipment or fabrication steps. External forces can effectively transport and position cells in preferred locations inside microfluidic channels. Except for centrifugal force-based positioning that can be used with any microfluidic channels, hydrodynamic and gravitational force-based positioning yield reproducible and biocompatible results when implemented with a microfluidic "module" that contains a barrier with embedded microgrooves. Primary rat cortical neurons, metastatic human breast cancer cells MDA-MB-231, NIH 3T3 mouse fibroblasts, and human umbilical vein endothelial cells (HUVECs) were compatible with the positioning processes. After positioning, cells attached, proliferated and migrated like control cells that were cultured on tissue culture dishes or glass coverslips. No apparent morphological differences were observed in positioned cells compared with control cells. Finally, to demonstrate a practical application of the methods, cells were placed in a single row along a wall inside a microfluidic chemotaxis chamber (MCC), and were exposed to stable concentration gradient of chemoattractant. Cell positioning allows that all cells get exposed to the same level of chemoattractant at the start of the experiment helping standardize cellular response.

Alzheimer's Disease Peptide Epitope Vaccine Reduces Insoluble but Not Soluble/oligomeric Abeta Species in Amyloid Precursor Protein Transgenic Mice

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2007  |  Pubmed ID: 18003852

Active vaccination of elderly Alzheimer's disease (AD) patients with fibrillar amyloid-beta peptide (Abeta42), even in the presence of a potent Th1 adjuvant, induced generally low titers of antibodies in a small fraction (approximately 20% responders) of those that received the AN-1792 vaccine. To improve the immunogenicity and reduce the likelihood of inducing adverse autoreactive T-cells specific for Abeta42, we previously tested in wild-type mice an alternative approach for active immunization: an epitope vaccine that selectively initiate B cell responses toward an immunogenic self-epitope of Abeta in the absence of anti-Abeta T cell responses. Here, we describe a second generation epitope vaccine composed of two copies of Abeta(1-11) fused with the promiscuous nonself T cell epitope, PADRE (pan human leukocyte antigen DR-binding peptide) that completely eliminates the autoreactive T cell responses and induces humoral immune responses in amyloid precursor protein transgenic 2576 mice with pre-existing AD-like pathology. Based on the titers of anti-Abeta(1-11) antibody experimental mice were divided into low, moderate and high responders, and for the first time we report a positive correlation between the concentration of anti-Abeta(1-11) antibody and a reduction of insoluble, cerebral Abeta plaques. The reduction of insoluble Abeta deposition was not associated with adverse events, such as CNS T cell or macrophage infiltration or microhemorrhages. Surprisingly, vaccination did not alter the levels of soluble Abeta. Alternatively, early protective immunization before substantial neuropathology, neuronal loss and cognitive deficits have become firmly established may be more beneficial and safer for potential patients, especially if they can be identified in a preclinical stage by the development of antecedent biomarkers of AD.

Experimental Investigation of Antibody-mediated Clearance Mechanisms of Amyloid-beta in CNS of Tg-SwDI Transgenic Mice

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Dec, 2007  |  Pubmed ID: 18057195

Novel amyloid precursor protein transgenic mice, which contain the Swedish as well as the vasculotropic Dutch and Iowa mutations (Tg-SwDI), were used to investigate the mechanisms of antibody-mediated clearance of amyloid-beta (Abeta) from the brain. Export of the Abeta-DI peptide across the blood-brain barrier is severely reduced because of the vasculotropic mutations. Therefore, antibody-mediated clearance of Abeta-DI is dependent on antibodies entering the brain. In this report, we immunized Tg-SwDI mice with various peptide antigens, including Abeta40-DI, Abeta42, and an Abeta epitope vaccine. Immunization of Tg-SwDI mice with substantial cortical diffuse and vascular fibrillar deposits failed to promote clearance of parenchymal or vascular amyloid deposits. We then immunized young Tg-SwDI mice before the accumulation of Abeta and saw no evidence that anti-Abeta antibodies could diminish deposition of parenchymal or vascular amyloid deposits. However, injection of anti-Abeta antibodies, affinity-purified from immunized Tg-SwDI mice, into the hippocampus induced a rapid clearance of diffuse Abeta deposits but not vascular amyloid deposits. These results further support the "peripheral sink hypothesis" as a legitimate mechanism of antibody-mediated clearance of Abeta when the blood-brain barrier remains intact. Thus, approaches that deliver immunotherapy to the brain may be more effective at clearing Abeta than immunization strategies in which the majority of the antibodies are in the periphery.

Amyloid-beta Peptide Binds to Microtubule-associated Protein 1B (MAP1B)

Neurochemistry International. May, 2008  |  Pubmed ID: 18079022

Extracellular and intraneuronal formation of amyloid-beta aggregates have been demonstrated to be involved in the pathogenesis of Alzheimer's disease. However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of targets have deleterious effects on cellular functions. In the present study we have shown for the first time that amyloid-beta 1-42 bound to a peptide comprising the microtubule binding domain of the heavy chain of microtubule-associated protein 1B by the screening of a human brain cDNA library expressed on M13 phage. This interaction may explain, in part, the loss of neuronal cytoskeletal integrity, impairment of microtubule-dependent transport and synaptic dysfunction observed previously in Alzheimer's disease.

A Two-year Study with Fibrillar Beta-amyloid (Abeta) Immunization in Aged Canines: Effects on Cognitive Function and Brain Abeta

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Apr, 2008  |  Pubmed ID: 18385314

Aged canines (dogs) accumulate human-type beta-amyloid (Abeta) in diffuse plaques in the brain with parallel declines in cognitive function. We hypothesized that reducing Abeta in a therapeutic treatment study of aged dogs with preexisting Abeta pathology and cognitive deficits would lead to cognitive improvements. To test this hypothesis, we immunized aged beagles (8.4-12.4 years) with fibrillar Abeta(1-42) formulated with aluminum salt (Alum) for 2.4 years (25 vaccinations). Cognitive testing during this time revealed no improvement in measures of learning, spatial attention, or spatial memory. After extended treatment (22 vaccinations), we observed maintenance of prefrontal-dependent reversal learning ability. In the brain, levels of soluble and insoluble Abeta(1-40) and Abeta(1-42) and the extent of diffuse plaque accumulation was significantly decreased in several cortical regions, with preferential reductions in the prefrontal cortex, which is associated with a maintenance of cognition. However, the amount of soluble oligomers remained unchanged. The extent of prefrontal Abeta was correlated with frontal function and serum anti-Abeta antibody titers. Thus, reducing total Abeta may be of limited therapeutic benefit to recovery of cognitive decline in a higher mammalian model of human brain aging and disease. Immunizing animals before extensive Abeta deposition and cognitive decline to prevent oligomeric or fibrillar Abeta formation may have a greater impact on cognition and also more directly evaluate the role of Abeta on cognition in canines. Alternatively, clearing preexisting Abeta from the brain in a treatment study may be more efficacious for cognition if combined with a second intervention that restores neuron health.

Exercise Alters the Immune Profile in Tg2576 Alzheimer Mice Toward a Response Coincident with Improved Cognitive Performance and Decreased Amyloid

Journal of Neuroinflammation. 2008  |  Pubmed ID: 18400101

Inflammation is associated with Abeta pathology in Alzheimer's disease (AD) and transgenic AD models. Previously, it has been demonstrated that chronic stimulation of the immune response induces pro-inflammatory cytokines IL-1beta and TNF-alpha which contribute to neurodegeneration. However, recent evidence has shown that inducing the adaptive immune response reduces Abeta pathology and is neuroprotective. Low concentrations of IFN-gamma modulate the adaptive immune response by directing microglia to differentiate to antigen presenting cells. Our objective was to determine if exercise could induce a shift from the immune profile in aged (17-19 months) Tg2576 mice to a response that reduces Abeta pathology.

Reducing AD-like Pathology in 3xTg-AD Mouse Model by DNA Epitope Vaccine - a Novel Immunotherapeutic Strategy

PloS One. 2008  |  Pubmed ID: 18461171

The development of a safe and effective AD vaccine requires a delicate balance between providing an adequate anti-Abeta antibody response sufficient to provide therapeutic benefit, while eliminating an adverse T cell-mediated proinflammatory autoimmune response. To achieve this goal we have designed a prototype chemokine-based DNA epitope vaccine expressing a fusion protein that consists of 3 copies of the self-B cell epitope of Abeta(42) (Abeta(1-11)) , a non-self T helper cell epitope (PADRE), and macrophage-derived chemokine (MDC/CCL22) as a molecular adjuvant to promote a strong anti-inflammatory Th2 phenotype.

Timing of Wingless Signalling Distinguishes Maxillary and Antennal Identities in Drosophila Melanogaster

Development (Cambridge, England). Jul, 2008  |  Pubmed ID: 18508864

The Drosophila adult head mostly derives from the composite eye-antenna imaginal disc. The antennal disc gives rise to two adult olfactory organs: the antennae and maxillary palps. Here, we have analysed the regional specification of the maxillary palp within the antennal disc. We found that a maxillary field, defined by expression of the Hox gene Deformed, is established at about the same time as the eye and antennal fields during the L2 larval stage. The genetic program leading to maxillary regionalisation and identity is very similar to the antennal one, but is distinguished primarily by delayed prepupal expression of the ventral morphogen Wingless (Wg). We find that precociously expressing Wg in the larval maxillary field suffices to transform it towards antennal identity, whereas overexpressing Wg later in prepupae does not. These results thus indicate that temporal regulation of Wg is decisive to distinguishing maxillary and antennal organs. Wg normally acts upstream of the antennal selector spineless (ss) in maxillary development. However, mis-expression of Ss can prematurely activate wg via a positive-feedback loop leading to a maxillary-to-antenna transformation. We characterised: (1) the action of Wg through ss selector function in distinguishing maxillary from antenna; and (2) its direct contribution to identity choice.

Cross-regulatory Protein-protein Interactions Between Hox and Pax Transcription Factors

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2008  |  Pubmed ID: 18755899

Homeotic Hox selector genes encode highly conserved transcriptional regulators involved in the differentiation of multicellular organisms. Ectopic expression of the Antennapedia (ANTP) homeodomain protein in Drosophila imaginal discs induces distinct phenotypes, including an antenna-to-leg transformation and eye reduction. We have proposed that the eye loss phenotype is a consequence of a negative posttranslational control mechanism because of direct protein-protein interactions between ANTP and Eyeless (EY). In the present work, we analyzed the effect of various ANTP homeodomain mutations for their interaction with EY and for head development. Contrasting with the eye loss phenotype, we provide evidence that the antenna-to-leg transformation involves ANTP DNA-binding activity. In a complementary genetic screen performed in yeast, we isolated mutations located in the N terminus of the ANTP homeodomain that inhibit direct interactions with EY without abolishing DNA binding in vitro and in vivo. In a bimolecular fluorescence complementation assay, we detected the ANTP-EY interaction in vivo, these interactions occurring through the paired domain and/or the homeodomain of EY. These results demonstrate that the homeodomain supports multiple molecular regulatory functions in addition to protein-DNA and protein-RNA interactions; it is also involved in protein-protein interactions.

Mannan-Abeta28 Conjugate Prevents Abeta-plaque Deposition, but Increases Microhemorrhages in the Brains of Vaccinated Tg2576 (APPsw) Mice

Journal of Neuroinflammation. 2008  |  Pubmed ID: 18823564

New pre-clinical trials in AD mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse responses that occurred during the clinical trials with AN-1792 vaccine formulation. Recently, we have pursued an alternative immunization strategy that replaces QS21 the Th1 type adjuvant used in the AN-1792 clinical trial with a molecular adjuvant, mannan that can promote a Th2-polarized immune response through interactions with mannose-binding and CD35/CD21 receptors of the innate immune system. Previously we established that immunization of wild-type mice with mannan-Abeta28 conjugate promoted Th2-mediated humoral and cellular immune responses. In the current study, we tested the efficacy of this vaccine configuration in amyloid precursor protein (APP) transgenic mice (Tg2576).

Gene Expression Changes in the Course of Normal Brain Aging Are Sexually Dimorphic

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2008  |  Pubmed ID: 18832152

Gene expression profiles were assessed in the hippocampus, entorhinal cortex, superior-frontal gyrus, and postcentral gyrus across the lifespan of 55 cognitively intact individuals aged 20-99 years. Perspectives on global gene changes that are associated with brain aging emerged, revealing two overarching concepts. First, different regions of the forebrain exhibited substantially different gene profile changes with age. For example, comparing equally powered groups, 5,029 probe sets were significantly altered with age in the superior-frontal gyrus, compared with 1,110 in the entorhinal cortex. Prominent change occurred in the sixth to seventh decades across cortical regions, suggesting that this period is a critical transition point in brain aging, particularly in males. Second, clear gender differences in brain aging were evident, suggesting that the brain undergoes sexually dimorphic changes in gene expression not only in development but also in later life. Globally across all brain regions, males showed more gene change than females. Further, Gene Ontology analysis revealed that different categories of genes were predominantly affected in males vs. females. Notably, the male brain was characterized by global decreased catabolic and anabolic capacity with aging, with down-regulated genes heavily enriched in energy production and protein synthesis/transport categories. Increased immune activation was a prominent feature of aging in both sexes, with proportionally greater activation in the female brain. These data open opportunities to explore age-dependent changes in gene expression that set the balance between neurodegeneration and compensatory mechanisms in the brain and suggest that this balance is set differently in males and females, an intriguing idea.

DNA Epitope Vaccine Containing Complement Component C3d Enhances Anti-amyloid-beta Antibody Production and Polarizes the Immune Response Towards a Th2 Phenotype

Journal of Neuroimmunology. Dec, 2008  |  Pubmed ID: 18838175

We have engineered a DNA epitope vaccine that expresses 3 self-B cell epitopes of Abeta(42) (3Abeta(1-11)), a non-self T helper (Th) cell epitope (PADRE), and 3 copies of C3d (3C3d), a component of complement as a molecular adjuvant, designed to safely reduce CNS Abeta. Immunization of mice with 3Abeta(1-11)-PADRE epitope vaccine alone generated only moderate levels of anti-Abeta antibodies and a pro-inflammatory T helper (Th1 phenotype) cellular immune response. However, the addition of 3C3d to the vaccine construct significantly augmented the anti-Abeta humoral immune response and, importantly, shifted the cellular immune response towards the potentially safer anti-inflammatory Th2 phenotype.

Blocking Abeta42 Accumulation Delays the Onset and Progression of Tau Pathology Via the C Terminus of Heat Shock Protein70-interacting Protein: a Mechanistic Link Between Abeta and Tau Pathology

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2008  |  Pubmed ID: 19020010

The molecular alterations that induce tau pathology in Alzheimer disease (AD) are not known, particularly whether this is an amyloid-beta (Abeta)-dependent or -independent event. We addressed this issue in the 3xTg-AD mice using both genetic and immunological approaches and show that a selective decrease in Abeta(42) markedly delays the progression of tau pathology. The mechanism underlying this effect involves alterations in the levels of C terminus of heat shock protein70-interacting protein (CHIP) as we show that Abeta accumulation decreases CHIP expression and increases tau levels. We show that the Abeta-induced effects on tau were rescued by restoring CHIP levels. Our findings have profound clinical implications as they indicate that preventing Abeta accumulation will significantly alter AD progression. These data highlight the critical role CHIP plays as a link between Abeta and tau and identify CHIP as a new potential target not only for AD but for other neurodegenerative disorders characterized by tau accumulation.

Immunostimulant Adjuvant Patch Enhances Humoral and Cellular Immune Responses to DNA Immunization

DNA and Cell Biology. Jan, 2008  |  Pubmed ID: 17961074

The focus of this report is on the development of an improved DNA immunization protocol, which takes advantage of the strengths of DNA immunization, as well as those associated with adjuvant delivered by transcutaneous immunostimulatory (IS) patches. Because transcutaneous delivery of adjuvants to the skin at the vaccination site has been shown to amplify the immune response to protein antigens, we hypothesized that the same IS patch when placed on the skin at the site of DNA injection could further enhance the immune response to a DNA influenza vaccine. We have combined an influenza DNA vaccine, hemagglutinin fused with three copies of complement C3d, to enhance uptake and antigen presentation, with an IS patch containing heat-labile enterotoxin from Escherichia coli. Coadministration of a potent adjuvant in IS patches placed on the skin at the site of DNA vaccination dramatically amplifies anti-influenza antibody immune response. Supplementing DNA vaccines with IS patches may be a particularly valuable strategy because DNA vaccines can be rapidly modified in response to mutations in pathogens, and individuals with compromised immune systems such as transplant patients and the elderly will benefit from the enhanced antibody response induced by the IS patches.

Active and Passive Abeta-immunotherapy: Preclinical and Clinical Studies and Future Directions: Part I

CNS & Neurological Disorders Drug Targets. Mar, 2009  |  Pubmed ID: 19275632

Active and Passive Abeta-immunotherapy: Preclinical and Clinical Studies and Future Directions: Part II

CNS & Neurological Disorders Drug Targets. Apr, 2009  |  Pubmed ID: 19355929

A Fibril-specific, Conformation-dependent Antibody Recognizes a Subset of Abeta Plaques in Alzheimer Disease, Down Syndrome and Tg2576 Transgenic Mouse Brain

Acta Neuropathologica. Oct, 2009  |  Pubmed ID: 19360426

Beta-amyloid (Abeta) is thought to be a key contributor to the pathogenesis of Alzheimer disease (AD) in the general population and in adults with Down syndrome (DS). Different assembly states of Abeta have been identified that may be neurotoxic. Abeta oligomers can assemble into soluble prefibrillar oligomers, soluble fibrillar oligomers and insoluble fibrils. Using a novel antibody, OC, recognizing fibrils and soluble fibrillar oligomers, we characterized fibrillar Abeta deposits in AD and DS cases. We further compared human specimens to those obtained from the Tg2576 mouse model of AD. Our results show that accumulation of fibrillar immunoreactivity is significantly increased in AD relative to nondemented aged subjects and those with select cognitive impairments (p < 0.0001). Further, there was a significant correlation between the extent of frontal cortex fibrillar deposit accumulation and dementia severity (MMSE r = -0.72). In DS, we observe an early age of onset and age-dependent accumulation of fibrillar OC immunoreactivity with little pathology in similarly aged non-DS individuals. Tg2576 mice show fibrillar accumulation that can be detected as young as 6 months. Interestingly, fibril-specific immunoreactivity was observed in diffuse, thioflavine S-negative Abeta deposits in addition to more mature neuritic plaques. These results suggest that fibrillar deposits are associated with disease in both AD and in adults with DS and their distribution within early Abeta pathology associated with diffuse plaques and correlation with MMSE suggest that these deposits may not be as benign as previously thought.

Immunization with Amyloid-beta Attenuates Inclusion Body Myositis-like Myopathology and Motor Impairment in a Transgenic Mouse Model

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. May, 2009  |  Pubmed ID: 19439591

Inclusion body myositis (IBM), the most common muscle disease to afflict the elderly, causes slow but progressive degeneration of skeletal muscle and ultimately paralysis. Hallmark pathological features include T-cell mediated inflammatory infiltrates and aberrant accumulations of proteins, including amyloid-beta (Abeta), tau, ubiquitinated-proteins, apolipoprotein E, and alpha-synuclein in skeletal muscle. A large body of work indicates that aberrant Abeta accumulation contributes to the myodegeneration. Here, we investigated whether active immunization to promote clearance of Abeta from affected skeletal muscle fibers mitigates the IBM-like myopathological features as well as motor impairment in a transgenic mouse model. We report that active immunization markedly reduces intracellular Abeta deposits and attenuates the motor impairment compared with untreated mice. Results from our current study indicate that Abeta oligomers contribute to the myopathy process as they were significantly reduced in the affected skeletal muscle from immunized mice. In addition, the anti-Abeta antibodies produced in the immunized mice blocked the toxicity of the Abeta oligomers in vitro, providing a possible key mechanism for the functional recovery. These findings provide support for the hypothesis that Abeta is one of the key pathogenic components in IBM pathology and subsequent skeletal muscle degeneration.

Abeta DNA Vaccination for Alzheimer's Disease: Focus on Disease Prevention

CNS & Neurological Disorders Drug Targets. Apr, 2010  |  Pubmed ID: 20205639

Pre-clinical and clinical data suggest that the development of a safe and effective anti-amyloid-beta (Abeta) immunotherapy for Alzheimer's disease (AD) will require therapeutic levels of anti-Abeta antibodies, while avoiding proinflammatory adjuvants and autoreactive T cells which may increase the incidence of adverse events in the elderly population targeted to receive immunotherapy. The first active immunization clinical trial with AN1792 in AD patients was halted when a subset of patients developed meningoencephalitis. The first passive immunotherapy trial with bapineuzumab, a humanized monoclonal antibody against the end terminus of Abeta, also encountered some dose dependent adverse events during the Phase II portion of the study, vasogenic edema in 12 cases, which were significantly over represented in ApoE4 carriers. The proposed remedy is to treat future patients with lower doses, particularly in the ApoE4 carriers. Currently there are at least five ongoing anti-Abeta immunotherapy clinical trials. Three of the clinical trials use humanized monoclonal antibodies, which are expensive and require repeated dosing to maintain therapeutic levels of the antibodies in the patient. However in the event of an adverse response to the passive therapy antibody delivery can simply be halted, which may provide a resolution to the problem. Because at this point we cannot readily identify individuals in the preclinical or prodromal stages of AD pathogenesis, passive immunotherapy is reserved for those that already have clinical symptoms. Unfortunately those individuals have by that point accumulated substantial neuropathology in affected regions of the brain. Moreover, if Abeta pathology drives tau pathology as reported in several transgenic animal models, and once established if tau pathology can become self propagating, then early intervention with anti-Abeta immunotherapy may be critical for favorable clinical outcomes. On the other hand, active immunization has several significant advantages, including lower cost and the typical immunization protocol should be much less intrusive to the patient relative to passive therapy, in the advent of Abeta-antibody immune complex-induced adverse events the patients will have to receive immuno-supperssive therapy for an extended period until the anti Abeta antibody levels drop naturally as the effects of the vaccine decays over time. Obviously, improvements in vaccine design are needed to improve both the safety, as well as the efficacy of anti-Abeta immunotherapy. The focus of this review is on the advantages of DNA vaccination for anti-Abeta immunotherapy, and the major hurdles, such as immunosenescence, selection of appropriate molecular adjuvants, universal T cell epitopes, and possibly a polyepitope design based on utilizing existing memory T cells in the general population that were generated in response to childhood or seasonal vaccines, as well as various infections. Ultimately, we believe that the further refinement of our AD DNA epitope vaccines, possibly combined with a prime boost regime will facilitate translation to human clinical trials in either very early AD, or preferably in preclinical stage individuals identified by validated AD biomarkers.

Linear and Conformation Specific Antibodies in Aged Beagles After Prolonged Vaccination with Aggregated Abeta

Neurobiology of Disease. Sep, 2010  |  Pubmed ID: 20451612

Previously we showed that anti-Abeta peptide immunotherapy significantly attenuated Alzheimer's-like amyloid deposition in the central nervous system of aged canines. In this report we have characterized the changes that occurred in the humoral immune response over 2.4years in canines immunized repeatedly with aggregated Abeta(1-42) (AN1792) formulated in alum adjuvant. We observed a rapid and robust induction of anti-Abeta antibody titers, which were associated with an anti-inflammatory T helper type 2 (Th2) response. The initial antibody response was against dominant linear epitope at the N-terminus region of the Abeta(1-42) peptide, which is identical to the one in humans and vervet monkeys. After multiple immunizations the antibody response drifted toward the elevation of antibodies that recognized conformational epitopes of assembled forms of Abeta and other types of amyloid. Our findings indicate that prolonged immunization results in distinctive temporal changes in antibody profiles, which may be important for other experimental and clinical settings.

Low Concentrations of Anti-Aβ Antibodies Generated in Tg2576 Mice by DNA Epitope Vaccine Fused with 3C3d Molecular Adjuvant Do Not Affect AD Pathology

Human Gene Therapy. Nov, 2010  |  Pubmed ID: 20528468

It has been demonstrated that an active vaccination strategy with protein- or DNA-based epitope vaccines composed of the immunodominant self B cell epitope of amyloid-β₄₂ (Aβ₄₂) and a non-self T helper (Th) cell epitope is an immunotherapeutic approach to preventing or treating Alzheimer's disease (AD). As a DNA-based epitope vaccine, we used a plasmid encoding three copies of Aβ(1-11) and Th cell epitope, PADRE (p3Aβ(1-11)-PADRE). We have previously reported that three copies of component of complement C3d (3C3d) acts as a molecular adjuvant significantly enhancing immune responses in wild-type mice of the H2(b) haplotype immunized with p3Aβ(1-11)-PADRE. Here, we tested the efficacy of p3Aβ(1-11)-PADRE and the same vaccine fused with 3C3d (p3Aβ(1-11)-PADRE-3C3d) in a transgenic (Tg) mouse model of AD (Tg2576) of the H2(bxs) immune haplotype. The overall responses to both vaccines were very weak in Tg2576 mice despite the fact that the 3C3d molecular adjuvant significantly enhanced the anti-Aβ response to 3Aβ(1-11)-PADRE. Importantly, generation of low antibody responses was associated with the strain of amyloid precursor protein Tg mice rather than with a molecular adjuvant, as a p3Aβ(1-11)-PADRE-3C3d vaccine induced significantly higher antibody production in another AD mouse model, 3xTg-AD of the H2(b) haplotype. Finally, this study demonstrated that low concentrations of antibodies generated by both DNA vaccines were not sufficient for the reduction of Aβ pathology in the brains of vaccinated Tg2576 animals, confirming previous reports from preclinical studies and the AN-1792 clinical trials, which concluded that the concentration of anti-Aβ antibodies may be essential for the reduction of AD pathology.

Reductions in Amyloid-beta-derived Neuroinflammation, with Minocycline, Restore Cognition but Do Not Significantly Affect Tau Hyperphosphorylation

Journal of Alzheimer's Disease : JAD. 2010  |  Pubmed ID: 20555131

Cognitive decline in Alzheimer's disease (AD) occurs as a result of the buildup of pathological proteins and downstream events including an elevated and altered inflammatory response. Inflammation has previously been linked to increased abnormal phosphorylation of tau protein. To determine if endogenous amyloid-beta (Abeta)-induced neuroinflammation drives tau phosphorylation in vivo, we treated 8-month-old 3xTg-AD with minocycline, an anti-inflammatory agent, to assess how it influenced cognitive decline and development of pathology. 4 months of treatment restored cognition to non-transgenic performance. Inflammatory profiling revealed a marked decrease in GFAP, TNFalpha, and IL6 and an increase in the CXCL1 chemokines KC and MIP1a. Minocycline also reduced levels of insoluble Abeta and soluble fibrils. Despite reducing levels of the tau kinase cdk5 coactivator p25, minocycline did not have wide effects on tau pathology with only one phospho-epitope showing reduction with treatment (S212/S214). The sum of these findings shows that reduction of the inflammatory events in an AD mouse model prevents cognitive deficits associated with pathology, but that endogenous Abeta-derived neuroinflammation does not contribute significantly to the development of tau pathology.

Anti-11[E]-pyroglutamate-modified Amyloid β Antibodies Cross-react with Other Pathological Aβ Species: Relevance for Immunotherapy

Journal of Neuroimmunology. Dec, 2010  |  Pubmed ID: 20864186

N-truncated/modified forms of amyloid beta (Aß) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as animal models of AD, and represent highly desirable therapeutic targets. In the present study we have focused on N-truncated/modified Aβ peptide bearing amino-terminal pyroglutamate at position 11 (AβN11(pE)). We identified two B-cell epitopes recognized by rabbit anti-AβN11(pE) polyclonal antibodies. Interestingly, rabbit anti-AβN11(pE) polyclonal antibodies bound also to full-length Aβ1-42 and N-truncated/modified AβN3(pE), suggesting that the three peptides may share a common B-cell epitope. Importantly, rabbit anti-AβN11(pE) antibodies bound to naturally occurring Aβ aggregates present in brain samples from AD patients. These results are potentially important for developing novel immunogens for targeting N-truncated/modified Aβ aggregates as well, since the most commonly used immunogens in the majority of vaccine studies have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Aβ, which is absent in N-amino truncated peptides.

Aging and Cerebrovascular Dysfunction: Contribution of Hypertension, Cerebral Amyloid Angiopathy, and Immunotherapy

Annals of the New York Academy of Sciences. Oct, 2010  |  Pubmed ID: 20955427

Age-related cerebrovascular dysfunction contributes to ischemic stroke, intracerebral hemorrhages (ICHs), microbleeds, cerebral amyloid angiopathy (CAA), and cognitive decline. Importantly, there is increasing recognition that this dysfunction plays a critical secondary role in many neurodegenerative diseases, including Alzheimer's disease (AD). Atherosclerosis, hypertension, and CAA are the most common causes of blood-brain barrier (BBB) lesions. The accumulation of amyloid beta (Aβ) in the cerebrovascular system is a significant risk factor for ICH and has been linked to endothelial transport failure and blockage of perivascular drainage. Moreover, recent anti-Aβ immunotherapy clinical trials demonstrated efficient clearance of parenchymal amyloid deposits but have been plagued by CAA-associated adverse events. Although management of hypertension and atherosclerosis can reduce the incidence of ICH, there are currently no approved therapies for attenuating CAA. Thus, there is a critical need for new strategies that improve BBB function and limit the development of β-amyloidosis in the cerebral vasculature.

Restricted V Gene Usage and VH/VL Pairing of Mouse Humoral Response Against the N-terminal Immunodominant Epitope of the Amyloid β Peptide

Molecular Immunology. Nov-Dec, 2010  |  Pubmed ID: 20970857

Over the last decade, the potential of antibodies as therapeutic strategies to treat Alzheimer's disease (AD) has been growing, based on successful experimental and clinical trials in transgenic mice. Despite, undesirable side effects in humans using an active immunization approach, immunotherapy still remains one of the most promising treatments for AD. In this study, we analyzed the V genes of twelve independently isolated monoclonal antibodies raised against the N-terminal immunodominant epitope of the amyloid β peptide (Aβ or A beta). Surprisingly, we found a high and unusual level of restriction in the VH/VL pairing of these antibodies. Moreover, these antibodies mostly differ in their heavy chain complementary determining region 3 (HCDR3) and the residues in the antibodies which contact Aβ are already present in the germline V-genes. Based on these observations and or co-crystal structures of antibodies with Aβ, the aim of the current study was to better understand the role of antibody V-domains, HCDR3 regions, key contact residue (H58) and germline encoded residues in Aβ recognition. For that purpose, we designed and produced a range of recombinant Fab constructs. All the Fabs were tested and compared by surface plasmon resonance on Aβ(1-16), Aβ(1-42) high molecular weight and Aβ(1-42) low molecular weight soluble oligomers. Although all the Fabs recognized the Aβ(1-16) peptide and the Aβ(1-42) high molecular weight soluble oligomers, they did not bind the Aβ(1-42) low molecular weight soluble oligomers. Furthermore, we demonstrated that: (1) an aromatic residue at position H58 in the antibody is essential in the recognition of Aβ and (2) Fabs based on germline V-genes bind to Aβ monomers with a low affinity. These findings may have important implications in designing more effective therapeutic antibodies against Aβ.

An Amyloid β(42)-dependent Deficit in Anandamide Mobilization is Associated with Cognitive Dysfunction in Alzheimer's Disease

Neurobiology of Aging. May, 2011  |  Pubmed ID: 21546126

The endocannabinoids and their attending cannabinoid (CB)(1) receptors have been implicated in the control of cognition, but their possible roles in dementias are still unclear. In the present study, we used liquid chromatography/mass spectrometry to conduct an endocannabinoid-targeted lipidomic analysis of postmortem brain samples from 38 Alzheimer's disease (AD) patients and 17 control subjects, matched for age and postmortem interval. The analysis revealed that midfrontal and temporal cortex tissue from AD patients contains, relative to control subjects, significantly lower levels of the endocannabinoid anandamide and its precursor 1-stearoyl, 2-docosahexaenoyl-sn-glycero-phosphoethanolamine-N-arachidonoyl (NArPE). No such difference was observed with the endocannabinoid 2-arachidonoyl-sn-glycerol or 15 additional lipid species. In AD patients, but not in control subjects, statistically detectable positive correlations were found between (1) anandamide content in midfrontal cortex and scores of the Kendrick's Digit Copy test (p = 0.004, r = 0.81; n = 10), which measures speed of information processing; and (2) anandamide content in temporal cortex and scores of the Boston Naming test (p = 0.027, r = 0.52; n = 18), which assesses language facility. Furthermore, anandamide and NArPE levels in midfrontal cortex of the study subjects inversely correlated with levels of the neurotoxic amyloid peptide, amyloid β-protein (Aβ)(42), while showing no association with Aβ(40) levels, amyloid plaque load or tau protein phosphorylation. Finally, high endogenous levels of Aβ(42) in Swedish mutant form of amyloid precursor protein (APP(SWE))/Neuro-2a cells directly reduced anandamide and NArPE concentrations in cells lysates. The results suggest that an Aβ(42)-dependent impairment in brain anandamide mobilization contributes to cognitive dysfunction in AD.

Cancer-testis Antigen, BORIS Based Vaccine Delivered by Dendritic Cells is Extremely Effective Against a Very Aggressive and Highly Metastatic Mouse Mammary Carcinoma

Cellular Immunology. 2011  |  Pubmed ID: 21641588

Here, we analyze for the first time the immunological and therapeutic efficacy of a dendritic cell (DC) vaccine based on a cancer-testis antigen, Brother of regulator of imprinted sites (BORIS), an epigenetically acting tumor-promoting transcription factor. Vaccination of mice with DC loaded with truncated form of BORIS (DC/mBORIS) after 4T1 mammary tumor implantation induced strong anti-cancer immunity, inhibited tumor growth (18.75% of mice remained tumor-free), and dramatically lowered the number of spontaneous clonogenic metastases (50% of mice remained metastases-free). Higher numbers of immune effector CD4 and CD8 T cells infiltrated the tumors of vaccinated mice vs. control animals. Vaccination significantly decreased the number of myeloid-derived suppressor cells (MDSCs) infiltrating the tumor sites, but not MDSCs in the spleens of vaccinated animals. These data suggest that DC-based mBORIS vaccination strategies have significant anti-tumor activity in a therapeutic setting and will be more effective when combined with agents to attenuate tumor-associated immune suppression.

Loss of Muscarinic M1 Receptor Exacerbates Alzheimer's Disease-like Pathology and Cognitive Decline

The American Journal of Pathology. Aug, 2011  |  Pubmed ID: 21704011

Alzheimer's disease (AD) is pathologically characterized by tau-laden neurofibrillary tangles and β-amyloid deposits. Dysregulation of cholinergic neurotransmission has been implicated in AD pathogenesis, contributing to the associated memory impairments; yet, the exact mechanisms remain to be defined. Activating the muscarinic acetylcholine M(1) receptors (M(1)Rs) reduces AD-like pathological features and enhances cognition in AD transgenic models. To elucidate the molecular mechanisms by which M(1)Rs affect AD pathophysiological features, we crossed the 3xTgAD and transgenic mice expressing human Swedish, Dutch, and Iowa triple-mutant amyloid precursor protein (Tg-SwDI), two widely used animal models, with the M(1)R(-/-) mice. Our data show that M(1)R deletion in the 3xTgAD and Tg-SwDI mice exacerbates the cognitive impairment through mechanisms dependent on the transcriptional dysregulation of genes required for memory and through acceleration of AD-related synaptotoxicity. Ablating the M(1)R increased plaque and tangle levels in the brains of 3xTgAD mice and elevated cerebrovascular deposition of fibrillar Aβ in Tg-SwDI mice. Notably, tau hyperphosphorylation and potentiation of amyloidogenic processing in the mice with AD lacking M(1)R were attributed to changes in the glycogen synthase kinase 3β and protein kinase C activities. Finally, deleting the M(1)R increased the astrocytic and microglial response associated with Aβ plaques. Our data highlight the significant role that disrupting the M(1)R plays in exacerbating AD-related cognitive decline and pathological features and provide critical preclinical evidence to justify further development and evaluation of selective M(1)R agonists for treating AD.

Therapeutic Modulation of Cerebral Microhemorrhage in a Mouse Model of Cerebral Amyloid Angiopathy

Stroke; a Journal of Cerebral Circulation. Nov, 2011  |  Pubmed ID: 21903962

The aging brain demonstrates frequent MRI and pathological evidence of cerebral microbleeds, which are often associated with cerebral amyloid angiopathy. To develop new therapeutic strategies for this disorder, we studied cerebral microhemorrhage in a well-characterized mouse model of cerebral amyloid angiopathy.

Frontal Cortex Neuropathology in Dementia Pugilistica

Journal of Neurotrauma. Oct, 2011  |  Pubmed ID: 22017610

Dementia pugilistica (DP) is associated with chronic traumatic brain injury and leads to a "punch drunk" syndrome characterized by impairments in memory and executive function, behavioral changes and motor signs. Microscopic features include the accumulation of neurofibrillary tangles (NFT), beta-amyloid (Aβ) and TDP-43 pathology. Here we describe detailed clinical and neuropathological information about a 55 year old retired boxer (ApoE3/4), who presented with executive dysfunction and behavioral impairments. At autopsy, significant Aβ pathology was primarily in the form of diffuse plaques. Tau pathology was extensive and was determined to be of Braak & Braak stage VI. Frontal white matter showed evidence of glial tau inclusions (astrocytes and oligodendroglia). Cerebrovascular pathology was minimal with patchy amyloid angiopathy. Inflammation was another key feature, including microglial activation and significant C1q labeling of neurons and NFTs was seen. TDP-43 positive pathology was also observed. Inflammation may be a key inciting as well as propagating feature of DP neuropathology.

Elevated Stearoyl-CoA Desaturase in Brains of Patients with Alzheimer's Disease

PloS One. 2011  |  Pubmed ID: 22046234

The molecular bases of Alzheimer's disease (AD) remain unclear. We used a lipidomic approach to identify lipid abnormalities in the brains of subjects with AD (N = 37) compared to age-matched controls (N = 17). The analyses revealed statistically detectable elevations in levels of non-esterified monounsaturated fatty acids (MUFAs) and mead acid (20:3n-9) in mid-frontal cortex, temporal cortex and hippocampus of AD patients. Further studies showed that brain mRNAs encoding for isoforms of the rate-limiting enzyme in MUFAs biosynthesis, stearoyl-CoA desaturase (SCD-1, SCD-5a and SCD-5b), were elevated in subjects with AD. The monounsaturated/saturated fatty acid ratio ('desaturation index')--displayed a strong negative correlation with measures of cognition: the Mini Mental State Examination test (r = -0.80; P = 0.0001) and the Boston Naming test (r = -0.57; P = 0.0071). Our results reveal a previously unrecognized role for the lipogenic enzyme SCD in AD.

Blocking IL-1 Signaling Rescues Cognition, Attenuates Tau Pathology, and Restores Neuronal β-catenin Pathway Function in an Alzheimer's Disease Model

Journal of Immunology (Baltimore, Md. : 1950). Dec, 2011  |  Pubmed ID: 22095718

Inflammation is a key pathological hallmark of Alzheimer's disease (AD), although its impact on disease progression and neurodegeneration remains an area of active investigation. Among numerous inflammatory cytokines associated with AD, IL-1β in particular has been implicated in playing a pathogenic role. In this study, we sought to investigate whether inhibition of IL-1β signaling provides disease-modifying benefits in an AD mouse model and, if so, by what molecular mechanisms. We report that chronic dosing of 3xTg-AD mice with an IL-1R blocking Ab significantly alters brain inflammatory responses, alleviates cognitive deficits, markedly attenuates tau pathology, and partly reduces certain fibrillar and oligomeric forms of amyloid-β. Alterations in inflammatory responses correspond to reduced NF-κB activity. Furthermore, inhibition of IL-1 signaling reduces the activity of several tau kinases in the brain, including cdk5/p25, GSK-3β, and p38-MAPK, and also reduces phosphorylated tau levels. We also detected a reduction in the astrocyte-derived cytokine, S100B, and in the extent of neuronal Wnt/β-catenin signaling in 3xTg-AD brains, and provided in vitro evidence that these changes may, in part, provide a mechanistic link between IL-1 signaling and GSK-3β activation. Taken together, our results suggest that the IL-1 signaling cascade may be involved in one of the key disease mechanisms for AD.

Delivery of a DNA Vaccine for Alzheimer's Disease by Electroporation Versus Gene Gun Generates Potent and Similar Immune Responses

Neuro-degenerative Diseases. Feb, 2012  |  Pubmed ID: 22301697

Background: Induction of a humoral response against amyloid-β peptide may be beneficial for Alzheimer's disease (AD) patients and may alleviate the onset and progression of AD. DNA-based vaccination provides a unique alternative method of immunization for treatment and prevention of AD. Currently, the two major delivery methods used for enhancing DNA uptake and immune responses to DNA vaccines in humans are electroporation (EP) and gene gun (GG). Objective: The goal of this translational study was to evaluate the efficacy of an AD DNA epitope vaccine (DepVac) delivered intramuscularly by EP or intradermally by GG. Methods: Humoral and cellular immune responses to immunization with DepVac were evaluated by ELISA and ELISPOT, respectively. Functional activity of the antibodies was also assessed. Results: EP- and GG-mediated immunizations with DepVac induced similar anti-amyloid-β (Aβ) antibody and T cell responses. Anti-Aβ antibodies bound to amyloid plaques in AD brain tissue and to toxic forms of Aβ(42) peptide. Conclusion: Both delivery methods are effective at promoting potent antibodies specific for Aβ.