USF Libraries
USF Digital Collections

Immunomodulation as a potential therapeutic approach for Alzheimer's disease

MISSING IMAGE

Material Information

Title:
Immunomodulation as a potential therapeutic approach for Alzheimer's disease
Physical Description:
ix, 219 leaves : ill. (some col.) ; 29 cm.
Language:
English
Creator:
Nikolic, William Veljko
Publication Date:

Subjects

Subjects / Keywords:
Alzheimer Disease -- therapy   ( mesh )
Antigens, CD40 -- therapeutic use   ( mesh )
CD40 Ligand -- therapeutic use   ( mesh )
Cord Blood Stem Cell Transplantation -- therapeutic use   ( mesh )
Immunotherapy -- adverse effects   ( mesh )
Mice, Transgenic   ( mesh )
Inflammation
CD40
Immunization
Human umbilical cord blood cells
Microglia
Dissertations, Academic -- Molecular Medicine -- Doctoral -- USF   ( lcsh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: Alzheimer's disease (AD) is the most prevalent form of progressive dementia and is characterized by the accumulation of amyloid beta (Aβ) peptide in the brain and in the cerebral vessels forming cerebral amyloid angiopathy (CAA). As previously reported, an active immunization strategy of mice with Aβ₁₋₄₂ peptide results in decreased Th1 and increased Th2 cytokine responses as well as an effectively clearance of CNS Aβ. This approach has also yielded favorable results for many patients, unfortunately, a small percentage of these study participants developed severe aseptic meningoencephalitis likely secondary to CNS invasion of activated T-cells. We have previously demonstrated that disruption of CD40-40L pathway reduces Aβ plaque load, promotes Th2 response, and rescues from cognitive impairments. However, direct blockage of the CD40 pathway by passive vaccination with anti-CD40L antibody leads to immunosupression.Therefore, in its current form this therapeutic strategy poses an unacceptable risk to the recipient of treatment, aged individual. For those reasons, the identification and characterization of alternative modulators/inhibitors of CD40 signaling may be necessary for the development of safe and effective AD immunotherapy. This proposal introduces novel immunomodulatory therapies that are based on previous vaccination strategies or cell based therapies across medial field. We showed that transcutaneous vaccination can both be efficacious and safe, thus clearly demonstrating that the right combination of the antigens, adjuvants, and the routes of administration are crucial for the right vaccine. Furthermore, we demonstrated that the effects of current Aβ vaccine strategies could be enhanced by a simultaneous blockade of CD40-40L signaling.As an alternative approach, we explored the possibility of cell-based therapies and showed that human umbilical cord blood cells, which are currently used as a treatment for systemic lupus erythematosus and leukemia, and currently investigated against stroke, amyotropic lateral sclerosis, age-related macular degeneration, multiple sclerosis, and Parkinson's disease, and showed that not just they improved the AD like pathology in transgenic animals but altered both the brain and peripheral inflammation levels. Lastly, we discussed the involvement of microglia, one of the key players in both AD pathogenesis and Aβ clearance and suggesed that microglia in actuality has a continuum of physiological activation states that contribute to proinflammation, antiinflammation, and phagocytosis.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2008.
Bibliography:
Includes bibliographical references.
Additional Physical Form:
Also available online.
Statement of Responsibility:
by William Veljko Nikolic.
General Note:
Includes vita.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 002061865
oclc - 528809416
usfldc doi - E14-SFE0002539
usfldc handle - e14.2539
System ID:
SFS0026856:00001


This item is only available as the following downloads:


Full Text
xml version 1.0 encoding UTF-8 standalone no
record xmlns http:www.loc.govMARC21slim xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.loc.govstandardsmarcxmlschemaMARC21slim.xsd
leader nam 2200373Ka 4500
controlfield tag 001 002061865
005 20100225150001.0
008 100225s2008 xx a b 000 0 eng d
datafield ind1 8 ind2 024
subfield code a E14-SFE0002539
035
(OCoLC)528809416
040
FHJ
c FHJ
049
FHJA
096
WT 155
b N693i 2008
1 100
Nikolic, William Veljko.
0 245
Immunomodulation as a potential therapeutic approach for Alzheimer's disease /
by William Veljko Nikolic.
260
2008.
300
ix, 219 leaves :
ill. (some col.) ;
29 cm.
500
Includes vita.
502
Dissertation (Ph.D.)--University of South Florida, 2008.
504
Includes bibliographical references.
590
Co-advisor: Jun Tan, Ph.D.
Co-advisor: Huntington Potter, Ph.D.
520
ABSTRACT: Alzheimer's disease (AD) is the most prevalent form of progressive dementia and is characterized by the accumulation of amyloid beta (A) peptide in the brain and in the cerebral vessels forming cerebral amyloid angiopathy (CAA). As previously reported, an active immunization strategy of mice with A peptide results in decreased Th1 and increased Th2 cytokine responses as well as an effectively clearance of CNS A. This approach has also yielded favorable results for many patients, unfortunately, a small percentage of these study participants developed severe aseptic meningoencephalitis likely secondary to CNS invasion of activated T-cells. We have previously demonstrated that disruption of CD40-40L pathway reduces A plaque load, promotes Th2 response, and rescues from cognitive impairments. However, direct blockage of the CD40 pathway by passive vaccination with anti-CD40L antibody leads to immunosupression.Therefore, in its current form this therapeutic strategy poses an unacceptable risk to the recipient of treatment, aged individual. For those reasons, the identification and characterization of alternative modulators/inhibitors of CD40 signaling may be necessary for the development of safe and effective AD immunotherapy. This proposal introduces novel immunomodulatory therapies that are based on previous vaccination strategies or cell based therapies across medial field. We showed that transcutaneous vaccination can both be efficacious and safe, thus clearly demonstrating that the right combination of the antigens, adjuvants, and the routes of administration are crucial for the right vaccine. Furthermore, we demonstrated that the effects of current A vaccine strategies could be enhanced by a simultaneous blockade of CD40-40L signaling.As an alternative approach, we explored the possibility of cell-based therapies and showed that human umbilical cord blood cells, which are currently used as a treatment for systemic lupus erythematosus and leukemia, and currently investigated against stroke, amyotropic lateral sclerosis, age-related macular degeneration, multiple sclerosis, and Parkinson's disease, and showed that not just they improved the AD like pathology in transgenic animals but altered both the brain and peripheral inflammation levels. Lastly, we discussed the involvement of microglia, one of the key players in both AD pathogenesis and A clearance and suggesed that microglia in actuality has a continuum of physiological activation states that contribute to proinflammation, antiinflammation, and phagocytosis.
530
Also available online.
2 650
Alzheimer Disease
x therapy.
Antigens, CD40
therapeutic use.
CD40 Ligand
therapeutic use.
Cord Blood Stem Cell Transplantation
therapeutic use.
Immunotherapy
adverse effects.
Mice, Transgenic.
653
Inflammation
CD40
Immunization
Human umbilical cord blood cells
Microglia
690
Dissertations, Academic
z USF
Molecular Medicine
Doctoral.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.2539



PAGE 1

Immunomodulation As A Potential Therapeutic Approach For Alzheimers Disease by William Veljko Nikolic A dissertation submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy Department of Molecular Medicine College of Medicine University of South Florida Co-Major Professor: Jun Tan, M.D., Ph.D. Co-Major Professor: Huntington Potter, Ph.D. Paula Bickford, Ph.D. Inge Wefes, Ph.D. Andreas Seyfang, Ph.D. Date of Approval: June 13, 2008 Keywords: Inflammation, CD40, immunization, human umbilical cord blood cells, microglia Copyright 2008, William Veljko Nikolic i

PAGE 2

ACKNOWLEDGEMENTS Thanks are due first to my major professor, Dr. Jun Tan, for his great insights, perceptiveness, and mentorship. My sincere thanks go to co-major professor, Dr. Huntington Potter and other committee members; Dr. Paula Bickford, Dr. Andreas Seyfang, and Dr. Inge Wefes. I would further like to thank my family for their continuous and overwhelming support. Lastly, I would like to recognize those individuals who have directly or indirectly made this Ph.D. possible, especially: Kavon Rezai-Zadeh, Jared Ehrhart, Dr. Huayan Hou, Dr. Yuin Bai, Dr. Takashi Mori, Dr. Terrence Town, Dr. Brian Giunta, and Jin Zeng. ii

PAGE 3

TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES iv LIST OF ABBREVIATIONS vi ABSTRACT viii CHAPTER ONE: INTRODUCTION 1 1.1. The impact of Alzeimers disease 1 1.2. Pathology of Alzheimers disease 2 1.3. Microglia and central nervous system 3 1.4. Classical roles of peripheral innate immune cells 4 1.4.1. The macrophage: prototypical phagocyte 5 1.4.2. The dendritic cell: professional antigen presenting cell 6 1.5. Microglial activation after toll-like receptor stimulation: a mixed response 8 1.6. Adaptive response of activated microglia in demyelinating disease via CD40-40 ligand interaction 11 1.6.1. Brain inflammation in demyelinating disease 11 1.6.2. CD40-40 ligand interaction in experimental autoimmune encephalitis 12 1.7. Activation of microglia after CD40 ligation in Alzheimers disease: a shift from innate to adaptive response 14 1.7.1. Alzheimers disease and microglial responses to A 14 1.7.2. Microglial response to A in context of CD40 ligation 15 1.8. Immunotherapy and Alzheimers disease 16 CHAPTER TWO: PERIPHERALLY ADMINISTERED HUMAN UMBILICAL CORD BLOOD CELLS REDUCE PARENCHYMAL AND VASCULAR -AMYLOID DEPOSITS AND SUPPRESS CD40-CD40L INTERACTION 21 CHAPTER THREE: CD40L DISRUPTION ENHANCES A VACCINE-MEDIATED REDUCTION OF CEREBRAL AMYLOIDOSIS WHILE MINIMIZING CEREBRAL AMYLOID ANGIOPATHY AND INFLAMMATION 75 i

PAGE 4

CHAPTER FOUR: TRANSCUTANEOUS A PEPTIDE IMMUNIZATION OF TRANSGENIC ALZHEIMERS MICE RESULTS IN REDUCED CEREBRAL A DEPOSITS IN THE ABSENCE OF T-CELL INFILTRATION AND MICROHEMORRHAGE 142 CHAPTER FIVE DISCUSSION 196 5.1. Microglia and central nervous system 196 5.2. Immunotherapy for Alzheimers disease 200 5.2.1. Immunotherapy: transcutaneous vaccination 202 5.2.2. Immunotherapy: human umbilical cold blood cells 205 REFERENCES 208 ABOUT THE AUTHOR End Page ii

PAGE 5

LIST OF TABLES Table 1 Phagocytic and antigen presenting cell responses of immune cells. 10 iii

PAGE 6

LIST OF FIGURES Figure 1. Cerebral A/-amyloid pathology is reduced in PSAPP and Tg2576 mice peripherally infused with HUCBC. 36 Figure 2. -amyloid associated microgliosis and astrocytosis are reduced in HUCBC infused-PSAPP mice. 42 Figure 3. HUCBC infusion results in CD40-dependent increased plasma Alevels in PSAPP mice. 49 Figure 4. HUCBC infusion promotes anti-inflammatory/Th2 responses and decreases sCD40L in the CNS. 52 Figure 5. HUCBC modulate microglial CD40 expression and promote A microglial/macrophage phagocytic activity. 56 Figure 6. Evaluation of the effects of CD40 deficiency on A antibody generation and A efflux in A 1-42 -immunized mice. 91 Figure 7. Cerebral Alevels are significantly reduced in A 1-42 -immunized PSAPP mice heterozygous for CD40. 95 Figure 8. -amyloid pathology is reduced in A 1-42 -immunized PSAPP mice heterozygous for CD40. 97 Figure 9. PSAPP/CD40 -/mice have increased anti-inflammatory IL-10 cytokine and decreased plasma soluble CD40L (sCD40L) after A 1-42 vaccination. 103 Figure 10. Peripheral and cerebral A levels are reduced in A 1-42 -immunized PSAPP mice treated with CD40L neutralizing antibody. 106 Figure 11. Cerebral -amyloid deposits and cerebral amyloid angiopathy are reduced in A 1-42 -immunized PSAPP or Tg2576 mice treated with CD40L neutralizing antibody. 111 Figure 12. CD40L blockade inhibits APC-like microglial activation in iv

PAGE 7

A 1-42 vaccinated PSAPP mice and promotes anti-inflammatory cellular immunity. 118 Figure 13. A-specific neurotoxic inflammatory responses are reduced in A 1-42 -immunized PSAPP mice deficient for CD40. 126 Figure 14. Generation of immune responses in wild-type C57BL/6 mice t.c. immunized with aggregated A 1-42 peptide plus CT. 157 Figure 15. A/CT t.c. immunization resulted in LC recruitment into dermal layers. 162 Figure 16. Increased systemic Aafter A 1-42 /CT t.c. immunization of PSAPP mice. 167 Figure 17. Reduction of cerebral A/-amyloid pathology in PSAPP mice t.c. immunized with A 1-42 /CT. 171 Figure 18. Simultaneous analysis of plasma and brain soluble A levels on a mouse-by-mouse basis. 176 Figure 19. Absence of T-cell infiltration or brain microhemorrhage in A/CT t.c. immunized mice. 180 Figure 20. Apolipoprotein B protein was undetectable in brain tissue homogenates derived from both A/CT and CT t.c.-immunized mice. 182 Figure 21. Model for innate versus adaptive microglial activation responses. 198 v

PAGE 8

LIST OF ABBREVIATION AD Alzheimers disease A Amyloid beta CAA Cerebral amyloid angiopathy Th1 T helper type 1 cell Th2 T helper type 2 cell CNS Central nervous system APP Amyloid precursor protein M Macrophage TLR Toll-like receptor PAMP Pathogen-associated molecular patterns MHC Major histocompatibility complex IL Interleukin TNFTumor necrosis factor alpha APC Antigen presenting cell DC Dendritic cell IFN Interferon EAE Autoimmune encephalomyelitis MS Multiple sclerosis vi

PAGE 9

NSAID Non-steroidal anti-inflammatory drugs CD40L CD40 ligand HUCBC Human umbilical cord blood cells HAMNC Human adult mononuclear cells CC Cingulate cortex H Hippocampus EC Entorhinal cortex GFAP Glial fibrial acidic protein ELISA Enzyme-linked immunosorbent assay IgG Immunoglobulin G IgM Immunoglobulin M TGF Tumor growth factor BBB Blood-brain barrier NK Natural killer cells t.c. Transcutaneous CT Cholera toxin LC Langerhans cells vii

PAGE 10

Immunomodulation As A Potential Therapeutic Approach for Alzheimers Disease William Veljko Nikolic ABSTRACT Alzheimers disease (AD) is the most prevalent form of progressive dementia and is characterized by the accumulation of amyloid beta (A) peptide in the brain and in the cerebral vessels forming cerebral amyloid angiopathy (CAA). As previously reported, an active immunization strategy of mice with A 1-42 peptide results in decreased Th1 and increased Th2 cytokine responses as well as an effectively clearance of CNS A. This approach has also yielded favorable results for many patients, unfortunately, a small percentage of these study participants developed severe aseptic meningoencephalitis likely secondary to CNS invasion of activated T-cells. We have previously demonstrated that disruption of CD40-40L pathway reduces A plaque load, promotes Th2 response, and rescues from cognitive impairments. However, direct blockage of the CD40 pathway by passive vaccination with anti-CD40L antibody leads to immunosupression. Therefore, in its current form this therapeutic strategy poses an unacceptable risk to the recipient of treatment, aged individual. For those reasons, the identification and characterization of alternative modulators/inhibitors of CD40 signaling may be necessary for the development of safe and effective AD immunotherapy. This proposal introduces novel immunomodulatory therapies that are based on previous vaccination strategies or cell based therapies across medial field. We showed viii

PAGE 11

that transcutaneous vaccination can both be efficacious and safe, thus clearly demonstrating that the right combination of the antigens, adjuvants, and the routes of administration are crucial for the right vaccine. Furthermore, we demonstrated that the effects of current A vaccine strategies could be enhanced by a simultaneous blockade of CD40-40L signaling. As an alternative approach, we explored the possibility of cell-based therapies and showed that human umbilical cord blood cells, which are currently used as a treatment for systemic lupus erythematosus and leukemia, and currently investigated against stroke, amyotropic lateral sclerosis, age-related macular degeneration, multiple sclerosis, and Parkinsons disease, and showed that not just they improved the AD like pathology in transgenic animals but altered both the brain and peripheral inflammation levels. Lastly, we discussed the involvement of microglia, one of the key players in both AD pathogenesis and A clearance and suggesed that microglia in actuality has a continuum of physiological activation states that contribute to proinflammation, antiinflammation, and phagocytosis. ix

PAGE 12

CHAPTER ONE INTRODUCTION 1.1 The impact of Alzheimers disease First described in 1906 by Alois Alzheimer, the Alzheimers disease (AD) is the leading cause of dementia among elderly. Dementia is a collective name for progressive degenerative brain syndromes, which affect memory, behavior, emotions, and thinking. Currently, about 10 percent of Americans over the age of 65 and half of those over age of 85 have been diagnosed with AD, which equates to over 5.2 million Americans that suffer from this disease. According to 2008 Alzheimers Association Facts and Figures report, AD is the seventh-leading cause of death in U.S. and where every 71 seconds someone develops AD. The report also estimates that the direct and indirect costs of Alzheimer's and other dementias to Medicare, Medicaid and businesses amount to more than $148 billion each year, which makes it a third most expensive disease to threat in the U.S. Generally speaking, Alzheimers disease can be divided into three basic stages. The early stage is characterized by mild cognitive decline, i.e. wordor name-finding problems, decline in ability to organize, and performance issues in social or work settings. The middle stage is characterized by severe cognitive decline (confusion, disorientation, and delusions), disruption in normal sleep/walking cycle, and increasing 1

PAGE 13

episodes of urinary or fecal incontinence. The late and final stage of AD is characterized by the loss of capacity of recognizable speech, individuals lose the ability to work, lose the ability to walk without assistance, then the ability to sit without support, the ability to smile, the ability to hold their head up, and individuals need help with eating and toileting and there is general incontinence of urine. 1.2 Pathology of Alzheimers disease Brain regions being affected early are entorhinal cortex, hippocampus, and basal forebrain. These are small specialized structures that play a crucial role in memory. Over time, the disease destroys large areas of the brain that control vital body functions and as a result AD patients typically die from pneumonia or lack of nutrition. Pathologically speaking, major AD characteristics are senile plaques (caused by amyloid beta peptide deposition), neurofibrilary tangles (caused by tau protein deposition), and neuronal loss (characterized by dystrophic neurites). Amyloid or senile plaque is primarily composed of 39-43 amino acid peptide amyloid beta (A). A is derived by a proteolytic cleavage of amyloid precursor protein (APP). There are A is normally water soluble, however it does have a tendency to aggregate, ex. especially longer forms of the peptide (A 1-40 and A 1-42 ) as regular -plated sheet conformations and come out as insoluble amyloid plaques. These plaques are normally detergent resistant. A can also be deposited as diffused deposits, often believed to be the intermediate step before the more compact amyloid plaques form. In recent years, A deposition has been identified in the vascular wall. This is know as a cerebral amyloid angiopathy (CAA), a pathology that was identified in as much as 83 percent of AD patients (Ellis et al., 1996). Neurofibrilary 2

PAGE 14

tangles are intraneuronal inclusions of abnormal fibers that consist of paired helical filaments. The filaments consist of microtubule-associated hyperphosphorylated tau proteins. Lastly, neuronal loss is thought to originate from amyloid plaques, inflammatory reactions due to cytokine releases, oxidative injury that disrupt neuronal metabolic and ionic homestasis, and impaired neuronal trafficking due to hyperphosphorylated tau filaments. 1.3 Microglia and central nervous system Microglia are somewhat enigmatic central nervous system (CNS) cells that have been traditionally regarded as CNS macrophages (Ms). They represent about 10% of the adult CNS cell population (Pessac et al., 2001). In mice, microglial progenitors can be detected in neural folds at the early stages of embryogenesis. Murine microglia are thought to originate from the yolk sac at a time in embryogenesis when monocyte/macrophage progenitors (of hematopoeitic origin) are also found (Pessac et al., 2001) (Alliot et al., 1999). Based on this observation, it is now generally accepted that adult mouse microglia originate from monocyte/macrophage precursor cells migrating from the yolk sac into the developing CNS, most likely independently of CCR2 (Mildner et al., 2007). Once CNS residents, these newly migratory cells actively proliferate during development, thereby giving rise to the resident CNS microglial cell pool. More recently however, it has been shown that bone marrow-derived cells can enter the CNS and become cells that phenotypically resemble microglia in the adult mouse (Eglitis and Mezey, 1997) (Brazelton et al., 2000) (Mezey et al., 2000). Interestingly, under conditions of CNS damage such as stroke, cholinergic fiber degeneration, or motor 3

PAGE 15

neuron injury, Priller and colleagues found that green fluorescent protein-labeled bone marrow cells could enter the CNS and take up a microglial phenotype (Priller et al., 2001). On other hand it was shown that bone-marrow derived cells do not contribute at all to the CD11b + /Iba-1 + population when the CNS was protected from irradiation, suggesting that proliferation of endogenous microglia is the most plausible explanation for the increase in Iba-1 + cell number (Mildner et al., 2007). This latter view could be further supported with citings that local microglia proliferate following lesioned CNS with intact blood-brain barrier (Priller et al., 2006) (Remington et al., 2007). Microglia normally exist in a quiescent (resting) state in the healthy CNS, and are morphologically characterized by small soma and ramified processes. However, upon activation in response to invading viruses or bacteria or CNS injury, microglia undergo morphological changes including shortening of cellular processes and enlargement of their soma (sometimes referred to as an amoeboid phenotype). Activated microglia also up-regulate a myriad of cell surface activation antigens and produce innate cytokines and chemokines (discussed in detail below). As the microglial lineage originates from peripheral myeloid precursor cells, it is helpful to consider the activation states of such peripheral innate immune cells to better understand the nature of microglial activation. 1.4 Classical roles of peripheral innate immune cells It is now widely accepted that both innate and adaptive arms of the immune system play important roles in maintaining immune homeostasis. However, little attention was paid to the evolutionarily much older innate immune system until the late Charlie Janeway proposed the involvement of innate mechanisms in vertebrate immunity. 4

PAGE 16

Specifically, Janeway pioneered the idea that lymphocyte activation could be critically regulated by the evolutionarily ancient system of antigen clearance by phagocytic cells of myeloid origin. Together with Ruslan Mezhitov, they originated the concept that these phagocytic innate immune cells recognize pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors, the most notable examples being a set of phylogenetically conserved, germ-line encoded Toll-like receptors (TLRs, currently 11-12 members, (Qureshi and Medzhitov, 2003) (Yamamoto et al., 2004) (Zhang et al., 2002) (Tabeta et al., 2004), resulting in expression of cell-surface activation molecules [for example, major histocompatibility complex (MHC) class I and II, B7.1, B7.2, and CD40] and secretion of innate cytokines (e.g., tumor necrosis factor(TNF-), interleukin (IL)-1, IL-6, IL-12, and IL-18 (Janeway and Medzhitov, 2002) (Medzhitov and Janeway, 2000a). Once activated, the innate arm of the immune response calls adaptive immune cells into action, and both branches act in concert to promote neutralization and clearance of invading pathogens. Thus, innate immune cells are able to discriminate non-infectious self from infectious non-self and thereby form the first line of defense against invading bacteria and viruses (for review see (Medzhitov and Janeway, 2000b) (Medzhitov and Janeway, 2002) (Medzhitov and Janeway, 2002) (Iwasaki and Medzhitov, 2004). 1.4.1 The macrophage: prototypical phagocyte Macrophages (Ms) are quintessential phagocytes whose primary role is to engulf pathogens such as invading bacteria and to remove debris and detritus, i.e., 5

PAGE 17

remnants of apoptotic cells. Tissue Ms develop when blood monocytes enter into the various organs and tissues and differentiate into specialized, site-specific Ms depending on their anatomical location, such as alveolar Ms (lung), histiocytes (connective tissue), kupffer cells (liver), mesangial cells (kidney), osteoclasts (bone), or microglia (brain) (Goldsby et al., 2002). Resting Ms are both weak phagocytes and weak lymphocyte activators (Adler et al., 1994). Upon activation however, for example in response to TLR stimulation by PAMPs, their phagocytic potential greatly enhances (Blander and Medzhitov, 2004) and they up-regulate cell-surface co-stimulatory molecules and produce pro-inflammatory innate cytokines as mentioned above. Typically, engulfment of the pathogen by phagocytosis triggers a respiratory burst involving production of reactive oxygen species such as superoxide and peroxinitrite that kill the pathogen (Adler et al., 1994) (Forman and Torres, 2001). In addition, activated M up-regulate cell-surface Fc receptors that aid in phagocytosis of pathogens opsonized by antibodies produced by plasma cells (Tsukada et al., 1994) (Blom et al., 2003). On the other hand, in response to debris from apoptotic cells, the M mounts a phagocytic response essentially in the absence of pro-inflammatory cytokines (Gregory and Devitt, 2004). The most likely reason for this anti-inflammatory phagocytic response is that pro-inflammatory cytokines such as TNFpromote bystander injury which may further damage tissues in which the apoptotic cells reside. Thus, Ms are highly capable of innate activation characterized by a strong phagocytic response sometimes accompanied by pro-inflammatory cytokine production (for a review see (Fujiwara and Kobayashi, 2005). 6

PAGE 18

1.4.2 The dendritic cell: professional antigen presenting cell Whereas Ms have limited ability to process and present antigen to T cells, dendritic cells (DCs) are considered professional antigen presenting cells (APCs). DCs can be found under the epithelia and in most organs where they capture and process non-self antigens, migrate to lymphoid organs, and present antigen in the context of MHC to CD4+ and CD8+ T lymphocytes. With their many finger-like cellular processes, DCs are morphologically optimized to simultaneously display antigen to many T cells. Like Ms, DCs respond to invading pathogens by recognizing PAMPs through TLRs, and subsequently phagocytose and process antigen. DCs then up-regulate cell-surface co-stimulatory molecules and secrete innate cytokines and chemokines (typically at levels an order of magnitude higher than those secreted by Ms) to promote recruitment and activation of CD4+ and/or CD8+ T lymphocytes. There are three generally accepted classifications of DCs in mice: plasmacytoid (p) DCs (CD11c lo CD11b lo B220+, CD8-), lymphoid (l) DCs (CD11c + CD11b-, CD8+), and myeloid (m) DCs (CD11c+, CD11b+, B220-, CD8-, there are several subtypes,) (Banchereau et al., 2000). In humans, there are clearly two distinct subsets of DCs: pDCs (CD11c-, CD11b-, CD14-, CD45RA+) and monocyte DCs (CD11c+, CD11b+, CD14+, CD45RA-) (for a review see (Shortman and Liu, 2002). DC classes differ from each other predominately in tissue distribution, production of specific cytokines, TLR expression, and ability to promote innate versus adaptive immune responses (for a review see (Iwasaki and Medzhitov, 2004). For example, freshly isolated human pDCs express TLR7 and 9, whereas mDCs express 7

PAGE 19

TLR1, 2, 3, 5, 6, and 8 (Hornung et al., 2002) (Jarrossay et al., 2001) (Kadowaki et al., 2001). Stimulation of human pDCs or monocytic DCs with synthetic TLR7 ligands induces the secretion of interferon (IFN)(important for anti-viral innate immunity) or IL-12 [a key inducer of the adaptive T helper (Th) type I response], respectively (Ito et al., 2002). Similarly, stimulation of TLR9 via DNA containing unmethylated CpG motifs results in IFNsecretion by pDCs and IL-12 production by murine mDCs (Hemmi et al., 2003). Despite these relative differences between DC classes, the major role of DCs on the whole remains; they act as potent APCs capable of strongly activating T lymphocytes. Their APC capacity is much stronger than that of Ms, as DCs are able to directly activate nave T cells whereas Ms are not (Iwasaki and Medzhitov, 2004). Thus, by virtue of their ability to promote T cell activation responses, DCs are highly capable of adaptive activation. Activation markers of phagocytosis and APC responses in various innate immune cells are presented in the Table 1. 1.5 Microglial activation after toll-like receptor stimulation: a mixed response Recent evidence indicates that microglia, like their peripheral innate immune cell counterparts, express a wide array of TLRs, including mRNA for TLRs 1-9 in mice (Olson and Miller, 2004) and in humans (Bsibsi et al., 2002). Furthermore, many of these TLRs have been shown to be functional, allowing microglial recognition of a variety of PAMPs. For example, Kielian and coworkers found that heat-killed Staphylococcus aureus and its cell wall product peptidoglycan (PGN) are able to stimulate innate activation of microglia characterized by pro-inflammatory cytokine and 8

PAGE 20

chemokine production (Kielian et al., 2002). Those authors found that the effect of PGN was critically dependent on TLR2, as TLR2-deficient mice demonstrated reduced cytokine and chemokine production after PGN challenge (Kielian et al., 2005). Furthermore, murine microglia respond to the TLR9 agonist, unmethylated CpG-DNA, by secreting numerous pro-inflammatory innate cytokines (probably responsible for neurotoxicity in oganotypic brain slice cultures treated with CpG-DNA (Iliev et al., 2004), by up-regulating co-stimulatory cell surface molecules, and by promoting adaptive activation by secreting IL-12 to affect T cell activation (Dalpke et al., 2002). In two recent studies, murine microglial pro-inflammatory responses to bacterial lipopolysaccharide (LPS), a known TLR4 ligand, resulted in dramatic injury to cultured oligodendrocytes (Lehnardt et al., 2002) and neurons (Lehnardt et al., 2003), further 9

PAGE 21

10

PAGE 22

demonstrating microglial bystander injury after TLR stimulation (probably mediated by over-production of innate pro-inflammatory cytokines). It has recently been shown that microglia respond to poly I:C [a synthetic double-stranded (ds) RNA analog thought to be recognized by TLR3, (Alexopoulou et al., 2001)] by producing pro-inflammatory cytokines and chemokines (Jack et al., 2005), and microglial pro-inflammatory responses to dsRNA seem to be dependent on TLR3, as TLR3-deficient microglia have blunted innate cytokine responses in vitro and markedly reduced cell surface activation markers in brain after poly I:C stimulation (Town et al., submitted). Finally, infection with West Nile virus, a retrovirus that produces dsRNA during its life cycle, results in profound microglial activation as assessed by pro-inflammatory cytokine production in vitro and cell surface activation markers in vivo, effects that are dramatically reduced in TLR3-deficient animals (Wang et al., 2004). In peripheral innate immune cells, TLR responses to PAMPs seem to be dependent on at least four different TLR intracellular adapter molecules: MyD88 (involved in TLR1, 2, 4, 6, 7, 8, and 9 signaling), TRIF/TICAM-1 (mediates TLR3 and 4 signaling), TIRAP/Mal (involved in TLR1, 2, 4, and 6 responses) and TIRP/TRAM/TICAM-2 (mediates TLR4 signaling). These adapter molecules bind to the intracellular leucine-rich repeat region of the TLR and promote recruitment of additional factors such as IRAKs and TRAF6 that allow for activation of transcription factors including IRF-3 and NF-B, which are responsible for activation of numerous innate cytokines and cell-surface activation antigen genes (for review see (Vogel et al., 2003) (Hemmi and Akira, 2005). It is still unclear how different TLR responses in innate immune cells (i.e., promotion of innate versus adaptive responses) can be achieved when 11

PAGE 23

many TLRs share intracellular signaling molecules. While little work has been done on intracellular signaling following TLR stimulation in microglia, it is likely that microglia utilize the same signaling cascades described for Ms and DCs. 1.6 Adaptive response of activated microglia in demyelinating disease via CD40-CD40 ligand interaction 1.6.1 Brain inflammation in demyelinating disease Experimental autoimmune encephalomyelitis (EAE) is a mouse model of the human disease multiple sclerosis (MS), an autoimmune disease characterized by inflammatory CNS demyelinating lesions accompanied by motor disturbances. EAE can be induced in different strains of mice by subcutaneous or intraperioteneal inoculation with adjuvant plus epitopes found in myelin such proteolipid protein, myelin basic protein, or myelin oligodendrocyte glycoprotein. The disease is critically dependent on activation of pro-inflammatory CD4+ T helper type I (Th1) cells by APCs, and these auto-aggressive Th1 cells can be adoptively transferred to non-diseased recipient mice that subsequently develop disease. EAE is characterized by paralysis, typically beginning in the tail and hind limbs and progressing to the fore limbs. In the SJL mouse strain, animals develop a relapsing-remitting form of the disease while C57BL/6 mice manifest paralysis that progressively worsens until death. Upon histopathological analysis, brains from EAE mice generally show infiltration of Th1 cells (and other lymphocytes including Ms and DCs) and activation of microglia, typically in white matter regions where demyelinating lesions are found (for review see (Olsson, 1995) 12

PAGE 24

(Swanborg, 1995) (Cornet et al., 1998). 1.6.2 CD40-CD40 ligand interaction in experimental autoimmune encephalomyelitis Immune/inflammatory cells receiving a primary stimulus (i.e., MHC-T cell receptor interaction between APCs and T lymphocytes, respectively) typically require co-stimulatory signals via other pairs of molecules in order to become activated [for instance, the B7-CD28 and/or CD40-CD40 ligand (L) dyads in APC/T-cell activation; (van Kooten and Banchereau, 2000)]. CD40L is a key immunoregulatory molecule that plays a co-stimulatory role in the activation of immune cells from both the innate and adaptive arms of the immune system, and is typically expressed by activated CD4+ and some CD8+ T cell subsets (Grewal and Flavell, 1998). CD40 receptor, a member of TNF and nerve growth factor super-family, is expressed on many professional and non-professional APCs, including dendritic cells, B cells, monocytes/macrophages and microglial cells (O'Keefe et al., 2002) (Carson et al., 1998) (Havenith et al., 1998) (Tan et al., 1999b) (Tan et al., 1999a). Nearly 10 years ago, activated Th cells that expressed CD40 ligand (CD40L) were found in brains of MS patients, and these cells were found in close apposition to CD40-bearing cells in active demyelinating lesions (Gerritse et al., 1996). The authors determined that the CD40-expressing cells were either macrophages or microglia based on staining for acid phosphatase or CD11b. To evaluate whether the CD40-CD40L interaction was pathogenic in EAE, Gerritse and co-workers administered a CD40L neutralizing antibody to SJL mice that were given proteolipid protein with adjuvant to induce EAE. Strikingly, EAE was prevented in a prophylactic treatment regimen of anti-CD40L, and, when EAE was 13

PAGE 25

induced in another cohort of animals, CD40L antibody treatment significantly reduced disease severity in an active treatment paradigm (Gerritse et al., 1996). It was later shown that genetic deficiency in CD40L (Grewal et al., 1996) or antibody-mediated blockade of CD40L (Howard et al., 1999) resulted in attenuation of Th1 differentiation and effector function, including marked inhibition of the Th1 cytokine IFNand reduced numbers of encephalitogenic effector T cells. In an effort to further understand the nature of the CD40-CD40L interaction responsible for promotion of EAE, Becher and colleagues used a bone marrow reconstitution system to determine which CD40-expressing cells were responsible for promoting EAE (Becher et al., 2001). In that report, the authors showed that CD40 expression by parenchymal microglia was responsible for recruitment/retention of encephalitogenic T cells in EAE. Strikingly, treatment of microglia with a combination of granulocyte macrophage-colony stimulating factor and CD40L has been shown to promote differentiation of these cells into cells that (1) express the pan-DC marker CD11c, (2) morphologically resemble DCs, and (3) secrete the Th1-promoting cytokine IL-12 p70 (Fischer and Reichmann, 2001). Such CD11c+ CD11b+ DC-like microglia were found in EAE brain lesions in inflammatory foci containing T cells, and exhibited potent stimulation of allogeneic T cell proliferation versus CD11cCD11b+ microglia (Fischer and Reichmann, 2001). Although their origin was not determined, it was recently shown that CNS DCs (possibly DC-like microglia) are responsible for activation of nave T cells in response to endogenous myelin epitopes (termed epitope spreading), and this process was initiated in the CNS as opposed to the peripheral lymphoid organs (McMahon et al., 2005). Thus, in the context of EAE, CD40-CD40L interaction on microglia seems to promote adaptive 14

PAGE 26

function of these cells, resulting in a DC-like activated microglia phenotype that promotes encephalitogenic Th1 cell differentiation and effector function. 1.7 Activation of microglia after CD40 ligation in Alzheimer disease: a shift from innate to adaptive response 1.7.1 Alzheimer disease and microglial responses to -amyloid It has been suggested that activated microglia play a key role in AD pathogenesis as they secrete pro-inflammatory innate cytokines such as TNFand IL-1, which have been shown to promote neuronal injury at high levels (Meda et al., 1995) (Barger and Harmon, 1997) (McGeer and McGeer, 1998). Furthermore, there is a large body of evidence that non-steroidal anti-inflammatory drug (NSAID) use is associated with reduced risk for AD in humans (Shapshak et al., 2004) (in t' Veld et al., 2001) (Zandi et al., 2002), for a review see (Szekely et al., 2004), and NSAID treatment of AD mice results in reduced -amyloid plaque burden concomitant with ameliorated microglial activation (Matsushima et al., 1994) (Matsushima et al., 1994) (Lim et al., 2001). Work done in Maxfields laboratory showed that challenge of microglia with labeled A peptides promotes phagocytosis but poor degradation of soluble or fibrillar A via scavenger receptors (Paresce et al., 1996) (Paresce et al., 1997) (Chung et al., 1999). Using knockout mice, his laboratory showed that the class A scavenger receptor (type I and II) is the predominant scavenger receptor responsible for A uptake by microglia, with other scavenger receptors playing a more minor role (including the class B 15

PAGE 27

scavenger receptor CD36) (Chung et al., 1999). 1.7.2 Microglial responses to -amyloid in the context of CD40 ligation We previously showed that, while murine microglial challenge with soluble A peptides alone does not elicit TNFsecretion, co-stimulation provided in the form of CD40 ligation (either via CD40L or an agonistic CD40 antibody) results in TNFproduction being synergistically affected (Tan et al., 1999a). Further, microglia cultured from AD mice deficient in CD40L demonstrate reduced TNFsecretion versus CD40L-sufficient AD mouse microglia. This form of microglial activation in CD40L-sufficient AD mice is pathogenic, as CD40L-deficient AD mice demonstrate reduced activated (CD11b+) microglia, an effect that is associated with mitigated abnormal hyper-phosphorylation of tau protein (a key indicator of neuronal stress) (Tan et al., 1999a). Furthermore, genetic ablation of CD40L or administration of a CD40L-neutralizing antibody markedly reduces -amyloid plaques in mouse models of AD, effects that are associated with mitigated astrocytosis and microgliosis (Tan et al., 2002a), for review see (Town et al., 2001) (Tan et al., 2002b). More recently, overproduction of microglia-associated CD40 and of astrocyte-derived CD40L was found in and around -amyloid plaques in AD patient brains (Togo et al., 2000) (Calingasan et al., 2002), raising the possibility that the CD40-CD40L interaction may contribute to AD pathogenesis by promoting brain inflammation. In order to better understand the form of microglial activation affected by A plus CD40L stimulation, we examined innate and adaptive activation of murine microglia challenged with A in the presence or absence of CD40L co-stimulation (Townsend et 16

PAGE 28

al., 2005). When microglia were challenged with fluorescent-tagged synthetic human A alone, they mounted a time-dependent phagocytic response (from 15 min to 60 min) which could be enhanced by Fc receptor stimulation using an anti-human A antibody. This phagocytic response to Aalone was not associated with production of the pro-inflammatory innate cytokines TNF-, IL-6, or IL-1, a result similar to that seen when microglia are challenged with apoptotic cells and mount an anti-inflammatory, pro-phagocytic innate response (Minghetti et al., 2005). Importantly, CD40L treatment opposed this phagocytic response, as determined by measuring both cell-associated Aandfree extracellular A As mentioned above, Maxfields laboratory demonstrated that microglia slowly degrade phagocytosed A peptides (Paresce et al., 1996) (Paresce et al., 1997) (Chung et al., 1999). We examined the ability of microglia to degrade A peptides by first pulsing them with Aand then chasing these cells after 1 hour of culture in the presence or absence of CD40L stimulation. Using this experimental approach, we found that CD40L also retarded microglial clearance of the peptide. We further assessed putative modulation of microglial Aphagocytosis by cytokines known to promote effector T cell function, and found that the pro-inflammatory Th1-type cytokines IFNand TNFinhibited Aphagocytosis whereas the anti-inflammatory Th2-type cytokines IL-4 and IL-10 boosted this response. 1.8 Immunotherapy and Alzheimers disease Alzheimers disease is described by many pathological characteristics and yet the predominant theory of the disease process in the amyloid hypothesis. This 17

PAGE 29

hypothesis suggests that A deposition can directly through diffuse or compact plaques, or indirectly through inflammatory cascade, result in progressive synaptic and neuritic injury. This injury causes alterations in intracellular kinases functions that further contribute to tau protein hyperphosphorylation and the formation of neurofibrilary tangles. The combination of these events and their propagation is believed to contribute to neuronal dysfunction and loss which will contribute to dementia observed in Alzheimers disease (Hardy and Selkoe, 2002). Thus, methods developed to clear or prevent formation of A in the brains of AD patients represent a possible treatment modality. One promising approach involves utilization of active A immunization strategies, which produce dramatic reductions in A pathology in animal studies (Schenk et al., 1999). However, a phase IIa clinical trial was abandoned after about 6% of A-immunized AD patients developed aseptic meningoencephalitis (Nicoll et al., 2003; Orgogozo et al., 2003) that appeared to involve brain inflammatory reactions mediated by T-cells and microglia (Monsonego et al., 2001; Schenk and Yednock, 2002; Greenberg et al., 2003; Monsonego et al., 2003a). Interestingly, a 12 month post-vaccination period analysis revealed an inverse correlation between titers of amyloid plaque-reactive antibodies and rate of cognitive decline (Hock et al., 2003), suggesting clinical efficacy. Despite the discontinuation of the clinical trials, A vaccination studies have continued in effort to identify an immunization approach that is both safe and effective. Current approaches have focused on minimizing T-cell mediated inflammatory responses in efforts to prevent CNS invasion of auto-aggressive T-cells, while promoting A antibody-mediated clearance mechanisms (Chackerian et al., 2006; Maier et al., 2006; 18

PAGE 30

Okura et al., 2006; Nikolic et al., 2007). A possible avenue to both enhance A clearance and down-regulate CNS inflammatory responses (including invasion of reactive T-cells) involves modulation of the CD40 receptor (CD40)-CD40 ligand (CD40L) system. In the periphery, a variety of innate immune cells known as antigen-presenting cells (APCs) express CD40, including dendritic cells, B-cells, and monocytes/macrophages. In the CNS, CD40 is expressed by resident cells including microglia, neurons, and astrocytes, as well as by peripherally-derived APCs (Tan et al., 2002a; Town et al., 2005). Recently, the CD40-CD40L interaction was determined to play a central role in promoting and maintaining dendritic cell APC phenotype during infections (Straw et al., 2003). In the context of CNS immunity, the CD40-CD40L interaction is required for microglial maturation into functional APCs (Ponomarev et al., 2006). We have recently shown that CD40L treatment of primary cultures of microglia inhibits phagocytosis of A antibody opsonized, as well as non-opsonized A species (Townsend et al., 2005). A blockade of the CD40-CD40L system down-regulates T-cell/microglia-mediated injury in the context of experimental autoimmune encephalitis (EAE) (Howard et al., 1999; Howard et al., 2002). Altogether, these studies suggest that blockade of the CD40-CD40L interaction could enhance A vaccination-mediated A clearance mechanisms, while minimizing pro-inflammatory T-cell-mediated damage in the CNS. However, a complete blockade of CD40-40L interaction would lead to distortion of immune functioning and hence an autoimmunity would emerge. Therefore, the need for an immunomodulator that can partially block the CD40-40L interaction and yet still allow normal immune functioning would be needed. Human umbilical cord blood cells (HUCBC) have been shown to 19

PAGE 31

oppose the pro-inflammatory T helper cell type 1 (Th1) response, as demonstrated in an animal model of stroke where HUCBC infusion promoted a strong anti-inflammatory T-helper 2 (Th2) response (Vendrame et al., 2004). Importantly, this effect was associated with reduced infarct volume and rescue of behavioral deficit (Vendrame et al., 2004). HUCBC infusion has also shown therapeutic benefit in other neuroinflammatory conditions including multiple sclerosis, amyotrophic lateral sclerosis, neurodegenerative macular degeneration, and Parkinsons disease (El-Badri et al., 2006; Garbuzova-Davis et al., 2006; Henning et al., 2006). In AD preclinical models, administration of these cells to the PSAPP mouse model of AD was associated with extension of lifespan although high doses were administered in this paradigm (Ghorpade et al., 2001). So perhaps a proper immunomodulator together with active A vaccine working in tangent could be a right step towards future Alzheimers disease treatment. 20

PAGE 32

CHAPTER TWO PERIPHERALLY ADMINISTERED HUMAN UMBILICAL CORD BLOOD CELLS REDUCE PARENCHYMAL AND VASCULAR -AMYLOID DEPOSITS AND SUPPRESS CD40-CD40L INTERACTION 2.1 Abstract Modulation of immune/inflammatory responses by diverse strategies including amyloid(A immunization, non-steroidal anti-inflammatory drugs, and manipulation of microglial activation states has been shown to reduce Alzheimers disease (AD)-like pathology and cognitive deficits in AD transgenic mouse models. Human cord blood cells (HUCBC) have unique immunomodulatory potential, and we wished to test whether these cells might alter AD-like pathology after infusion into the PSAPP mouse model of AD. Here, we report a marked reduction of A levels/-amyloid plaques and associated astrocytosis following multiple low dose infusions of HUCBC. HUCBC infusions also reduced cerebral vascular A deposits in the Tg2576 AD mouse model. Interestingly, these effects were associated with suppression of the CD40-CD40L interaction as evidenced by decreased circulating and brain soluble CD40L (sCD40L) and elevated systemic IgM levels, attenuated CD40L-induced inflammatory responses, and reduced surface expression of CD40 on microglia. Importantly, deficiency of CD40 abolishes the 21

PAGE 33

effect of HUCBC on elevated plasma A levels. Moreover, microglia isolated from HUCBC-infused PSAPP mice demonstrated increased phagocytosis of A. Further, sera from HUCBC-infused PSAPP mice significantly increased microglial phagocytosis of A 1-42 peptide while inhibiting IFN--induced microglial CD40 expression. Increased microglial phagocytic activity in this scenario was inhibited by addition of recombinant CD40L protein. These data suggest that HUCBC infusion confers mitigation of AD-like pathology by disrupting CD40L activity. 2.2 Introduction Alzheimers disease (AD) is the most common progressive dementia, and is pathologically characterized by deposition of amyloid--peptide (A) in the brain parenchyma. A plaques are potent activators of both microglia and astrocytes, central nervous system (CNS)-resident immunocompetent cells that respond to cerebral amyloidosis by chronic, pro-inflammatory activation (Benzing et al., 1999). While it was once thought that activation of microglia and astrocytes in the AD brain was an epiphenomenon and not a pathoetiological contributor to AD, more recent studies have suggested that the A-mediated inflammatory cascade is an etiological perpetrator in AD For example, therapeutic strategies aimed at manipulating this inflammatory cascade, including A immunization (Schenk et al., 1999; Bard et al., 2000; Nicoll et al., 2003), non-steroidal anti-inflammatory drugs (NSAID) (Matsushima et al., 1994; in t' Veld et al., 2001; Szekely et al., 2004), and modulation of microglial activation (Tan et al., 1999a; Town et al., 2001; Tan et al., 2002b; Todd Roach et al., 2004; Laporte et al., 22

PAGE 34

2006), are able to reduce AD-like pathology and improve behavioral impairment in Alzheimers transgenic mouse models and, in some cases, reduce AD pathology in humans. We previously showed that the CD40-CD40 ligand (CD40L) interaction plays a critical role in A-induced pro-inflammatory microglial activation (Tan et al., 1999a). Moreover, we have demonstrated that disruption of this signaling pathway reduces cerebral A deposits in the Tg2576 mouse model of AD and improves cognitive deficits in PSAPP AD mice (Town et al., 2001; Tan et al., 2002a; Tan et al., 2002b; Todd Roach et al., 2004). The implication of CD40-CD40L interaction in AD-associated brain inflammatory process is supported from studies demonstrating increased expression of CD40 and CD40L in and around -amyloid plaques in AD brain (Togo et al., 2000; Calingasan et al., 2002). Recently, Desideri and colleagues (Desideri et al., 2006) reported circulating soluble CD40L (sCD40L) levels are significantly increased in AD patients vs. healthy elderly controls, further supporting a role for this receptor/ligand dyad in the pathogenesis of AD. Human umbilical cord blood cells (HUCBC) have been shown to oppose the pro-inflammatory T helper cell type 1 (Th1) response, as demonstrated in an animal model of stroke where HUCBC infusion promoted a strong anti-inflammatory T-helper 2 (Th2) response (Vendrame et al., 2004). Importantly, this effect was associated with reduced infarct volume and rescue of behavioral deficit (Vendrame et al., 2004). HUCBC infusion has also shown therapeutic benefit in other neuroinflammatory conditions including multiple sclerosis, amyotrophic lateral sclerosis, neurodegenerative macular 23

PAGE 35

degeneration, and Parkinsons disease (El-Badri et al., 2006; Garbuzova-Davis et al., 2006; Henning et al., 2006). In AD preclinical models, administration of these cells to the PSAPP mouse model of AD was associated with extension of lifespan although high doses were administered in this paradigm (Ghorpade et al., 2001). Based on these lines of evidence, we investigated whether multiple low-dose administration of HUCBC to AD transgenic mouse models could reduce AD-like pathology through suppression of deleterious inflammatory responses involving the CD40 pathway. To address this possibility, we infused both double transgenic PSAPP mice and Tg2576 AD mouse models with HUCBC and then examined cerebral parenchymal and vascular A levels/deposits, astrocytosis, microgliosis, and CD40 pathway-associated molecules. 2.3 Materials and methods 2.3.1 Animals and administration of human umbilical cord cells HUCBC (95-98% mononuclear cells) were provided by Saneron CCEL Therapeutics, Inc. (Tampa, FL). Transgenic PSAPP (APPswe, PSEN1dE9) and Tg2576 mice were obtained from the Jackson Laboratory (Jankowsky et al., 2001; Garcia-Alloza et al., 2006) and Taconic Inc. (Hsiao et al., 1996), respectively, and were intravenously (i.v.) treated with HUCBC (100,000 cells/mouse) or PBS bi-weekly for the first two months and monthly for the remaining four months (n = 10/group, 5/5). Mice were treated starting at 7 months of age (after appreciable A deposits), and blood was 24

PAGE 36

collected by sub-mandibular bleeding at 0, 2, 4 and 6 months to monitor plasma cytokines, sCD40L and A levels throughout the study. We analyzed brains of these mice for A deposits and gliosis at 13 months of age (when these mice manifest well-established AD-like pathology, including A deposits and gliosis). For PSAPP mice deficient in CD40, we treated these mice and controls at 8 weeks of age (preliminary studies showed that we can clearly detect plasma A levels at this age) with HUCBC. Blood samples were collected by sub-mandibular bleeding at the 2 nd month after the treatment. Animals were housed and maintained in the College of Medicine Animal Facility at the University of South Florida (USF), and all experiments were performed in compliance with protocols approved by USF Institutional Animal Care and Use Committee. 2.3.2Immunohistochemistry analysis Mice were anesthetized with isofluorane and transcardially perfused with ice-cold physiological saline containing heparin (10 U/mL). Brains were rapidly isolated and quartered using a mouse brain slicer (Muromachi Kikai, Tokyo, Japan). The first and second anterior quarters were homogenized for Western blot analysis, and the third and fourth posterior quarters were used for microtome or cryostat sectioning (Tan et al., 2002a). Brains were then fixed in 4% paraformaldehyde in PBS at 4C overnight and routinely processed in paraffin in a core facility at the Department of Pathology (USF College of Medicine). Five coronal sections from each brain (5 m thickness) were cut with a 150 m interval [for cingulate cortex (CC) bregma -0.10 mm to -0.82 mm; for 25

PAGE 37

entorhinal cortex (EC) and hippocampus (H), bregma -2.92 mm to -3.64 mm]. Sections were routinely deparaffinized and hydrated in a graded series of ethanol before preblocking for 30 min at ambient temperature with serum-free protein block (Dako Cytomation, Carpinteria, CA). A immunohistochemical staining was performed using anti-human amyloidantibody (4G8) in conjunction with the VectaStain Elite ABC kit (Vector Laboratories, Burlingame, CA) coupled with diaminobenzidine substrate. Congo red staining was done according to standard practice using 10% (w/v) filtered Congo red dye cleared with alkaline alcohol. These sections were rinsed three times for 5 min each in 70% ethanol, hydrated for 5 min in PBS, and mounted in Vectashield fluorescence mounting media (Vector Laboratories). -amyloid plaques positive for 4G8 or Congo red were visualized under bright field using an Olympus BX-51 microscope. A burden was determined by quantitative image analysis. Briefly, images of five 5-m sections (150 m apart) through each anatomic region of interest (hippocampus and neocortex) were captured and a threshold optical density was obtained that discriminated staining form background. Manual editing of each field was used to eliminate artifacts. Data are reported as percentage of immunolabeled area captured (positive pixels divided by total pixels captured). Quantitative image analysis was performed by a single examiner (TM) blinded to sample identities. 2.3.3 Immunofluorescence analysis Double immunofluorescence for A and CD40 was performed using rat anti-mouse CD40 (1:1000; Pharmingen) and rabbit anti-pan A (1:100; Biosource 26

PAGE 38

International, Inc) with overnight incubation followed by incubation at ambient temperature with goat anti-rat IgG FITC (1:50; PharMingen) and donkey anti-rabbit Alexa Fluor 555 (1:500; Invitrogen) for 45 min. Double immunofluorescence for A and activated astrocytes was performed using a biotinylated human amyloidmonoclonal antibody (4G8; 1:100, Signet Laboratories, Dedham, MA) and GFAP polyclonal antibody (1:500, DAKO). Normal rabbit, normal mouse serum (isotype control), or phosphate buffered saline (PBS, 0.1 M, pH 7.4) were used instead of primary antibody or ABC reagent as a negative control. Quantitative image analysis was done based on a previous method (Tan et al., 2002a) with minor modifications. Images were acquired as digitized tagged-image format files to retain maximum resolution using an Olympus BX60 microscope with an attached digital camera system (DP-70, Olympus, Tokyo, Japan), and digital images were routed into a Windows PC for quantitative analyses using SimplePCI software (Compix, Inc. Imaging Systems, Cranberry Township, PA). The cingulate cortex region was captured from the image of the cortex adjacent to the sagittal fissure, and the entorhinal cortex region was captured from the image of the cortex ventral to the rhinal fissure. In images from cingulate and entorhinal regions, the cortical edge was not included in order to capture the full anatomic region of interest. The hippocampal region was captured from between a portion of the CA1 subfield of the pyramidal cell layer and the lacunosum molecular layer. The anatomical locations and boundaries of the regions analyzed were based on those previously defined (Obregon et al., 2006). Images of five 5 m sections through each anatomic region of interest were captured, and a threshold optical density was obtained that discriminated staining from 27

PAGE 39

background. Each anatomic region of interest was manually edited to eliminate artifacts. For burden analyses, data are represented as percentage of immunolabeled area captured (positive pixels) relative to the full area captured (total pixels). 2.3.4 Flow cytometric and Western blot analyses of CD40 expression For flow cytometric analysis of microglial CD40 expression, primary cultured microglial cells were plated in 6-well tissue culture plates at 5 x 10 5 cells/well and incubated with IFN(100 ng/mL) in the presence or absence of serum derived from HUCBCor PBS-infused individual PSAPP mice. Twelve hours after incubation, microglial cells were washed with flow buffer [PBS containing 0.1% (w/v) sodium azide and 2% (v/v) FCS] and re-suspended in 250 l of ice-cold flow buffer for fluorescence activated cell sorting (FACS) analysis, according to methods described previously (Tan et al., 1999d). Briefly, cells were pre-incubated with anti-mouse CD16/CD32 monoclonal antibody (clone 2.4G2, PharMingen) for 10 min at 4C to block non-specific binding to Fc receptors. Cells were then centrifuged at 5,000 x g, washed 3 times with flow buffer, and then incubated in flow buffer with hamster anti-mouse CD40-FITC or isotype control antibody-FITC (1:100 dilution; PharMingen). After 30 min incubation at room temperature, cells were washed twice with flow buffer, re-suspended in 250 L of flow buffer and analyzed by a FACScan TM instrument (Becton Dickinson). A minimum of 10,000 cells were accepted for FACS analysis. Cells were gated based on morphological characteristics using CellQuest TM software (Beckton Dickinson) such that apoptotic and necrotic cells were not accepted for FACS analysis. Percentages of positive (CD4028

PAGE 40

expressing) cells were calculated as follows: for each treatment, the mean fluorescence value for the isotype-matched control antibody was subtracted from the mean fluorescence value for the CD40-specific antibody. For Western immunoblotting analysis of brain CD40 expression, mouse brain homogenates were prepared from HUCBCand PBS-infused PSAPP mice as previously described (Tan et al., 2002a). An aliquot corresponding to 100 g of total protein of each sample was separated by SDS-PAGE and transferred electrophoretically to immunoblotting PVDF membranes. Nonspecific antibody binding was blocked with 5% nonfat dry milk for 1 hr at room temperature in Tris-buffered saline (TBS; 20 mM Tris and 500 mM NaCl, pH 7.5). Subsequently, membranes were first hybridized with rabbit anti-CD40 antibody (1:1,500 dilution; StressGen) for 2 hrs and then washed 3 times in TBS and immunoblotting was by an anti-rabbit HRP-conjugated IgG secondary antibody as a tracer. The luminol reagent was used to develop the blots. To demonstrate equal loading, the same membranes were then stripped with -mercaptoethanol stripping solution (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM -mercaptoethanol) and re-probed with mouse monoclonal antibody to actin. Densitometric analysis was done as previously described (Tan et al., 2002a) using a FluorS Multiimager with Quantity One software (BioRad). 2.3.5 and cytokine ELISAs Mouse brains were isolated under sterile conditions on ice and placed in ice cold lysis buffer as previously described (Tan et al., 2002a). Brains were then sonicated on ice 29

PAGE 41

for approximately 3 minutes, allowed to stand for 15 minutes at 4C, and centrifuged at 15,000 rpm for 15 minutes. This fraction represented the detergent-soluble fraction. Detergent-insoluble A1-40, 42 species were further subjected to acid extraction of brain homogenates in 5 M guanidine buffer (Johnson-Wood et al., 1997), followed by a 1:5 dilution in lysis buffer. A1-40, 42 were detected in brain homogenates prepared with lysis buffer or in plasma samples at a 1:10 or 1:5 dilution, respectively, in dilution buffer (PBS + 1% BSA + PMSF). A1-40, 42 was quantified in these samples using our own A1-40, 42 ELISA kits (Rezai-Zadeh et al., 2005) and further evaluated with commercially available A1-40, 42 ELISA kits (IBL-America) in accordance with the manufacturers instructions, except that standards included 0.5 M guanidine buffer in some cases to facilitate A aggregation. A1-40, 42 were represented as pg/mL of plasma and pg/mg of total protein (mean SD). Cell suspensions of splenocytes from individual mice were prepared as previously described (Town et al., 2002) and passed in 0.5 mL aliquots into 24-well plates at 3 6 /mL. These cells were treated for 48 hrs with concanavalin A (Con A, 5 g/mL). Supernatants were then collected and assayed by IL-10, TNF-, and IL-12(p70) cytokine ELISA kits in strict accordance with the manufacturer's instruction (R&D Systems). The Bio-Rad protein assay (Bio-Rad) was performed to measure total cellular protein from each of the cell groups under consideration just prior to quantification of cytokine release by ELISA, and cytokine secretion is expressed as pg/mg total cellular protein (mean SD). To verify whether stimulation of splenocytes produced any between-groups differences on cell death that might account for altered cytokine profiles, LDH release 30

PAGE 42

assay was carried out as described (Town et al., 2002), and LDH was not detected in any of the wells studied. ELISAs for IgM and IgG antibodies were carried out as previously described (Nikolic et al., 2007). Optical densities were determined by a microplate reader at 450 nm. The ratio of IgM to IgG was calculated using optical density values and then the average ratio for each group was determined (mean SD). Brain tissue-derived (from the detergent-soluble brain homogenate fraction) and serum-derived (plasma) samples were analyzed for sCD40L (Bener MedSystems Burlingame, CA), IL-4, IL-10, IL-2, IFN-, TNF-, IL-1, IL-12 (p70), and TGFcytokines by Bioplex assays (Bio-Rad Laboratories, Hercules, CA) according to the manufacturers protocol. 2.3.6 Microglial phagocytosis assay Primary cultures of murine microglia were established as previously described (Tan et al., 1999a; Townsend et al., 2005). For fluorometic analysis of FITC-A 1-42 primary murine microglia were seeded at 1 x 10 5 cells/well (n = 6 for each condition) in 24-well tissue-culture plates containing 0.5 mL of complete RPMI 1640 medium. These cells were treated for 60 min with aged A 1-42 conjugated with FITC (Biosource International) (Townsend et al., 2005). In the presence of FITC-A 1-42 microglia were then co-treated with serum (1/200, 1/400, 1/800 dilution) derived from HUCBCor PBS-infused individual PSAPP mice in the presence or absence of CD40L protein (2 g/mL). Microglia were rinsed 3 times in A-free complete medium, and media were exchanged with fresh A-free complete medium for 10 min both to allow for removal of non-incorporated A and to promote concentration of the A into phagosomes. Extracellular 31

PAGE 43

and cell associated FITC-A were quantified using an MSF reader (SpectraMax, Molecular Devices) with an emission wavelength of 538 nm and an excitation wavelength of 485 nm. A standard curve from 0 nM to 500 nM of FITC-A was run for each plate. Total cellular proteins were quantified using the Bio-Rad protein assay. The mean fluorescence values for each sample were determined by fluorometic analysis. Relative fold change values were calculated as the mean fluorescence value for each experimental sample over control. In this manner, both extracellular and cell associated FITC-A were quantified. To determine the extent to which cell death might have influenced phagocytic activity in the various treatment groups, we performed LDH release assay, and no significant cell death was detected over the 3 h time-frame in any of the treatment groups (p > 0.05). Primary culture peripheral macrophages were collected from 3-month old wild type mice by infusing their peritoneal cavity with ice-cold PBS following a four day i.p. immunization with 1mL of 3% (w/v) brewers thyoglycollate resuspended in PBS. Cells were pooled following the isolation in order to decrease the variables. Further, they were plated in a culture medium (RPMI-1640; 10% fetal bovine serum and antibiotics) to give 1.5 10 6 cells/well in 6 well plates. Cells were incubated overnight at 37 C under 5% CO 2 in a humidified incubator, and non-adherent cells were removed by washing twice with PBS at 37 C. Following the removal of non-adherent cells, the remaining cells were tested for A phagocytosis as described above with the addition of 1:200, 1:400, and 1:800 dilution of sera derived from HUCBC-treated mice, sera derived from PBS-treated mice, as well as supernatants from cultured HUCBC cells. 32

PAGE 44

2.3.7 Statistical Analysis Data are presented as mean SD. All statistics were calculated using one-way analysis of variance (ANOVA) for multiple comparisons. A p value of < 0.05 was considered significant. The statistical package for the social sciences release 10.0.5 (SPSS Inc., Chicago, Illinois) was used for all data analysis. 2.4 Results 2.4.1 Cerebral parenchymal and vascular -amyloid plaques are reduced in AD transgenic mice peripherally infused with HUCBC Previous work in a mouse model of stroke has shown that HUCBC infusion results in significant reduction in infarct volume as well as rescue of behavioral deficits associated with decreased pro-inflammatory cytokine production (Vendrame et al., 2004). We sought to determine whether HUCBC (95-98% mononuclear cells) infusion could impact A-associated pathology in PSAPP double transgenic mice. These animals were intravenuously (i.v.) injected with HUCBC (100,000 cells/mouse) beginning at 7 months of age (when -amyloid deposits have already accumulated). At 13 months of age, mice were sacrificed and evaluated for changes in AD-like pathology. We chose to administer multiple low doses of HUCBC because, in our pilot studies, we observed that this strategy was superior compared to a single high dose of HUCBC on reducing cerebral amyloidosis in Tg2576 mice (data not shown). HUCBC infusion in PSAPP mice resulted 33

PAGE 45

in marked reduction of cerebral -amyloid pathology as assayed by A antibody (4G8) immunohistochemistry (Figure 1A) and Congo red histochemistry (Figure 1C). Quantitative image analysis revealed statistically significant differences for each brain region examined (P < 0.001) between PSAPP mice infused with HUCBC (PSAPP/HUCBC) and PSAPP mice peripherally infused with PBS (PSAPP/PBS) for both A antibody (Figure 1B) and Congo red staining (Figure 1D). Furthermore, ELISA analysis of brain extracts showed that levels of both detergent-soluble and -insoluble A 1-40 -42 peptides were reduced in PSAPP mice infused with HUCBC (by 62% and 70%, respectively; Figure 1E). A t-test for independent samples revealed significant between-groups differences for each group examined (P < 0.001). Given that peripheral administration of HUCBC reduces cerebral parenchymal A deposits and brain A levels in PSAPP mice, we wished to investigate the impact of HUCBC infusion on cerebral amyloid angiopathy (CAA), which is characterized by A deposits in the cerebral vasculature and is known to occur in 83% of AD patients (Ellis et al., 1996). For this analysis, we used the Tg2576 mouse model of AD, which is known to manifest copious A deposits in cerebral vessels at 15 to 20 months of age (Kaul et al., 2000; Christie et al., 2001; Li et al., 2003; Friedlich et al., 2004; Robbins et al., 2006). We peripherally infused these mice with HUCBC or controls (PBS vehicle treatment or no treatment) (n = 10, 5/5 per group) at 12 months of age using the identical procedure above. Six months thereafter, these mice were sacrificed for analyses of cerebral parenchymal or vascular -amyloid deposits by Congo red histochemistry. As shown in Figure 1F, Tg2576 mice receiving HUCBC treatment demonstrated reduction 34

PAGE 46

of both cerebral parenchymal and vascular Congo red deposits compared with controls. Quantitative image analysis revealed statistically significant differences between Tg2576/HUCBC and Tg2576/PBS or non-treated control groups when examining total (78%), vascular (86%), or parenchymal (74%) Congo red staining (P < 0.001; Figure 1G). No significant difference was revealed between Tg2576/PBS and non-treated Tg2576 control mice (P > 0.05). In addition, we also analyzed cerebral A levels/-amyloid deposits by A ELISA and A antibody immunohistochemistry, and we obtained statistically significant results similar to those observed in HUCBC-infused PSAPP mice (P < 0.001; data not shown). 35

PAGE 47

Figure 1 36

PAGE 48

37

PAGE 49

38

PAGE 50

39

PAGE 51

Figure 1. Cerebral A/-amyloid pathology is reduced in PSAPP and Tg2576 mice peripherally infused with HUCBC. Mouse paraffin-embedded coronal brain sections from the cingulate cortex (CC), hippocampus (H), and entorhinal cortex (EC) were stained with monoclonal human A antibody, (A) 4G8 or (C) Congo red. Percentages (plaque area/total area) of (B) A antibody-immunoreactive deposits or of (D) Congo red-stained deposits were calculated by quantitative image analysis (mean SD; n = 10, 5/5 per group). (E) A ELISA analysis was carried out for both levels of detergent-soluble A 1-40, 42 (top panel) or 5 M guanidine-extracted A 1-40, 42 (bottom panel). Data are represented as mean SD of A 1-40, 42 (pg/mg protein). Mouse paraffin-embedded coronal brain sections from hippocampal regions of Tg2576 mice were stained with (F) Congo red. Positions of the hippocampal subfields CA1, CA3, and dentate gyrus (DG) are indicated in the upper left panel. Arrows indicate A deposit-affected vessels. (G) Percentages (% labeled area) of Congo red-stained plaques/vessels were quantified by image analysis (mean SD; n = 10, 5/5), and percentage reduction is indicated. 40

PAGE 52

2.4.2 Reduced CD40-positive microglia and GFAP-positive astrocytes in PSAPP mice peripherally infused with HUCBC It has previously been suggested that brain inflammation resulting from activated microglia and astrocytes contributes to -amyloid plaque formation (Frackowiak et al., 1992; Potter et al., 2001), and we have previously shown that ligation of microglial CD40 enables activation in response to A peptides (Tan et al., 1999a; Tan et al., 1999b; Tan et al., 1999c). To investigate whether HUCBC could inhibit brain inflammation, we examined co-localization of -amyloid deposits with CD40-positive microglia [an in vivo microgliosis marker (Togo et al., 2000)] or reactive [glial fibrillary acidic protein (GFAP)-positive] astrocytes by immunohistochemistry and Western blot analyses in PSAPP mice. As shown in Figure 2A, CD40-positive microglial cells were reduced in the PSAPP/HUCBC-infused group. Quantitative image analysis revealed statistically significant reductions when comparing PSAPP/HUCBC-infused and PSAPP/PBS-infused groups for both A and CD40 staining in hippocampal dentate gyrus and CA1 regions (**P < 0.001) (Figure 2B). Western blot analysis of CD40 expression showed a statistically significant decrease in brain homogenates from HUCBC-infused PSAPP mice (P < 0.001) (Figure 2C). Furthermore, immunohistochemistry/histochemistry and immunofluorescence analyses showed reductions in -amyloid-associated astrocytosis in PSAPP/HUCBC mice vs. PSAPP/PBS-treated mice (Figure 2D), and morphometry revealed reductions for neocortex and hippocampus by 84% and 86%, respectively in PSAPP/HUCBC mice (P < 0.001) (Figure 2E). 41

PAGE 53

Figure 2 42

PAGE 54

43

PAGE 55

44

PAGE 56

45

PAGE 57

Figure 2. -amyloid associated microgliosis and astrocytosis are reduced in HUCBC infused-PSAPP mice. (A) Immunofluorescence was performed on mouse brain coronal paraffin sections prepared from PSAPP mice infused with HUCBC or PBS. Red signal indicates A positive (top panels); green indicates CD40 positive (middle panels), and merged images (bottom panels) reveal co-localization of CD40 and A DAPI (blue) was used as a nuclear counterstain. (B) Immunofluorescence intensity for A and CD40 was determined. (C) Western blot analysis shows reduced CD40 expression in brain homogenates from PSAPP/HUCBC vs. PSAPP/PBS mice as indicated (actin was used as an internal reference control). Densitometry analysis shows the ratio of CD40 to actin as indicated below the figure. (D) Immunohistochemistry analysis shows GFAP and A double staining (top panel), and immunofluorescence (bottom panel) reveals co-localization of GFAP (red signal) and A (green signal). (E) Morphometric analysis results (mean GFAP/-amyloid double positive plaques per mouse SD) are shown for the neocortex and the hippocampus of PSAPP/HUCBC vs. PSAPP/PBS mice. Percent reduction of plaques double positive for GFAP and A in PSAPP/HUCBC mice is indicated. 46

PAGE 58

2.4.3 Increased plasma A levelscorrelate with decreased CD40-CD40L interaction in HUCBC-infused PSAPP mice We have previously shown that administration of neutralizing CD40L antibody to PSAPP mice results in increased levels of plasma A concomitant with reduced cerebral A/-amyloid pathology, suggesting that depletion of CD40L promotes brain-to-blood clearance of A (Tan et al., 2002a). It is well-known that the CD40-CD40L interaction promotes pro-inflammatory Th1 and opposes anti-inflammatory Th2 immune responses (Grewal and Flavell, 1998; Mackey et al., 1998). In addition, HUCBC treatment has been shown to be an immunoregulator in an animal model of stroke (Vendrame et al., 2004; Newman et al., 2006). We investigated whether reduction of cerebral A levels/-amyloid deposits in HUCBC-infused PSAPP mice might 1) result from increased brain-to-blood clearance of A, and 2) be associated with suppression of the pro-inflammatory CD40-CD40L interaction. We probed individual blood samples from PSAPP mice infused with HUCBC or PBS for A 1-40, 42 and soluble CD40L (sCD40L). ELISA revealed increased plasma A 1-40, 42 levels in PSAPP/HUCBC mice which inversely correlated with decreased levels of plasma sCD40L in these animals (Figures 3A-C). One-way ANOVA followed by post hoc comparison revealed significant differences between PSAPP/HUCBC-infused and PSAPP/PBS-infused mice for plasma A levels and plasma sCD40L levels at each time point indicated (Figures 3A-C) (**P <0.001). It is well established that CD40-CD40L interaction on B cells is required for IgM to IgG antibody class switching. Therefore, we went on to evaluate the functional consequence of HUCBC-mediated suppression of the CD40-CD40L interaction on IgM 47

PAGE 59

and IgG titers in mouse blood samples obtained at the time of sacrifice. ELISA data revealed a significantly increased ratio of IgM to IgG in PSAPP/HUCBC mice when compared to control (Figure 3D, *P < 0.05), suggesting that the CD40 signaling pathway is functionally suppressed in HUCBC-infused PSAPP mice. It has been recently reported that CD40 deficiency in APP transgenic mice confers a decrease in A/-amyloid loads (Laporte et al., 2006). Although to a lesser extent than PSAPP/CD40 +/+ mice, we also found PSAPP/CD40 -/mice do clearly manifest -amyloid deposits (data not shown), allowing us to test whether administration of HUCBC to PSAPP/CD40 -/mice resulted in further amelioration of amyloidosis in these animals. Thus, we treated PSAPP/CD40 -/at 8 weeks of age with HUCBC or PBS (control) and assayed circulating A levels, which correlate with cerebral amyloid levels in transgenic AD mice (DeMattos et al., 2002a). Results indicate no further benefit of HUCBC in PSAPP/CD40 -/mice on enhanced A plasma levels (Figures 3E and F; P > 0.05), a presumed indicator of A brain-to-blood efflux. These data suggest that HUCBC mediate beneficial effects on reduction of amyloidosis via reducing CD40 pathway bioactivity, and are consistent with our previous studies showing that genetic or pharmacologic ablation of CD40-CD40L interaction mitigates AD-like pathology in transgenic mice (Tan et al., 1999a; Tan et al., 2002a). 48

PAGE 60

Figure 3 49 49

PAGE 61

Figure 3. HUCBC infusion results in CD40-dependent increased plasma Alevels in PSAPP mice. ELISA analysis results are shown from blood (plasma) for (A) A 1-40 (B) A 1-42 (C) sCD40L, and (D) IgM/IgG. Data are presented as mean SD (n = 10) for A 1-40 A 1-42 or sCD40L (pg/mL plasma). Arrows below the panels show the time for each peripheral infusion with HUCBC or PBS. (D) data are presented a ratio of IgM to IgG in blood (plasma) from mice at the 6 th month following the treatment. A ELISA analysis for (E) A 1-40 and (F) A 1-42 in blood (plasma) derived from PSAPP/CD40 +/+ or PSAPP/CD40 -/mice at the 2 nd month following the third HUCBC infusion. Data in (E and F), are presented as mean SD (n = 4, 2 /2) of A 1-40 or A 1-42 (pg/mL plasma). 50

PAGE 62

We hypothesized that, if HUCBC mediated reduced amyloidosis by reducing CD40 pathway activity, this should be associated with a shift from proto anti-inflammatory cytokines in HUCBC-infused PSAPP mice. Consistent with this hypothesis, we found that plasma levels of the anti-inflammatory cytokines IL-4 and IL-10 were increased in HUCBC-infused PSAPP mice (Figure 4A, **P < 0.001). Furthermore, primary splenocytes from HUCBC-infused PSAPP mice showed reduced pro-inflammatory TNFand IL-12 (p70) and increased anti-inflammatory IL-10 secretion. We also analyzed brain cytokine levels by ELISA, and results showed statistically significant increases in anti-inflammatory TGFand IL-10 levels in PSAPP/HUCBC-infused mouse brain homogenates (Figures 4B and C; **P < 0.001). Consistent with our data showing reduction in circulating sCD40L after HUCBC treatment, we also measured sCD40L in brain homogenates and found a significant decrease in PSAPP/HUCBC mice compared to control (Figure 4D, **P < 0.001). 51

PAGE 63

Figure 4 52 52

PAGE 64

Figure 4. HUCBC infusion promotes anti-inflammatory/Th2 responses and decreases sCD40L in the CNS. ELISA results are shown for (A) plasma-derived, (B) splenocyte culture-derived, (C) brain tissue derived cytokines, and (D) brain tissue derived sCD40L. Data are presented as mean SD (n = 10) values of cytokines (pg/mL plasma or medium) (D and E), or fold increase of cytokines over control (untreated) mice (C and D). 53

PAGE 65

2.4.4 HUCBC inhibit microglial CD40 expression and enhance in vitro phagocytosis of A peptides We and others have previously shown that microglial CD40 expression is important for CNS inflammatory responses (Tan et al., 1999a; Tan et al., 1999b; Tan et al., 1999c; Togo et al., 2000; Calingasan et al., 2002; Tan et al., 2002a; Tan et al., 2002b), and IFNis a strong inducer of microglial CD40 expression (Carson et al., 1998; Tan et al., 2000; Sokol et al., 2006). In order to investigate whether a soluble factor secreted following HUCBC infusion could modulate microglial expression of CD40, we treated primary microglial cells with serum from HUCBCor PBS-infused individual PSAPP mice in the presence of IFN(100 ng/mL) for 8 hrs. We then examined CD40 expression by FACS analysis. As shown in Figure 5A, sera derived from HUCBC-infused PSAPP mice significantly inhibited IFN--induced microglial CD40 expression compared to controls (P < 0.001). However, this effect was not directly mediated by HUCBC or human adult mononuclear cells (HAMNC), but was rather due to a soluble circulating factor produced by HUCBC-infused PSAPP mice (Figure 5A). We and others have shown that stimulation of microglial CD40 results in impaired A phagocytic activity (Townsend et al., 2005) and promotion of microgial neurotoxic inflammatory responses (Ponomarev et al., 2006). Thus, we wished to examine whether HUCBC could enhance microglial phagocytosis of A peptide. We prepared primary cultures of adult microglia from HUCBCand PBS-infused PSAPP mice according to previously described methods (Lue and Walker, 2002), and then subjected these cells to A phagocytosis assay using native or A antibody-opsonized 54

PAGE 66

fluorescent-tagged A 1-42 (FITC-A 1-42 ) according to our previously described methods (Townsend et al., 2005). As shown in Figure 5B, when measuring FITC-tagged A 1-42 in cell supernatants or lysates, one-way ANOVA followed by post-hoc comparison showed a significant increase in A uptake by microglia derived from HUCBCvs. PBS-infused PSAPP mice (**P < 0.001). Interestingly, the presence of A IgG [2.5 g/mL;(Townsend et al., 2005)] significantly enhanced A uptake by PSAPP/HUCBCvs. PSAPP/PBS-derived microglial cells ( ## P < 0.001). Given that sera from HUCBC-infused PSAPP mice suppressed IFN--induced microglial CD40 expression, we wished to test if the sera could increase microglial A phagocytosis. We incubated primary cultures of neonatal microglia with serum from individual PSAPP/HUCBCvs. PSAPP/PBS mice at 1:200, 1:400, and 1:800 dilutions in the presence of FITC-A 1-42 We found that sera at the 1:200 dilution markedly enhanced microglial phagocytosis of A 1-42 peptide, which was opposed by the presence of recombinant mouse CD40L protein at 2 g/mL (Figure 5C). In addition, we wished to test if sera-derived HUCBC-treated mice could increase peripheral macrophage phagocytic activity. We incubated both sera derived from HUCBC and PBS-treated animals at 1:200, 1:400, and 1:800 dilutions with primary macrophage cells from wild-type mice in six-well tissue-culture plates in the presence of 300 nM FITC-A 1-42 as described above. We found that sera at the 1:200 dilution significantly enhanced macrophage phagocytosis of A 1-42 peptide (Figure 5D) (**P < 0.01 with n = 4 for each mouse group presented). However these effects were not observed in cultured HUCBC media (data not shown). 55

PAGE 67

Figure 5 56

PAGE 68

57

PAGE 69

58

PAGE 70

Figure 5. HUCBC modulate microglial CD40 expression and promote A microglial/macrophage phagocytic activity. (A) FACS analysis for CD40 expression in primary wild-type neonatal microglial cells treated with cultured medium from HUCBC or human adult mononuclear cells (HAMNC), or serum from individual PSAPP/HUCBC or PSAPP/PBS mice following IFNchallenge. Data are presented as percentage of CD40 expressing cells (mean SD; n = 5). (B,C,D) Microglial/macrophage phagocytosis assay results for extracellular and cell-associated FITC-A 1-42 which was detected using a fluorometer. Data are represented as the relative fold of mean ( SD) fluorescence over control for each sample (n = 4 for each condition presented). Primary microglial cells from (B) adult PSAPP/HUCBC or PSAPP/PBS mice, (C) wild-type neonatal microglia, and (D) primary peripheral macrophage. 59

PAGE 71

2.5 Discussion Based on genetic, biochemical, and post-mortem evidence, A peptides are key etiological contributors to AD pathogenesis (Hardy and Selkoe, 2002). In addition to parenchymal A deposits, deposition of A in the cerebral vasculature (known as CAA) is a pathological feature of AD, and occurs with 83% frequency in AD patients (Ellis et al., 1996; Hardy and Selkoe, 2002; Jellinger, 2002; Green et al., 2005). A has been shown to mediate pro-inflammatory and neurodegenerative changes, and oligomeric forms of the peptide are neurotoxic (Malinin et al., 2005). It is well-documented that brain inflammatory mechanisms mediated by reactive glia are activated in response to A plaques (Benzing et al., 1999; Eikelenboom and van Gool, 2004; Rozemuller et al., 2005; Townsend et al., 2005). Expression profiles of two such pro-inflammatory molecules, CD40 and CD40L, are markedly increased in and around A plaques in AD patients and in mouse models of the disease (Togo et al., 2000; Calingasan et al., 2002), and genetic or pharmacologic blockade of CD40-CD40L interaction reduces AD-like pathology in transgenic AD mice (Tan et al., 2002a), suggesting an etiologic role of this receptor/ligand dyad in the disease (Town et al., 2001; Tan et al., 2002b). In a recent clinical report, it was found that circulating sCD40L levels are significantly increased in AD patients (Desideri et al., 2006), suggesting that peripheral as well as brain dysregulation of the CD40 pathway occurs in AD. We have previously shown that CD40 ligation promotes pro-inflammatory activation of microglia and reduces microglial phagocytosis of A peptide in vitro (Tan et al., 1999a; Townsend et al., 2005), supporting a mechanistic explanation for reduced AD-like pathology after blocking the CD4060

PAGE 72

CD40L interaction (Tan et al., 2002b). HUCBC have been shown to down-regulate pro-inflammatory Th1 response in an animal model of stroke (Vendrame et al., 2004), and have also shown therapeutic benefit in other neuroinflammatory/neurodegenerative conditions (El-Badri et al., 2006; Garbuzova-Davis et al., 2006; Henning et al., 2006). Based on this evidence, we sought to examine their putative therapeutic value in mitigating AD-like pathology in transgenic mice. After HUCBC infusion, treated mice exhibited diminished cerebral A/-amyloid pathology and down-regulation of pro-inflammatory responses in the brain and in the periphery. Based on the conspicuous role of CD40-CD40L interaction in mediating brain pro-inflammatory response and exacerbating AD-like pathology, we investigated whether HUCBC-mediated reduction of AD-like pathology might be associated with alteration in this receptor/ligand dyad. Our results show decreased expression of microglial CD40 and reduction in both CNS and peripheral sCD40L concomitant with HUCBC-induced diminished AD-like pathology, raising the possibility that disruption of CD40-CD40L interaction may be responsible for mitigation of AD-like pathology in this scenario. To directly address this hypothesis, we treated PSAPP mice deficient for CD40 with HUCBC and assayed circulating A levels as a marker of brain-to-blood A efflux, and results showed no further benefit of HUCBC in these mice. Here, we demonstrate that infusion of the HUCBC mononuclear fraction into PSAPP and Tg2576 mice results in reduced levels of both soluble and insoluble brain A 1-40, 42 concomitant with increased plasma A 1-40, 42 levels. Past studies have suggested brain-to-blood clearance mechanisms that selectively remove A from the brain, 61

PAGE 73

potentially reducing A levels in normal as well as AD patient brains (Shibata et al., 2000; DeMattos et al., 2002a; DeMattos et al., 2002b; Shiiki et al., 2004; Crossgrove et al., 2005). Experiments in rat models demonstrating clearance of A 1-40 peptide from the brain via the blood-brain-barrier (BBB) support this notion (Shibata et al., 2000; Shiiki et al., 2004; Terasaki and Ohtsuki, 2005). Vascular endothelial cells, which are important BBB constituents, express CD40 (Suo et al., 1998; Tan et al., 1999d; Town et al., 2001; Sokol et al., 2006), and we now show that sCD40L is reduced in blood plasma from HUCBC-treated PSAPP mice, raising the possibility that interruption of CD40-CD40L interaction at the level of cerebrovascular endothelial cells may promote brain-to-blood clearance of A. Further, reduced circulating sCD40L levels in HUCBC-treated PSAPP mice raises the possibility that inflammatory cytokines produced by CD40-CD40L interaction on endothelial cells are reduced, and this idea is consistent with our finding of a shift towards anti-inflammatory cytokines in the CNS after HUCBC infusion. Interestingly, we also demonstrate that CAA [which is present in 83% of AD patients (Ellis et al., 1996)] is reduced by 68% after HUCBC treatment in Tg2576 AD mice. This result shows that reduction in parenchymal A does not come at the cost of increased vascular A deposits, unlike a model in which transforming growth factor-1 overexpression reduces parenchymal plaques but increases vascular A deposits (Wyss-Coray et al., 1997; Wyss-Coray et al., 2001). In vitro HUCBC studies have shown that these cells secrete soluble factors that have beneficial effects (Vendrame et al., 2005; Newman et al., 2006). For example, supernatants from cultured HUCBC promote survival of NT2 neural cells and peripheral 62

PAGE 74

blood mononuclear cells cultured under conditions designed to induce cell stress and limit protein synthesis (El-Badri et al., 2006). Additionally, HUCBC have been shown to produce a number of neurotrophic factors and cytokines that modulate inflammatory responses, including nerve growth factor, colony stimulating factor-1 thrombopoietin and IL-11 (Suen et al., 1994; McGowan et al., 1999; Vendrame et al., 2004). Previous reports have shown that HUCBC entry into the brain is not required to promote neuroprotection (Borlongan et al., 2004), and that recovery following brain injury is mediated through peripheral responses (Townsend et al., 2005). We did not detect infiltration of HUCBCs into brain parenchyma, either at 4 hrs after HUCBC administration or at the time of mouse sacrifice (data not shown), making it unlikely that these cells were directly involved in ameliorating cerebral amyloidosis. Therefore, we hypothesized that a soluble factor produced after HUCBC infusion in the periphery was responsible for reduced AD-like pathology and inflammatory response. To test this, we 1) measured cytokines in blood plasma, spleen, and brains from HUCBCor PBS-treated PSAPP mice, 2) evaluated the impact of sera from these treated mice on IFN--induced microglial CD40 expression, and 3) assayed A phagocytosis in vitro in neonatal microglia treated with sera from HUCBC/PSAPP or PBS/PSAPP mice and in adult microglial cultures derived from these mice. Results generally show a shift from pro-inflammatory Th1-type cytokines towards anti-inflammatory Th2 cytokines in tissues from HUCBC-treated PSAPP mice. Further, sera from HUCBC-treated mice are able to reduce microglial CD40 expression and enhance A phagocytosis by these cells. Finally, adult microglia from HUCBC-treated PSAPP mice have increased capacity to 63

PAGE 75

phagocytose A. When taken together, the above results suggest that, in addition to promoting brain-to-blood A efflux, HUCBC infusion promotes production of a peripheral anti-inflammatory soluble factor that is likely able to cross the BBB and affect microglial A clearance. Previous reports have show that soluble factors, including heat-shock proteins and pro-inflammatory cytokines, are capable of modulating A phagocytosis by microglia (Kakimura et al., 2002; Koenigsknecht-Talboo and Landreth, 2005), and our previous work has shown that microglial CD40-CD40L interaction retards A phagocytosis/clearance (Townsend et al., 2005). Non-saturable BBB transport mechanisms have been described for a number of cytokines including TNF(which is transported via TNF receptors) and IL-1, and other soluble factors such as leukemia inhibitory factor, chemoattractant-1, and epithelial growth factor (Quan and Banks, 2007). Thus, it remains possible that soluble factors produced by the host in response to HUCBC treatment gain access to the brain via the BBB and encounter microglia. Ultimately, we propose that infused HUCBCs exert their effect on reducing cerebral amyloidosis by causing the host to secrete a soluble factor that acts by reducing sCD40L-CD40 interaction on microglia, which then promotes microglial clearance of A. This mechanism is supported by our observations of 1) reduced brain levels of sCD40L in HUCBC-infused PSAPP mice, 2) reduced CD40 expression on microglia cultured in the presence of HUCBC-infused PSAPP mouse sera, 3) increased A phagocytosis/removal by microglia cultured in the presence of HUCBC-infused PSAPP mouse sera or cultured from adult PSAPP/HUCBC mice, and 4) our previous observations that microglial CD40 64

PAGE 76

ligation shifts these cells away from a A phagocytic phenotype and towards a pro-inflammatory response (32). Future studies designed to identify this soluble factor are warranted, and may yield additional pharmacotherapeutic target(s). Additionally, our observation of no further therapeutic benefit of HUCBCs when administered to PSAPP/CD40-/mice establishes a CD40 pathway-dependent mechanism for HUCBC therapeutic benefit on reduction of cerebral amyloidosis. These results dovetail with our previous studies showing that CD40-CD40L interaction mitigates AD-like pathology in transgenic mice (Tan et al., 1999a; Tan et al., 2002a). It was recently shown that peripheral macrophages are able to infiltrate the brain and limit cerebral amyloidosis in AD mice after irradiation, suggesting that hematogenously-derived macrophages are efficient at phagocytosing and clearing A deposits (Simard et al., 2006). However, earlier studies have shown that brain-resident microglia are also able to phagocytose/clear A (Paresce et al., 1996; Paresce et al., 1997; Chung et al., 1999). We did not detect the presence of brain infiltrating macrophages in the current experimental paradigm. Specifically, we stained for CD40 (a marker for both macrophages and microglia), and noted the presence of process-bearing cells that morphologically resembled microglia in and around A plaques (see Figure 2A). Also, we did not observe vascular cuffing that would suggest the presence of infiltrating macrophages that are frequently observed in other CNS inflammatory conditions such as experimental autoimmune encephalomyelitis (Imrich and Harzer, 2001). Furthermore, our results provide evidence that both primary culture microglia and macrophages posses the ability for enhanced A phagocytosis following in vitro stimulation with sera derived 65

PAGE 77

from HUCBC-treated animals. This too is consistent with peripheral immunomodulation of CD40-CD40L interaction by HUCBC treatment. Additionally, given the difficulties inherent to discriminating macrophages from microglia, and the ability of peripheral macrophages to engraft into the CNS and take up a microglial phenotype after brain injury (Priller et al., 2001), it remains possible that peripheral macrophages may contribute to reduced cerebral amyloidosis after HUCBC treatment. In this report, we have shown that HUCBC infusion ameliorates AD-like pathology, including reductions in 1) cerebral Alevels/-amyloid pathology, 2) CAA, and 3) brain inflammation including CD40-positive activated microglia and GFAP-positive activated astrocytes. These effects of HUCBCs were associated with increased brain-to-blood efflux of A and a shift from pro-inflammatory Th1 to anti-inflammatory Th2 cytokines both in the brain and in the periphery, similar to what we observed after A immunization (Town et al., 2002; Town et al., 2005a; Town et al., 2005b). Further, HUCBC infusion of PSAPP mice reduces both CNS and circulating sCD40L levels, and sera from these mice is able to promote microglial A phagocytosis. When taken together, our results provide the basis for a novel immunomodulatory strategy for AD using HUCBC. 66

PAGE 78

2.6 References Bard, F., Cannon, C., Barbour, R., Burke, R. L., Games, D., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Lieberburg, I., Motter, R., Nguyen, M., Soriano, F., Vasquez, N., Weiss, K., Welch, B., Seubert, P., Schenk, D., Yednock, T., 2000. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med. 6, 916-9. Benzing, W. C., Wujek, J. R., Ward, E. K., Shaffer, D., Ashe, K. H., Younkin, S. G., Brunden, K. R., 1999. Evidence for glial-mediated inflammation in aged APP(SW) transgenic mice. Neurobiol Aging. 20, 581-9. Borlongan, C. V., Hadman, M., Sanberg, C. D., Sanberg, P. R., 2004. Central nervous system entry of peripherally injected umbilical cord blood cells is not required for neuroprotection in stroke. Stroke. 35, 2385-9. Calingasan, N. Y., Erdely, H. A., Altar, C. A., 2002. Identification of CD40 ligand in Alzheimer's disease and in animal models of Alzheimer's disease and brain injury. Neurobiol Aging. 23, 31-9. Carson, M. J., Reilly, C. R., Sutcliffe, J. G., Lo, D., 1998. Mature microglia resemble immature antigen-presenting cells. Glia. 22, 72-85. Christie, R., Yamada, M., Moskowitz, M., Hyman, B., 2001. Structural and functional disruption of vascular smooth muscle cells in a transgenic mouse model of amyloid angiopathy. Am J Pathol. 158, 1065-71. Chung, H., Brazil, M. I., Soe, T. T., Maxfield, F. R., 1999. Uptake, degradation, and release of fibrillar and soluble forms of Alzheimer's amyloid beta-peptide by microglial cells. J Biol Chem. 274, 32301-8. Crossgrove, J. S., Li, G. J., Zheng, W., 2005. The choroid plexus removes beta-amyloid from brain cerebrospinal fluid. Exp Biol Med (Maywood). 230, 771-6. DeMattos, R. B., Bales, K. R., Cummins, D. J., Paul, S. M., Holtzman, D. M., 2002a. Brain to plasma amyloid-beta efflux: a measure of brain amyloid burden in a mouse model of Alzheimer's disease. Science. 295, 2264-7. DeMattos, R. B., Bales, K. R., Parsadanian, M., O'Dell, M. A., Foss, E. M., Paul, S. M., Holtzman, D. M., 2002b. Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimer's disease. J Neurochem. 81, 229-36. Desideri, G., Cipollone, F., Necozione, S., Marini, C., Lechiara, M. C., Taglieri, G., Zuliani, G., Fellin, R., Mezzetti, A., di Orio, F., Ferri, C., 2006. Enhanced soluble CD40 ligand and Alzheimer's disease: Evidence of a possible pathogenetic role. Neurobiol 67

PAGE 79

Aging. Eikelenboom, P., van Gool, W. A., 2004. Neuroinflammatory perspectives on the two faces of Alzheimer's disease. J Neural Transm. 111, 281-94. El-Badri, N. S., Hakki, A., Saporta, S., Liang, X., Madhusodanan, S., Willing, A. E., Sanberg, C. D., Sanberg, P. R., 2006. Cord blood mesenchymal stem cells: Potential use in neurological disorders. Stem Cells Dev. 15, 497-506. Ellis, R. J., Olichney, J. M., Thal, L. J., Mirra, S. S., Morris, J. C., Beekly, D., Heyman, A., 1996. Cerebral amyloid angiopathy in the brains of patients with Alzheimer's disease: the CERAD experience, Part XV. Neurology. 46, 1592-6. Frackowiak, J., Wisniewski, H. M., Wegiel, J., Merz, G. S., Iqbal, K., Wang, K. C., 1992. Ultrastructure of the microglia that phagocytose amyloid and the microglia that produce beta-amyloid fibrils. Acta Neuropathol (Berl). 84, 225-33. Friedlich, A. L., Lee, J. Y., van Groen, T., Cherny, R. A., Volitakis, I., Cole, T. B., Palmiter, R. D., Koh, J. Y., Bush, A. I., 2004. Neuronal zinc exchange with the blood vessel wall promotes cerebral amyloid angiopathy in an animal model of Alzheimer's disease. J Neurosci. 24, 3453-9. Garbuzova-Davis, S., Gografe, S. J., Sanberg, C. D., Willing, A. E., Saporta, S., Cameron, D. F., Desjarlais, T., Daily, J., Kuzmin-Nichols, N., Chamizo, W., Klasko, S. K., Sanberg, P. R., 2006. Maternal transplantation of human umbilical cord blood cells provides prenatal therapy in Sanfilippo type B mouse model. Faseb J. 20, 485-7. Garcia-Alloza, M., Robbins, E. M., Zhang-Nunes, S. X., Purcell, S. M., Betensky, R. A., Raju, S., Prada, C., Greenberg, S. M., Bacskai, B. J., Frosch, M. P., 2006. Characterization of amyloid deposition in the APPswe/PS1dE9 mouse model of Alzheimer disease. Neurobiol Dis. 24, 516-24. Ghorpade, A., Persidskaia, R., Suryadevara, R., Che, M., Liu, X. J., Persidsky, Y., Gendelman, H. E., 2001. Mononuclear phagocyte differentiation, activation, and viral infection regulate matrix metalloproteinase expression: implications for human immunodeficiency virus type 1-associated dementia. J Virol. 75, 6572-83. Green, D. A., Masliah, E., Vinters, H. V., Beizai, P., Moore, D. J., Achim, C. L., 2005. Brain deposition of beta-amyloid is a common pathologic feature in HIV positive patients. Aids. 19, 407-11. Grewal, I. S., Flavell, R. A., 1998. CD40 and CD154 in cell-mediated immunity. Annu Rev Immunol. 16, 111-35. Hardy, J., Selkoe, D. J., 2002. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science. 297, 353-6. 68

PAGE 80

Henning, R. J., Burgos, J. D., Ondrovic, L., Sanberg, P., Balis, J., Morgan, M. B., 2006. Human umbilical cord blood progenitor cells are attracted to infarcted myocardium and significantly reduce myocardial infarction size. Cell Transplant. 15, 647-58. Hsiao, K., Chapman, P., Nilsen, S., Eckman, C., Harigaya, Y., Younkin, S., Yang, F., Cole, G., 1996. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science. 274, 99-102. Imrich, H., Harzer, K., 2001. On the role of peripheral macrophages during active experimental allergic encephalomyelitis (EAE). J Neural Transm. 108, 379-95. in t' Veld, B. A., Ruitenberg, A., Hofman, A., Launer, L. J., van Duijn, C. M., Stijnen, T., Breteler, M. M., Stricker, B. H., 2001. Nonsteroidal antiinflammatory drugs and the risk of Alzheimer's disease. N Engl J Med. 345, 1515-21. Jankowsky, J. L., Slunt, H. H., Ratovitski, T., Jenkins, N. A., Copeland, N. G., Borchelt, D. R., 2001. Co-expression of multiple transgenes in mouse CNS: a comparison of strategies. Biomol Eng. 17, 157-65. Jellinger, K. A., 2002. Alzheimer disease and cerebrovascular pathology: an update. J Neural Transm. 109, 813-36. Johnson-Wood, K., Lee, M., Motter, R., Hu, K., Gordon, G., Barbour, R., Khan, K., Gordon, M., Tan, H., Games, D., Lieberburg, I., Schenk, D., Seubert, P., McConlogue, L., 1997. Amyloid precursor protein processing and A beta42 deposition in a transgenic mouse model of Alzheimer disease. Proc Natl Acad Sci U S A. 94, 1550-5. Kakimura, J., Kitamura, Y., Takata, K., Umeki, M., Suzuki, S., Shibagaki, K., Taniguchi, T., Nomura, Y., Gebicke-Haerter, P. J., Smith, M. A., Perry, G., Shimohama, S., 2002. Microglial activation and amyloid-beta clearance induced by exogenous heat-shock proteins. Faseb J. 16, 601-3. Kaul, R., Plummer, F. A., Kimani, J., Dong, T., Kiama, P., Rostron, T., Njagi, E., MacDonald, K. S., Bwayo, J. J., McMichael, A. J., Rowland-Jones, S. L., 2000. HIV-1-specific mucosal CD8+ lymphocyte responses in the cervix of HIV-1-resistant prostitutes in Nairobi. J Immunol. 164, 1602-11. Koenigsknecht-Talboo, J., Landreth, G. E., 2005. Microglial phagocytosis induced by fibrillar beta-amyloid and IgGs are differentially regulated by proinflammatory cytokines. J Neurosci. 25, 8240-9. Laporte, V., Ait-Ghezala, G., Volmar, C. H., Mullan, M., 2006. CD40 deficiency mitigates Alzheimer's disease pathology in transgenic mouse models. J Neuroinflammation. 3, 3. 69

PAGE 81

Li, L., Cao, D., Garber, D. W., Kim, H., Fukuchi, K., 2003. Association of aortic atherosclerosis with cerebral beta-amyloidosis and learning deficits in a mouse model of Alzheimer's disease. Am J Pathol. 163, 2155-64. Lue, L. F., Walker, D. G., 2002. Modeling Alzheimer's disease immune therapy mechanisms: interactions of human postmortem microglia with antibody-opsonized amyloid beta peptide. J Neurosci Res. 70, 599-610. Mackey, M. F., Barth, R. J., Jr., Noelle, R. J., 1998. The role of CD40/CD154 interactions in the priming, differentiation, and effector function of helper and cytotoxic T cells. J Leukoc Biol. 63, 418-28. Malinin, N. L., Wright, S., Seubert, P., Schenk, D., Griswold-Prenner, I., 2005. Amyloid-beta neurotoxicity is mediated by FISH adapter protein and ADAM12 metalloprotease activity. Proc Natl Acad Sci U S A. 102, 3058-63. Matsushima, G. K., Taniike, M., Glimcher, L. H., Grusby, M. J., Frelinger, J. A., Suzuki, K., Ting, J. P., 1994. Absence of MHC class II molecules reduces CNS demyelination, microglial/macrophage infiltration, and twitching in murine globoid cell leukodystrophy. Cell. 78, 645-56. McGowan, E., Sanders, S., Iwatsubo, T., Takeuchi, A., Saido, T., Zehr, C., Yu, X., Uljon, S., Wang, R., Mann, D., Dickson, D., Duff, K., 1999. Amyloid phenotype characterization of transgenic mice overexpressing both mutant amyloid precursor protein and mutant presenilin 1 transgenes. Neurobiol Dis. 6, 231-44. Newman, M. B., Willing, A. E., Manresa, J. J., Sanberg, C. D., Sanberg, P. R., 2006. Cytokines produced by cultured human umbilical cord blood (HUCB) cells: implications for brain repair. Exp Neurol. 199, 201-8. Nicoll, J. A., Wilkinson, D., Holmes, C., Steart, P., Markham, H., Weller, R. O., 2003. Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat Med. 9, 448-52. Nikolic, W. V., Bai, Y., Obregon, D., Hou, H., Mori, T., Zeng, J., Ehrhart, J., Shytle, R. D., Giunta, B., Morgan, D., Town, T., Tan, J., 2007. Transcutaneous beta-amyloid immunization reduces cerebral beta-amyloid deposits without T cell infiltration and microhemorrhage. Proc Natl Acad Sci U S A. 104, 2507-12. Obregon, D. F., Rezai-Zadeh, K., Bai, Y., Sun, N., Hou, H., Ehrhart, J., Zeng, J., Mori, T., Arendash, G. W., Shytle, D., Town, T., Tan, J., 2006. ADAM10 activation is required for green tea (-)-epigallocatechin-3-gallate-induced alpha-secretase cleavage of amyloid precursor protein. J Biol Chem. 281, 16419-27. Paresce, D. M., Ghosh, R. N., Maxfield, F. R., 1996. Microglial cells internalize aggregates of the Alzheimer's disease amyloid beta-protein via a scavenger receptor. 70

PAGE 82

Neuron. 17, 553-65. Paresce, D. M., Chung, H., Maxfield, F. R., 1997. Slow degradation of aggregates of the Alzheimer's disease amyloid beta-protein by microglial cells. J Biol Chem. 272, 29390-7. Ponomarev, E. D., Shriver, L. P., Dittel, B. N., 2006. CD40 expression by microglial cells is required for their completion of a two-step activation process during central nervous system autoimmune inflammation. J Immunol. 176, 1402-10. Potter, H., Wefes, I. M., Nilsson, L. N., 2001. The inflammation-induced pathological chaperones ACT and apo-E are necessary catalysts of Alzheimer amyloid formation. Neurobiol Aging. 22, 923-30. Priller, J., Flugel, A., Wehner, T., Boentert, M., Haas, C. A., Prinz, M., Fernandez-Klett, F., Prass, K., Bechmann, I., de Boer, B. A., Frotscher, M., Kreutzberg, G. W., Persons, D. A., Dirnagl, U., 2001. Targeting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment. Nat Med. 7, 1356-61. Quan, N., Banks, W. A., 2007. Brain-immune communication pathways. Brain Behav Immun. 21, 727-35. Rezai-Zadeh, K., Shytle, D., Sun, N., Mori, T., Hou, H., Jeanniton, D., Ehrhart, J., Townsend, K., Zeng, J., Morgan, D., Hardy, J., Town, T., Tan, J., 2005. Green tea epigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and reduces cerebral amyloidosis in Alzheimer transgenic mice. J Neurosci. 25, 8807-14. Robbins, E. M., Betensky, R. A., Domnitz, S. B., Purcell, S. M., Garcia-Alloza, M., Greenberg, C., Rebeck, G. W., Hyman, B. T., Greenberg, S. M., Frosch, M. P., Bacskai, B. J., 2006. Kinetics of cerebral amyloid angiopathy progression in a transgenic mouse model of Alzheimer disease. J Neurosci. 26, 365-71. Rozemuller, A. J., van Gool, W. A., Eikelenboom, P., 2005. The neuroinflammatory response in plaques and amyloid angiopathy in Alzheimer's disease: therapeutic implications. Curr Drug Targets CNS Neurol Disord. 4, 223-33. Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Liao, Z., Lieberburg, I., Motter, R., Mutter, L., Soriano, F., Shopp, G., Vasquez, N., Vandevert, C., Walker, S., Wogulis, M., Yednock, T., Games, D., Seubert, P., 1999. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature. 400, 173-7. Shibata, M., Yamada, S., Kumar, S. R., Calero, M., Bading, J., Frangione, B., Holtzman, D. M., Miller, C. A., Strickland, D. K., Ghiso, J., Zlokovic, B. V., 2000. Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at 71

PAGE 83

the blood-brain barrier. J Clin Invest. 106, 1489-99. Shiiki, T., Ohtsuki, S., Kurihara, A., Naganuma, H., Nishimura, K., Tachikawa, M., Hosoya, K., Terasaki, T., 2004. Brain insulin impairs amyloid-beta(1-40) clearance from the brain. J Neurosci. 24, 9632-7. Simard, A. R., Soulet, D., Gowing, G., Julien, J. P., Rivest, S., 2006. Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron. 49, 489-502. Sokol, D. K., Chen, D., Farlow, M. R., Dunn, D. W., Maloney, B., Zimmer, J. A., Lahiri, D. K., 2006. High levels of Alzheimer beta-amyloid precursor protein (APP) in children with severely autistic behavior and aggression. J Child Neurol. 21, 444-9. Suen, Y., Lee, S. M., Schreurs, J., Knoppel, E., Cairo, M. S., 1994. Decreased macrophage colony-stimulating factor mRNA expression from activated cord versus adult mononuclear cells: altered posttranscriptional stability. Blood. 84, 4269-77. Suo, Z., Tan, J., Placzek, A., Crawford, F., Fang, C., Mullan, M., 1998. Alzheimer's beta-amyloid peptides induce inflammatory cascade in human vascular cells: the roles of cytokines and CD40. Brain Res. 807, 110-7. Szekely, C. A., Thorne, J. E., Zandi, P. P., Ek, M., Messias, E., Breitner, J. C., Goodman, S. N., 2004. Nonsteroidal anti-inflammatory drugs for the prevention of Alzheimer's disease: a systematic review. Neuroepidemiology. 23, 159-69. Tan, J., Town, T., Paris, D., Mori, T., Suo, Z., Crawford, F., Mattson, M. P., Flavell, R. A., Mullan, M., 1999a. Microglial activation resulting from CD40-CD40L interaction after beta-amyloid stimulation. Science. 286, 2352-5. Tan, J., Town, T., Paris, D., Placzek, A., Parker, T., Crawford, F., Yu, H., Humphrey, J., Mullan, M., 1999b. Activation of microglial cells by the CD40 pathway: relevance to multiple sclerosis. J Neuroimmunol. 97, 77-85. Tan, J., Town, T., Saxe, M., Paris, D., Wu, Y., Mullan, M., 1999c. Ligation of microglial CD40 results in p44/42 mitogen-activated protein kinase-dependent TNF-alpha production that is opposed by TGF-beta 1 and IL-10. J Immunol. 163, 6614-21. Tan, J., Town, T., Suo, Z., Wu, Y., Song, S., Kundtz, A., Kroeger, J., Humphrey, J., Crawford, F., Mullan, M., 1999d. Induction of CD40 on human endothelial cells by Alzheimer's beta-amyloid peptides. Brain Res Bull. 50, 143-8. Tan, J., Town, T., Mullan, M., 2000. CD45 inhibits CD40L-induced microglial activation via negative regulation of the Src/p44/42 MAPK pathway. J Biol Chem. 275, 37224-31. Tan, J., Town, T., Crawford, F., Mori, T., DelleDonne, A., Crescentini, R., Obregon, D., 72

PAGE 84

Flavell, R. A., Mullan, M. J., 2002a. Role of CD40 ligand in amyloidosis in transgenic Alzheimer's mice. Nat Neurosci. 5, 1288-93. Tan, J., Town, T., Mullan, M., 2002b. CD40-CD40L interaction in Alzheimer's disease. Curr Opin Pharmacol. 2, 445-51. Terasaki, T., Ohtsuki, S., 2005. Brain-to-blood transporters for endogenous substrates and xenobiotics at the blood-brain barrier: an overview of biology and methodology. NeuroRx. 2, 63-72. Todd Roach, J., Volmar, C. H., Dwivedi, S., Town, T., Crescentini, R., Crawford, F., Tan, J., Mullan, M., 2004. Behavioral effects of CD40-CD40L pathway disruption in aged PSAPP mice. Brain Res. 1015, 161-8. Togo, T., Akiyama, H., Kondo, H., Ikeda, K., Kato, M., Iseki, E., Kosaka, K., 2000. Expression of CD40 in the brain of Alzheimer's disease and other neurological diseases. Brain Res. 885, 117-21. Town, T., Tan, J., Mullan, M., 2001. CD40 signaling and Alzheimer's disease pathogenesis. Neurochem Int. 39, 371-80. Town, T., Vendrame, M., Patel, A., Poetter, D., DelleDonne, A., Mori, T., Smeed, R., Crawford, F., Klein, T., Tan, J., Mullan, M., 2002. Reduced Th1 and enhanced Th2 immunity after immunization with Alzheimer's beta-amyloid(1-42). J Neuroimmunol. 132, 49-59. Town, T., Nikolic, V., Tan, J., 2005a. The microglial "activation" continuum: from innate to adaptive responses. J Neuroinflammation. 2, 24. Town, T., Tan, J., Flavell, R. A., Mullan, M., 2005b. T-cells in Alzheimer's disease. Neuromolecular Med. 7, 255-64. Townsend, K. P., Town, T., Mori, T., Lue, L. F., Shytle, D., Sanberg, P. R., Morgan, D., Fernandez, F., Flavell, R. A., Tan, J., 2005. CD40 signaling regulates innate and adaptive activation of microglia in response to amyloid beta-peptide. Eur J Immunol. 35, 901-10. Vendrame, M., Cassady, J., Newcomb, J., Butler, T., Pennypacker, K. R., Zigova, T., Sanberg, C. D., Sanberg, P. R., Willing, A. E., 2004. Infusion of human umbilical cord blood cells in a rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct volume. Stroke. 35, 2390-5. Vendrame, M., Gemma, C., de Mesquita, D., Collier, L., Bickford, P. C., Sanberg, C. D., Sanberg, P. R., Pennypacker, K. R., Willing, A. E., 2005. Anti-inflammatory effects of human cord blood cells in a rat model of stroke. Stem Cells Dev. 14, 595-604. Wyss-Coray, T., Borrow, P., Brooker, M. J., Mucke, L., 1997. Astroglial overproduction 73

PAGE 85

of TGF-beta 1 enhances inflammatory central nervous system disease in transgenic mice. J Neuroimmunol. 77, 45-50. Wyss-Coray, T., Lin, C., Yan, F., Yu, G. Q., Rohde, M., McConlogue, L., Masliah, E., Mucke, L., 2001. TGF-beta1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice. Nat Med. 7, 612-8. 74

PAGE 86

CHAPTER THREE CD40L DISRUPTION ENHANCES A VACCINE-MEDIATED REDUCTION OF CEREBRAL AMYLOIDOSIS WHILE MINIMIZING CEREBRAL AMYLOID ANGIOPATHY AND INFLAMMATION D. Obregon 1 H. Hou 1 ,Y. Bai 1 W.V. Nikolic 1 T. Mori 1,3 Deyan Luo 1 J. Zeng 1 J. Ehrhart 1 F. Fernandez 1 D. Morgan 2 B. Giunta 1 T. Town 1,4 and J. Tan 1,2,3 1 Neuroimmunology Laboratory, Institute for Research in Psychiatry, Department of Psychiatry and Behavioral Medicine; 2 Department of Molecular Pharmacology and Physiology, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, Florida 33613 3 Institute of Medical Science, Saitama Medical School, 1981 Kamoda, Kawagoe, Saitama 350-8550, Japan 4 Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, New Haven, Connecticut 06520-8011 75

PAGE 87

3.1 Abstract Amyloid(A immunization efficiently reduces amyloid plaque load and memory impairment in transgenic mouse models of Alzheimers disease (AD) (Schenk et al., 1999; Morgan et al., 2000). Active Aimmunization has also yielded favorable results in a subset of AD patients (Hock et al., 2003). However, a small percentage of patients developed severe aseptic meningoencephalitis associated with brain inflammation and infiltration of T-cells (Nicoll et al., 2003; Orgogozo et al., 2003). We and others have shown that blocking the CD40-CD40 ligand (L) interaction mitigates A-induced inflammatory responses and enhances Aclearance (Tan et al., 2002b; Townsend et al., 2005). Here, we utilized genetic and pharmacologic approaches to test whether CD40-CD40L blockade could enhance the efficacy of A 1-42 immunization, while limiting potentially damaging inflammatory responses. We show that genetic or pharmacologic interruption of CD40-CD40L interaction enhanced A 1-42 immunization efficacy to reduce cerebral amyloidosis in the PSAPP and Tg2576 mouse models of AD. Potentially deleterious pro-inflammatory immune responses, cerebral amyloid angiopathy (CAA) and cerebral microhemorrhage were reduced or absent in these combined approaches. Pharmacologic blockade of CD40L decreased T-cell neurotoxicity to A-producing neurons. Further reduction of cerebral amyloidosis in A-immunized PSAPP mice completely deficient for CD40 occurred in the absence of A immunoglobulin G (IgG) antibodies or efflux of A from brain to blood, but was rather correlated with anti-inflammatory cytokine profiles and reduced plasma soluble CD40L. These results suggest CD40-CD40L blockade promotes anti-inflammatory cellular immune responses, 76

PAGE 88

likely resulting in promotion of microglial phagocytic activity and A clearance while precluding generation of neurotoxic A-reactive T-cells. Thus, combined approaches of A immunotherapy and CD40-CD40L blockade may provide for safer and more effective Avaccine. 3.2 Introduction Amyloid(A), a proteolytic product of amyloid precursor protein (APP), is a key molecule in the pathogenesis and progression of Alzheimers disease (AD) (Blennow et al., 2006). Overproduction of soluble and aggregated A species drives cerebral amyloidosis including -amyloid plaque formation, a hallmark pathological feature of AD. Thus, methods developed to clear or prevent formation of A in the brains of AD patients represent a possible treatment modality. One promising approach involves utilization of active A immunization strategies, which produce dramatic reductions in A pathology in animal studies (Schenk et al., 1999). However, a phase IIa clinical trial was abandoned after about 6% of A-immunized AD patients developed aseptic meningoencephalitis (Nicoll et al., 2003; Orgogozo et al., 2003) that appeared to involve brain inflammatory reactions mediated by T-cells and microglia (Monsonego et al., 2001; Schenk and Yednock, 2002; Greenberg et al., 2003; Monsonego et al., 2003). Interestingly, a 12 month post-vaccination period analysis revealed an inverse correlation between titers of amyloid plaque-reactive antibodies and rate of cognitive decline (Hock et al., 2003), suggesting clinical efficacy. Despite the discontinuation of the clinical trials, A vaccination studies have continued in effort to identify an immunization 77

PAGE 89

approach that is both safe and effective. Current approaches have focused on minimizing T-cell mediated inflammatory responses in efforts to prevent CNS invasion of auto-aggressive T-cells, while promoting A antibody-mediated clearance mechanisms (Chackerian et al., 2006; Maier et al., 2006; Okura et al., 2006; Nikolic et al., 2007). A possible avenue to both enhance A clearance and down-regulate CNS inflammatory responses (including invasion of reactive T-cells) involves modulation of the CD40 receptor (CD40)-CD40 ligand (CD40L) system. CD40 is a ~ 45-50 kDa cell surface molecule, which is a member of the tumor necrosis factor-TNF-/nerve growth factor (NGF) receptor super-family. In the periphery, a variety of innate immune cells known as antigen-presenting cells (APCs) express CD40, including dendritic cells, B-cells, and monocytes/macrophages. In the CNS, CD40 is expressed by resident cells including microglia, neurons, and astrocytes, as well as by peripherally-derived APCs (Tan et al., 2002a; Town et al., 2005). CD40L (also known as CD154), is expressed as a membrane-anchored molecule by activated T-cells and astrocytes, and can also be secreted as a smaller soluble protein (van Kooten and Banchereau, 2000). The CD40-CD40L interaction acts as an accessory co-stimulatory pathway involved in key immune cell processes including: activation, maturation/differentiation, growth/proliferation, and regulation of apoptosis (Town et al., 2001a). We have previously shown that CD40 ligation is a molecular trigger for pro-inflammatory microglial activation in response to A peptides (Tan et al., 1999). Further, genetic or pharmacologic blockade of the CD40-CD40L interaction reduces -amyloid pathology in the brains of transgenic mouse models of AD (Tan et al., 1999; Tan 78

PAGE 90

et al., 2002a). Increased CD40 and CD40L immunoreactivity has been found in and around -amyloid plaques in AD brain (Togo et al., 2000; Calingasan et al., 2002), further suggesting that CD40-CD40L interaction may contribute to A and -amyloid plaque pathology. Recently, the CD40-CD40L interaction was determined to play a central role in promoting and maintaining dendritic cell APC phenotype during infections (Straw et al., 2003). In the context of CNS immunity, the CD40-CD40L interaction is required for microglial maturation into functional APCs (Ponomarev et al., 2006). We have recently shown that CD40L treatment of primary cultures of microglia inhibits phagocytosis of A antibody opsonized, as well as non-opsonized A species (Townsend et al., 2005). Associated with reduced A phagocytic capacity, CD40L treatment up-regulated cell surface markers indicative of an APC phenotype including CD45, CD86, MHC II, and promoted the release of pro-inflammatory molecules including interleukin (IL)-1 and TNF-(Townsend et al., 2005). Thus, CD40-CD40L interaction may act as a molecular switch necessary to drive pro-inflammatory microglial APC phenotype maturation at the cost of reducing phagocytosis (Townsend et al., 2005; Ponomarev et al., 2006). Additionally, blockade of the CD40-CD40L system down-regulates T-cell/microglia-mediated injury in the context of experimental autoimmune encephalitis (EAE) (Howard et al., 1999; Howard et al., 2002). Altogether, these studies suggest that blockade of the CD40-CD40L interaction could enhance A vaccination-mediated A clearance mechanisms, while minimizing pro-inflammatory T-cell-mediated damage in the CNS. To investigate this hypothesis, we studied transgenic PSAPP mice overexpressing 79

PAGE 91

mutant human presenilin-1 (DeltaE9), and Swedish mutant human APP (APP Swe ), which develop AD-like pathology (Jankowsky et al., 2001). We took a genetic approach to CD40 blockade by crossing these mice with CD40 -/mice to yield: CD40 wild-type (PSAPP), CD40 heterozygous deficient (PSAPP/CD40 +/), and CD40 homozygous deficient (PSAPP/CD40 -/) animals. We then vaccinated these mice over a course of 4 months utilizing aggregated A 1-42 peptide or vehicle. We also took a pharmacologic approach by administering CD40L neutralizing antibody to A 1-42 -vaccinated PSAPP mice. Results from both strategies showed enhanced reduction of cerebral amyloidosis as evidenced by reductions in A load, -amyloid plaque burden, and cerebral amyloid angiopathy (CAA). Moreover, we report reduced cerebral microhemorrhage and inflammatory immune responses as measured by cytokine analysis and T-cell induced neurotoxicity to Aproducing neurons. These results were associated with inhibition of microglial APC phenotype. Interestingly, homozygous CD40 deficient A 1-42 -immunized PSAPP mice displayed reduced cerebral amyloidosis in the absence of immunoglobulin G (IgG) antibodies or efflux of A from brain to blood. These effects were correlated with reduced plasma CD40 ligand (CD40L) and increased anti-inflammatory cytokine levels. Altogether these data suggest that disruption of the CD40-CD40L interaction enhances A 1-42 -immunization mediated A clearance mechanisms by promoting anti-inflammatory cellular immunity to support microglial clearance of A. 3.3 Materials and methods 80

PAGE 92

3.3.1 Reagents Anti-human Amonoclonal antibody (4G8) was purchased from Signet Laboratories. A 1-42 peptide was obtained from Biosource International (Camarillo, CA). A 1-42 peptide was added to 0.9% saline (4 mg/mL), vortexed, and incubated for 24 h at 37 C. This solution was aliquoted, frozen and stored at 80 C. Immediately prior to use, A 1-42 aliquots were thawed and then mixed with adjuvant or PBS at 1:1 (v/v). Complete and incomplete Freund's adjuvant were purchased from Sigma. DuoSet TM enzyme-linked ELISA kits (including TNF-, transforming growth factor-1 (TGF-1), IL-1, and IL-10) were obtained from R&D Systems (Minneapolis, MN). Purified rat anti-mouse MHC class II antibody was obtained from PharMingen (San Diego, CA). Congo red and concanavalin A (Con A) were obtained from Sigma. A 1-40 and A 1-42 ELISA kits were purchased from IBL-America (Minneapolis, MN). Murine IgG and HRP-conjugated goat anti-mouse IgG were obtained from Pierce Biotechnology, Inc. (Rockford, IL). Goat anti-mouse IgM peroxidase conjugate antibody (A8786) was obtained from Sigma. 3.3.2 Mice Wild-type C57BL/6, PSAPP mice (APP Swe PSEN1dE9) and CD40 deficient (CD40 -/) mice were all obtained from Jackson Laboratories (Bar Harbor, ME). We crossed CD40 -/mice with PSAPP mice and characterized offspring by polymerase chain reaction-based genotyping for the mutant APP construct and mutant presenilin1 (PS1) gene (to examine PSAPP status) and neomycin selection vector (to type for CD40 81

PAGE 93

deficiency), followed by Western blot for brain APP and splenic CD40 protein, respectively. Animals were housed and maintained under specific pathogen-free conditions in the College of Medicine Animal Facility at the University of South Florida, and all experiments were in compliance with protocols approved by the University of South Florida Institutional Animal Care and Use Committee. The animals that we studied were PSAPP/CD40 +/+ PSAPP/CD40 +/, PSAPP/CD40 -/, CD40 -/, and their littermates. All of the mice included did not develop infections or neoplasms during the duration of this study. 3.3.3 Immunization strategies For our genetic approach to CD40-CD40L blockade, we crossed PSAPP and CD40 -/mice and, at 8 months of age, divided them into groups consisting of A 1-42 or vehicle (PBS)-vaccinated CD40 -/and wild-type mice (n = 8 for each group, 4/4), or PSAPP/CD40 +/+ (PSAPP/CD40 +/+ /A 1-42 PSAPP/CD40 +/+ /PBS), PSAPP/CD40 +/(PSAPP/CD40 +//A 1-42 PSAPP/CD40 +//PBS), or PSAPP/CD40 -/(PSAPP/CD40 -//A 1-42 PSAPP/CD40 -//PBS) mice (n = 16 for each group, 8/8). Immunization of these mice was performed at regular time intervals in a similar fashion to the methods described by Schenk et al. (Schenk et al., 1999). Briefly, 8 month-old mice were injected with A 1-42 (100 g/mouse) or PBS emulsified in monophosphoryl lipid A (detoxified endotoxin) from S. minnesota (MPL) and synthetic trehalose dicorynomycolate (TDM) biweekly until 9 months of age, and monthly injection with A 1-42 or PBS alone was performed thereafter. 82

PAGE 94

For our pharmacologic approach to CD40-CD40L blockade, we studied 8 month-old PSAPP mice divided into five groups (n = 16 for each group, 8/8) as follows: PBS-treated A 1-42 immunized PSAPP mice (PSAPP/A 1-42 /PBS), CD40L antibody-treated A 1-42 immunized PSAPP mice (PSAPP/A 1-42 /CD40L antibody), Isotype control IgG-treated A 1-42 immunized PSAPP mice (PSAPP/A 1-42 /IgG antibody), or CD40L antibody-treated nonA 1-42 immunized PSAPP mice (PSAPP/CD40L antibody). We immunized these mice with A 1-42 as described above and treated them with CD40L antibody (200 g/mouse) based on our previous report (Tan et al., 2002a). For all mice, blood samples were collected from the sub-mandibular vein just before immunization and then on a monthly basis thereafter 1-2 days prior to the succeeding monthly injection (except the final collection, which was taken one month after the final injection) throughout the course of immunization, and mice were sacrificed at 12 months of age. 3.3.4 Measurement of plasma IgG and IgM A antibodies by ELISA A antibodies in individual mouse plasma and brain homogenates were measured in duplicate according to previously described methods (Maier et al., 2005). Briefly, human A 1-42 peptide was coated at 1 g/mL in 50 mM carbonate buffer, pH 9.6 (coating buffer) on 96-well immunoassay plates overnight at 4 C. The plates were washed with 0.05% Tween 20 in PBS (washing buffer) five times and blocked with blocking buffer (PBS with 1% BSA, 5% horse serum) for 2 hrs at room temperature. Murine IgG or IgM was serially diluted in coating buffer (1,000-0 g/mL) to generate a standard curve. Mouse plasma and brain homogenate samples were diluted in blocking buffer at 83

PAGE 95

concentrations ranging from 1:400 to 1:102,400, added to the plates, and incubated for 2 hrs at room temperature. After 3 washes with washing buffer, a detection antibody (HRP-conjugated goat anti-mouse IgG, or HRP-conjugated goat anti-mouse IgM was diluted at 1:4,000), added to the plates and incubated for 1 hr at 37 C. Following 4 washes, tetramethylbenzidine substrate was added to the plates and incubated for 15 min at room temperature. Fifty L of stop solution (2 N H 2 SO 4 ) was added to each well of the plates. The optical density of each well was immediately determined by a microplate reader at 450 nm. A antibody data are reported as g per mL of plasma (mean SD). 3.3.5 Measurement of A species from plasma and brain homogenates by ELISA Mouse brains were isolated under sterile conditions on ice and placed in ice-cold lysis buffer (containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1mM EDTA, 1 mM EGTA, 1% v/v Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM -glycerolphosphate, 1 mM Na 3 VO 4 1 g/mL leupeptin, 1 mM PMSF) as previously described (Rezai-Zadeh et al., 2005). Brains were then sonicated on ice for approximately 3 min, allowed to stand for 15 min at 4C, and centrifuged at 15,000 rpm for 15 min. A 1-40 and A 1-42 species were detected by a 2-step extraction protocol, similar to previously published methods (Johnson-Wood et al., 1997; Rezai-Zadeh et al., 2005). Detergent-soluble A 1-40 and A 1-42 were directly detected in plasma and brain homogenates prepared with lysis buffer described above by a 1:4 or 1:10 dilution, respectively. Total A 1-40 and A 1-42 species were detected by acid extraction of brain homogenates in 5 M guanidine buffer, followed by a 1:10 dilution in lysis buffer. A 1-40 and A 1-42 were quantified in individual 84

PAGE 96

samples in duplicate using A 1-40 and A 1-42 ELISA kits in accordance with the manufacturers instructions (IBL-America), except that standards included 0.5 M guanidine buffer in some cases. A 1-40 and A 1-42 are represented as pg per mL of plasma or pg per mg of total protein (mean SD). 3.3.6 Brain and plasma cytokine analysis Enzyme-linked immunoabsorbance assay (ELISA) for detection of IL-1, IL-10, TGF-1, or TNFwas carried out for measurement of cytokines in mouse blood plasma, or brain. Tissues were obtained at the time of sacrifice, and were diluted in PBS and assayed using the kits described above in strict accordance with the manufacturer's instruction (R&D Systems). The Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) was performed to measure total cellular protein from each sample prior to quantification of cytokine release by ELISA, and cytokine secretion is expressed in pg/mg total protein. 3.3.7 Histology Mice were anesthetized with isofluorane and transcardially perfused with ice-cold physiological saline containing heparin (10 U/ml). Brains were rapidly isolated and quartered using a mouse brain slicer (Muromachi Kikai, Tokyo, Japan). The first and second anterior quarters were homogenized for Western blot analysis, and the third and fourth posterior quarters were used for microtome or cryostat sectioning as previously described (Tan et al., 2002a). Brains were then fixed in 4% paraformaldehyde in 0.9% 85

PAGE 97

saline at 4C overnight and routinely processed in paraffin in a core facility at the Department of Pathology (University of South Florida College of Medicine). Five coronal sections from each brain (5 m thickness) were cut with a 150 m interval. Paraffin sections were routinely deparaffinized and hydrated in a graded series of ethanol. All sections were pre-blockading for 30 min at ambient temperature with serum-free protein block (Dako Cytomation, Carpinteria, CA). A immunohistochemical staining was performed using anti-human amyloidantibody (clone 4G8; 1:100; Signet Laboratories) in conjunction with the VectaStain Elite ABC kit (Vector Laboratories, Burlingame, CA) coupled with the diaminobenzidine substrate. For microglia/macrophage immunostaining (MHC II, Iba, and CD45), sections were prepared as described above. Following pre-blocking, sections were treated overnight with anti-mouse MHC II (1:500) Iba (1:100) or CD45 (1:500) antibodies diluted in PBS (obtained from Santa Cruz (O'Keefe et al., 2002)), incubated with HRP-conjugated anti-mouse IgG, and developed. For congo red histochemistry, sections were routinely deparaffinized and rinsed in 70% (v/v) ethanol before staining with fresh-filtered 1% (w/v) congo red diluted in 70% ethanol for 5 min. These sections were rinsed three times for 5 min each in 70% ethanol, hydrated for 5 min in 0.9% saline, and mounted. -amyloid plaques and reactive microglia were visualized using an Olympus BX-51 microscope (Olympus, Tokyo, Japan). Quantitative image analysis was performed for 4G8 immunohistochemistry and congo red histochemistry. Images were obtained using an Olympus BX-51 microscope and digitized using an attached MagnaFire imaging system (Olympus, Tokyo, Japan). 86

PAGE 98

Briefly, images of five 5-m sections (150 m apart) through each anatomic region of interest (hippocampus or cortical areas) were captured and a threshold optical density was obtained that discriminated staining form background. Manual editing of each field was used to eliminate artifacts. Data are reported as percentage of immunolabeled area captured (positive pixels) divided by the full area captured (total pixels). Quantitative image analysis was performed by a single examiner (TM) blinded to sample identities. 3.3.8 Splenocyte cultures Cell suspensions of splenocytes from individual mice were prepared as previously described (Town et al., 2001b; Town et al., 2002) and passed in 0.5 mL aliquots into 24-well plates at 3 10 6 cells/mL. These cells were cultured for 48 h in the presence or absence of Con A (5 g/mL), or A 1-42 (20 g/mL). Supernatants were then collected and assayed by IFN-, IL-2, and IL-4 cytokine ELISA kits in strict accordance with the manufacturer's instruction (R&D Systems). The Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) was performed to measure total cellular protein from each well prior to quantification of cytokine release by ELISA, and cytokine secretion is expressed in pg/mg total cellular protein (mean SD). To verify whether stimulation of splenocytes produced any between-groups differences on cell death that might account for altered cytokine profiles, LDH release assay was carried out as described (Townsend et al., 2005) and LDH was not detected in any of the wells studied. 3.3.9 A-specific lymphocyte neurotoxicity assay 87

PAGE 99

Primary cultured neuronal cells were used as target cells in 51 Cr release assay for A-specific lymphocyte neurotoxicity (Tan et al., 1999). We co-cultured primary neuronal cells from PSAPP mice or their littermates with CD3 + T-cells (including CD4 + and CD8 + T-cells) isolated from primary cultures of splenocytes derived from A 1-42 /IgGor A 1-42 /CD40L antibody-treated PSAPP mice as described above. As in our previous studies (Tan et al., 1999; Town et al., 2002), primary neuronal cells were labeled with 51 Cr as target cells and co-cultured with T-cells as effectors. Four hour51 Cr release assay was then carried out. Total release represents the radioactivity released after lysis of target cells with 5% Triton X-100. 3.3.10 Statistical analysis All data were normally distributed; therefore, in instances of single mean comparisons, Levenes test for equality of variances followed by t-test for independent samples was used to assess significance. In instances of multiple mean comparisons, analysis of variance (ANOVA) was used, followed by post-hoc comparison using Bonferronis method. Alpha levels were set at 0.05 for all analyses. The statistical package for the social sciences release 10.0.5 (SPSS Inc., Chicago, Illinois) was used for all data analysis. 3.4 Results 3.4.1 CD40 deficiency modulates A antibody production after A vaccination 88

PAGE 100

It is well-established that B-cells require CD40 engagement by T-cell-derived CD40L to produce IgG antibodies in response to vaccination (Kawabe et al., 1994; Bishop and Hostager, 2003). To determine the effects of A 1-42 vaccination on A antibody production in the absence of CD40 expression, strainand gender-matched CD40 deficient (CD40 -/) and wild type mice (n = 8 per group, 4/4) were immunized. We employed a four-month vaccination strategy according to modified previous methods (Schenk et al., 1999) utilizing synthetic aggregated A 1-42 peptide or PBS with MPL/TDM as adjuvant. Mouse plasma samples were collected monthly over this four-month vaccination period and subjected to ELISA for measurement of A antibodies. Results indicated potent A IgG production in wild-type mice, whereas CD40 -/mice produced no detectable A IgG (Figure 6A, top panel). While CD40 -/mice did not produce detectable A IgG, they did produce A IgM that was not significantly different from wild-type mice [98.56 SD 7.45 vs. 86.98 SD 12.09 (g/mL at 1 month after the first immunization). To investigate the impact of CD40 deficiency on A 1-42 vaccination-induced humoral immune responses in a mouse model of AD, we vaccinated 8 month-old PSAPP mice with three CD40 genotypes (PSAPP/CD40 +/+ PSAPP/CD40 +/, and PSAPP/CD40 -/) with either A 1-42 or vehicle (PBS). Blood samples from all mice were individually collected once monthly over the four month vaccination period. As expected, A 1-42 vaccinated PSAPP/CD40 -/mice demonstrated no detectable A IgG, whereas PSAPP/CD40 +/and PSAPP/CD40 +/+ mice produced similar increases in A antibodies throughout the four-month A 1-42 vaccination program (Figure 6A, bottom panel). 89

PAGE 101

3.4.2 Increased plasma A 1-40 and A 1-42 in heterozygous CD40 deficient PSAPP mice after A vaccination Activation of A efflux from the CNS to the systemic circulatory system is a well-recognized clearance mechanism underlying A vaccination in AD mouse models (Sigurdsson et al., 2002; Lemere et al., 2003). To determine the effect of partial or complete CD40 deficiency on activation of A efflux after A 1-42 vaccination, we separately measured plasma A 1-40 and A 1-42 species by ELISA monthly over the four-month vaccination period. Importantly, PSAPP/CD40 +//A 1-42 mice exhibited dramatically increased plasma A 1-40 and A 1-42 compared to PSAPP/CD40 -//A 1-42 animals at the time points analyzed (Figure 6B, P < 0.001), pointing to a shift in A load from the CNS to the systemic circulation in this group. PSAPP/CD40 -//A 1-42 mice displayed very low levels of plasma A species, similar to unvaccinated mouse groups. The lack of elevated A plasma levels in PSAPP/CD40 -//A 1-42 mice can most likely be explained by the absence of A IgG in this mouse group, as homozygous CD40 deficiency conferred absence of A IgG production to either A species (Figure 6A, bottom panel). Interestingly, PSAPP/CD40 +//A 1-42 mice displayed similarly elevated plasma levels of A 1-40 and A 1-42 when compared with the PSAPP/CD40 +/+ /A 1-42 mouse group (P > 0.05). These data further suggest that A IgG production may be required for A efflux from the CNS to the periphery in this vaccination paradigm. 90

PAGE 102

Figure 6 91

PAGE 103

92

PAGE 104

Figure 6. Evaluation of the effects of CD40 deficiency on A antibody generation and A efflux in A 1-42 -immunized mice. Peripheral blood samples were collected monthly throughout the four-month A immunization course. (A) The graph shows antibody levels for wild-type vs. CD40 -/mice (top panel) and PSAPP mice deficient for CD40 vs. appropriate controls as indicated (bottom panel) following A 1-42 vaccination. PSAPP/CD40 +/+ /A 1-42 and PSAPP/CD40 +//A 1-42 mice produced similar elevations in A IgG antibodies, in contrast to PSAPP/CD40 -//A 1-42 PSAPP/CD40 +/+ /PBS, PSAPP/CD40 +//PBS, and PSAPP/CD40 -//PBS mice that produced undetectable levels of A IgG antibodies. Data are presented as mean SD of plasma A antibodies (g/mL). (B) Plasma A 1-40 and A 1-42 peptides were measured separately by ELISA. Data are represented as mean SD of A 1-40 (top panel) or A 1-42 (bottom panel). PSAPP/CD40 +/+ /A 1-42 and PSAPP/CD40 +//A 1-42 mice produced similar elevations in plasma A 1-40 and A 1-42 in contrast to PSAPP/CD40 -//A 1-42 PSAPP/CD40 +/+ /PBS, PSAPP/CD40 +//PBS, and PSAPP/CD40 -//PBS mice that produced minimal levels of plasma A 1-40 and A 1-42 93

PAGE 105

3.4.3 Reduced cerebral A 1-40 and A 1-42 in heterozygous CD40 deficient PSAPP mice vaccinated with A 1-42 As shown in Figure 7, results revealed that A 1-42 vaccination of mice completely (PSAPP/CD40 -/) and partially (PSAPP/CD40 +/) deficient for CD40 yielded decreased amounts of soluble (top panels) and insoluble (bottom panels) A 1-40 and A 1-42 in brain homogenates as measured by ELISA. Most importantly, a significantly greater reduction in both soluble and insoluble A 1-40 and A 1-42 levels was evident in the PSAPP/CD40 +//A 1-42 group compared to either PSAPP/CD40 +/+ /A 1-42 or PSAPP/CD40 -//A 1-42 groups (*P < 0.05; **P < 0.001). We further observed that -amyloid histopathology was also markedly reduced in the PSAPP/CD40 +//A 1-42 group as determined by A antibody immunohistochemical analysis (Figure 8A), and congo red staining (Figure 8B) of mouse coronal brain sections from A vaccinated mice. Quantitative analysis of results revealed significantly reduced A antibodyand congo red-positive -amyloid plaque burden in each brain region examined from PSAPP/CD40 +//A 1-42 mice as compared to either PSAPP/CD40 +/+ /A 1-42 or PSAPP/CD40 -//A 1-42 groups (Figure 8C, **P < 0.001). Together, these data indicate that CD40 heterozygosity confers the greatest reduction in A load in PSAPP mice when compared to all other groups following A 1-42 vaccination, and suggest that partial disruption of CD40 signaling could maximize A 1-42 vaccination efficacy. 94

PAGE 106

Figure 7 95 95

PAGE 107

Figure 7. Cerebral Alevels are significantly reduced in A 1-42 -immunized PSAPP mice heterozygous for CD40. Detergent-soluble A 1-40 and A 1-42 (A) and insoluble (5M guanidine-soluble) A 1-40 and A 1-42 peptides (B) were measured separately in brain homogenates by ELISA. Data are presented as mean SD of A 1-40 or A 1-42 (pg/mg protein). 96

PAGE 108

Figure 8 97 97

PAGE 109

98

PAGE 110

99

PAGE 111

Figure 8. -amyloid pathology is reduced in A 1-42 -immunized PSAPP mice heterozygous for CD40. Mouse coronal brain sections were embedded in paraffin and stained with monoclonal human A antibody (A), or were stained with congo red (B), and the hippocampus is shown. (C) Percentages [plaque area/total area; mean SD with n = 16 mice (8/8)] of A antibody-immunoreactive A plaques (top panel) and congo red-positive A deposits (bottom panel) were calculated by quantitative image analysis for each brain region (CC/H: cingulate cortex and hippocampus; EC: entorhinal cortex) as indicated. 100

PAGE 112

Interestingly, A plaque reduction in PSAPP/CD40 -//A 1-42 mice was significantly reduced when compared to PSAPP/CD40 +/+ /A 1-42, or PSAPP/CD40 -//PBS groups (Figure 8C, *P < 0.05). These data are additionally supported by ELISA analyses of both soluble and insoluble A 1-40 and A 1-42 in brain homogenates (Figure 7), and suggest other mechanisms besides A IgG production, such as cellular immune responses, might be involved in the observed reductions of cerebral A/-amyloid in PSAPP mice after A 1-42 vaccination. 3.4.4 A 1-42 vaccination results in markedly increased anti-inflammatory cytokines and reduced plasma soluble CD40L in PSAPP/CD40 -/mice Due to the observed lack of A IgG antibodies in PSAPP/CD40 -/mice following A 1-42 vaccination, we wished to investigate whether cellular immune responses could be involved in the reductions of cerebral A and -amyloid deposits in these animals. To test this hypothesis, we performed ELISA to examine anti-inflammatory cytokine profiles in brain homogenates from PSAPP/CD40 +/+ PSAPP/CD40 +/, and PSAPP/CD40 -/mice vaccinated with either A 1-42 or vehicle (PBS). As shown in Figure 9A, analysis of data revealed significantly (**P <0.001) elevated expression of brain IL-10 from either PSAPP/CD40 -//A 1-42 or PSAPP/CD40 -/+ /A 1-42 mice compared to PSAPP/CD40 +/+ /A 1-42 mice, but not for IL-1 P >0.05). Moreover, we found a significant decrease in plasma soluble CD40L (sCD40L) when comparing PSAPP/CD40 -//A 1-42 to either PSAPP/CD40 +/+ /A 1-42 or PSAPP/CD40 -//PBS mice (**P <0.001, Figure 9B). Reduced sCD40L in PSAPP/CD40 -//A 1-42 compared to PSAPP/CD40 +/+ /A 1-42 mice occurred in 101

PAGE 113

the absence of significantly different levels of A IgM antibodies (data not shown). These data indicate that reductions in cerebral A after A 1-42 vaccination of PSAPP/CD40 -//PBS mice is associated with a rise in the anti-inflammatory cytokines IL-4, IL-10 and TGF-1, and a decrease in plasma sCD40L levels. 102

PAGE 114

Figure 9 103

PAGE 115

Figure 9. PSAPP/CD40 -/mice have increased anti-inflammatory IL-10 cytokine and decreased plasma soluble CD40L (sCD40L) after A 1-42 vaccination. (A) ELISA analysis of cytokine levels in brain homogenates from the indicated mouse groups. Data are presented as mean SD of each cytokine (pg/mg total protein). (B) ELISA for plasma sCD40L levels in the indicated mouse groups. Data are presented as mean SD of plasma sCD40L protein (pg/mL). 104

PAGE 116

3.4.5 Neutralizing CD40L antibody increases circulating A 1-40 and A 1-42 levels and reduces cerebral amyloidosis in A 1-42 vaccinated PSAPP and Tg2576 mice To determine whether pharmacologic inhibition of CD40-CD40L interaction might produce a similar effect as genetic disruption on enhancing A 1-42 vaccination efficacy, we administered neutralizing CD40L antibody to PSAPP mice in combination with active A 1-42 vaccination described in Materials and Methods. Blood samples were individually collected from all mice at a monthly time interval. Similar to the effects observed in A 1-42 vaccinated PSAPP/CD40 +/mice, A 1-42 vaccinated PSAPP mice treated with CD40L antibody displayed elevations in plasma levels of A 1-40 and A 1-42 and A IgG antibodies that did not significantly differ from A 1-42 vaccinated PSAPP mice injected with an isotype-matched IgG control antibody (Figure 10A, top to bottom, respectively). The PSAPP/A 1-42 /CD40L antibody and PSAPP/A 1-42 /IgG groups produced greater plasma levels of A 1-40 and A 1-42 species and A antibodies than either PSAPP mice receiving PBS or treatment with CD40L antibody alone, confirming that 1) immunization of this cohort of animals was successful and 2) neutralizing CD40L did not hamper A antibody production (Figure 10A). Additionally, no significant differences were revealed for either plasma A 1-40 and A 1-42 levels or A antibodies when comparing PSAPP/A 1-42 /IgG to PSAPP/A 1-42 groups (P > 0.05; data not shown). 105

PAGE 117

Figure 10 106 106

PAGE 118

107

PAGE 119

108 Figure 10. Peripheral and cerebral A levels are reduced in A1-42-immunized PSAPP mice treated with CD40L neutralizing antibody. (A) ELISA analysis for plasma levels of A1-40 and A1-42 and A antibodies. Plasma A1-40 (top panel) and A1-42 (middle panel) were measured separately by ELISA. PSAPP/A1-42/CD40L antibody and PSAPP/A1-42/IgG mice produced similar elevations in plasma A1-40 and A1-42, in contrast to PSAPP/CD40L antibody and PSAPP/PBS mice which produced minimal levels of plasma A1-40 and A1-42. Data are represented as mean SD of A1-40 or A1-42 (pg/mL) in plasma. A antibody levels (bottom panel) were measured by ELISA. PSAPP/A1-42/CD40L antibody and PSAPP/A1-42/IgG mice produced similar elevations in plasma A IgG antibodies in contrast to PSAPP/CD40L antibody and PSAPP/PBS mice which had undetectable levels of plasma A IgG antibodies. Data are presented as mean SD of A antibodies (g/mL) in plasma. No significant difference in A antibody levels between PSAPP/A1-42/CD40L antibody and PSAPP/A1-42/IgG mice (P > 0.05) was observed. (B) Soluble A1-40 and A1-42 peptides (top panel) and insoluble A1-40 and A1-42 (bottom panel) in brain homogenates were measured separately by ELISA. Data are presented as mean SD of A1-40 or A1-42 peptides normalized to total protein (pg/mg).

PAGE 120

nftcur n of tivity could reduce CAA following A vaccination in the Tg2576 mouse model of AD. Since Tg2576 mice are known to produce CAA pathology at 15 to 20 months of age (Christie et al., 2001; Li et al., 2003; Friedlich et al., 2004; Kim et al., 2007), we initiated i.p. injection of these mice with CD40L antibody at 12 months of age (n = 16, 8/8) in conjunction with active A1-42 immunization using an identical procedure as described above. Four months later, we sacrificed these mice and examined CAA. As We next examined the effect of neutralizing CD40L antibody on cerebral A levels in PSAPP mice vaccinated with A 1-42 ELISA analysis revealed that PSAPP/A 1-42 /CD40L antibody mice display significantly reduced amounts of cerebral soluble and insoluble A 1-40 and A 1-42 peptides as compared with PSAPP/A 1-42 /IgG, PSAPP/CD40L antibody, or PSAPP/PBS groups (**P < 0.001; Figure 10B). Furthermore, PSAPP/A 1-42 /CD40L antibody mice showed a marked reduction in cerebral A deposits compared to PSAPP/A 1-42 /IgG mice or other control groups (**P < 0.001; Figs. 11A C). These data show additional reduction in cerebral A and -amyloid in the context of A 1-42 vaccination provided by pharmacologic blockade of CD40-CD40L interaction. Together, these data demonstrate that disruption of the CD40-CD40L interaction by 1) a genetic approach or 2) pharmacologic depletion of available CD40L by neutralizing antibody enhances reduction of cerebral amyloidosis after A 1-42 vaccination. Recent studies have show CAA and cerebral microhemorrhage oen ocin AD mice following intraperitoneal injection (i.p) active or passive A vaccination (Wilcock et al., 2004; Wilcock et al., 2007). Thus, we next investigated if disruptioCD40L ac 109

PAGE 121

shown nly 1-42/CD40L on, we also and 0L 1-42 mpared to A1-42-alone-immunized mice (P < 0.001; data not sho in Figs. 11D E, disruption of CD40L activity by the depleting antibody not opromotes additional reduction in total congo red after A 1-42 vaccination, but also further reduces vascular congo red signal. This was confirmed by statistical analysis, which revealed significant differences between Tg2576/A 1-42 /IgG and Tg2576/Aantibody groups (**P < 0.001) for both total and vascular congo red. In additianalyzed cerebral A levels/-amyloid deposits in these two groups by A ELISA immunochemistry. Similar to the effects observed in A 1-42 /CD40L antibody-vaccinated PSAPP mice, Tg2576 mice receiving both A 1-42 immunization and depleting CD4antibody displayed a marked decrease in cerebral soluble and insoluble A 1-40 and A levels and -amyloid load co wn). 110

PAGE 122

111 Figure 11

PAGE 123

112

PAGE 124

113

PAGE 125

114

PAGE 126

115

PAGE 127

116 Figure 11. Cerebral -amyloid deposits and cerebral amyloid angiopathy are reduced in A1-42-immunized PSAPP or Tg2576 mice treated with CD40L neutralizing antibody. Mouse paraffin-embedded coronal brain sections from were stained with rabbit Pan--amyloid antibody (A) or with congo red (B), and the hippocampus is shown. (C) Percentages (plaque area/total area; mean SD) of A antibody-immunoreactive deposits (top panel) or of congo red-stained sections (bottom panel) were calculated by quantitative image analysis. (D) Tg2576 received A1-42 vaccination plus neutralizing CD40L antibody or isotype-matched control IgG both A1-42, and brain sections were stained with congo red (hippocampus is shown). Positions of the hippocampal subfields CA1, CA3, and DG (dentate gyrus) are indicated in the upper left panel. Arrows indicate A deposit-affected vessels. (E) Percentages (% of area) of congo red-stained plaques were quantified by image analysis [mean SD with (n = 16, 8/8)].

PAGE 128

e tions t the e neutralization mitigated APC-like microglia, we fluorescently labeled brain sections with MHC II and CD45 antibodies. As shown in Figure 12C, Iba1 positive microglial cells were largely positive for CD45 and MHC II in A1-42/IgG immunized PSAPP mice, but not in A1-42 and CD40L antibody co-immunized PSAPP mice. These data indicate that depletion of functional CD40L results in decreased pro-inflammatory APC-like microglial activation in PSAPP/A1-42/CD40L antibody mice. It should be noted that Iba1 immunostaining does not distinguish resident microglia from peripherally-derived monocytes that may migrate to the CNS and take up a microglial phenotype. 3.4.6 CD40 pathway blockade decreases MHC II and CD45-positive microglia and increases anti-inflammatory cytokines in A 1-42 immunized PSAPP mice To evaluate the effects of CD40/CD40L blockade on pro-inflammatory APC-likmicroglial activation in the A 1-42 vaccination paradigm, we first stained brain secfrom PSAPP/A 1-42 /IgG and PSAPP/A 1-42 /CD40L antibody mice with Iba1 antibody (Figure 12A). We quantified Iba1 positive microglia/macrophages with/withouspindle-shaped morphology, as it has been previously reported that microglial cells become spindle-shaped when exposed to stimuli including LPS and Con A that promotthe pro-inflammatory APC phenotype (Washington et al., 1996; Bernhardi and Nicholls, 1999). Interestingly, disruption of CD40L activity significantly reduced spindle-shaped microglia/macrophages by morphologic analysis (**P < 0.001), but did not alter non-spindle-shaped cells (Figure 12B). To further evaluate whether CD40L 117

PAGE 129

Figure 12 118

PAGE 130

119

PAGE 131

120

PAGE 132

121

PAGE 133

122 Figure 12. CD40L blockade inhibits APC-like microglial activation in A1-42 vaccinated PSAPP mice and promotes anti-inflammatory cellular immunity. (A) Representative hippocampal sections from PSAPP/A1-42/IgG and PSAPP/A1-42/CD40L antibody mouse brains were stained with Iba1 antibody to illustrate both microglial load and morphology. (B) Quantitative image analysis of microglial load (Iba1 positive) and percentage of spindle-shaped Iba1 positive microglia is shown. (C) Representative hippocampal sections from PSAPP/A1-42/IgG and PSAPP/A1-42/CD40L antibody mouse brains were stained with Iba1 together with MHC II or CD45 antibodies to illustrate microglial load and activation status (DAPI was used as a nuclear counterstain). (D) Th1 and Th2 cytokine analysis by ELISA was conducted on mouse brain homogenates from PSAPP, PSAPP/A1-42/IgG and PSAPP/A1-42/CD40L antibody mice. Data are represented as mean SD of each cytokine in brain homogenates (pg/mg total protein) from PSAPP, PSAPP/A1-42/CD40L antibody or PSAPP/A1-42/IgG mice.

PAGE 134

ro-0 /IgG other or IL-1. ory 3.4.7 A1-42-immunized PSAPP mice treated with CD40L neutralizing antibody exhibit increases in anti-inflammatory cytokines and decreases in neurotoxic inflammatory responses in vitro To investigate A-specific T-cell immune responses after A1-42 immunization plus neutralizing CD40L antibody treatment, we established primary cultures of splenocytes from: PSAPP/A1-42/IgG, PSAPP/A1-42/CD40L antibody, PSAPP/CD40L antibody, and PSAPP/PBS mice. We then quantified key cytokines produced by activated T-cells (IFN-, IL-2, and IL-4) in supernatants by ELISA. Both non-specific To further determine the consequences of this phenomenon in terms of inflammatory responses, we analyzed brain homogenates from PSAPP/A 1-42 /CD40L antibody, PSAPP/A 1-42 /IgG, and other control groups by ELISA for expression of pinflammatory and anti-inflammatory cytokines. Analysis of results revealed a significantly (**P <0.001) greater expression of anti-inflammatory TGF-1 and IL-1cytokines from PSAPP/A 1-42 /CD40L antibody mice compared to the PSAPP/A 1-42mice (Figure 12D). Moreover, PSAPP/A 1-42 /CD40L antibody mice also produced significantly (P <0.001) greater expression of TGF-1 and IL-10 when compared tocontrols including PSAPP/IgG and PSAPP/A 1-42 /PBS mice (data not shown). No significant between-groups differences were revealed when considering TNF-These data indicate that CD40L blockade correlates with a rise in the anti-inflammatcytokines TGF-1 and IL-10, without affecting the pro-inflammatory cytokines TNFand IL-1. 123

PAGE 135

(Con A1-42 y T-cell 1-G nes (**P < 0.001) after in vitro A1-42 challenge. Of note, there were gnificant differences within mouse groups between IL-4 and either IFN-or IL-2 after 1-se groups (P > 0.05; data not shown). g mice ) and specific recall stimulation of primary cultured splenocytes with A resulted in increases in the pro-inflammatory T-cell cytokines, IFNand IL-2, in both PSAPP/A 1-42 /IgG and PSAPP/A 1-42 /CD40L antibody groups compared to the PSAPP/CD40L antibody group (Figure 13A). However, these pro-inflammatorcytokines were reduced in the PSAPP/A 1-42 /CD40L antibody group versus PSAPP/A42 /IgG mice. Moreover, PSAPP/A 1-42 /CD40L antibody mice demonstrated increased anti-inflammatory T helper type 2 cytokine IL-4 when compared to PSAPP/A 1-42 /Ig mice. These data indicate that disruption of CD40L activity in A 1-42 vaccinated mice reduces pro-inflammatory A-specific T-cell immune responses in favor of an anti-inflammatory response. One-way ANOVA followed by post hoc comparison revealed significant differences when comparing PSAPP/A 1-42 /CD40L antibody to PSAPP/CD40L antibody or PSAPP/A 1-42 /IgG mouse groups for levels of each of the three cytoki si A 1-42 recall challenge ( ## P < 0.001). Following challenge, no significant difference in cytokine release was observed from splenocytes between PSAPP/PBS and PSAPP/A42 /IgG control mou To determine whether CD40L neutralization could mitigate potentially damagineffects of A-specific T-cells, we co-cultured primary neuronal cells from PSAPPor their littermates with CD3 + T-cells (including CD4 + and CD8 + T-cells) isolated from the primary cultured splenocytes derived from PSAPP/A 1-42 /IgG or PSAPP/A 1-42 /CD40L antibody mice as described above. Per our previous reports (Tan et al., 2000; 124

PAGE 136

r ccinated roCD40L activity. Tan et al., 2002a; Town et al., 2002), we labeled primary neuronal cells with 51 Cr astarget cells and co-cultured them with T-cells as effectors, and carried out four-hour 51 Crelease assay. ANOVA showed main effects of effector:target ratio for A 1-42 vaPSAPP mouse-derived T-cells (effectors) and PSAPP mouse-derived neuronal cells (target cells) (**P <0.001; n = 8 mice for PSAPP/A 1-42 /IgG and PSAPP/A 1-42 /CD40L antibody groups; n = 5 mice for both control groups) (Figure 13B), but not when unvaccinated PSAPP mouse-derived T-cells were used (control 1) or when contl littermate (non-transgenic mouse)-derived neuronal cells (control 2) were used (P > 0.05). ANOVA followed by post-hoc comparison revealed a significant difference across ratios between PSAPP/A 1-42 /IgG and PSAPP/A 1-42 /CD40L antibody T-cells (**P < 0.001), indicating an overall decrease in percentage of cell lysis as a result of disrupting 125

PAGE 137

Figure 13 126

PAGE 138

127

PAGE 139

128 Figure 13. A-specific neurotoxic inflammatory responses are reduced in A1-42-immunized PSAPP mice deficient for CD40. (A) Splenocytes were individually isolated and cultured from mice as indicated after A1-42 immunization and either CD40L antibody treatment or PBS injection (control). These cells were stimulated with Con A (5 g/mL) or A1-42 (20 g/mL) for 48 hrs. Cultured supernatants were collected from these cells for IFN-, IL-2, and IL-4 cytokine analyses by ELISA. Data are represented as mean SD (n = 10) of each cytokine in supernatants (pg/mg total intracellular protein). (B) A specific T cell-mediated neuronal cell injury was determined by 51Cr release assay. Data are reported as mean 51Cr release values SD, and n = 8 for each condition presented. PSAPP/A1-42/IgG mouse group, effectors: A1-42/IgG-immunized PSAPP mouse-derived T cells; target cells: PSAPP-mouse-derived primary neuronal cells. PSAPP/A1-42/CD40L antibody mouse group, effectors: A1-42/CD40L antibody-immunized PSAPP mouse-derived T cells; target cells: PSAPP-mouse-derived primary neuronal cells. Control 1, effectors: unvaccinated PSAPP mouse-derived T cells; target cells: PSAPP mouse-derived neuronal cells. Control 2, effectors: A1-42-immunized PSAPP mouse-derived T cells; target cells: non-transgenic mouse-derived primary neuronal cells.

PAGE 140

999). To e o-ryt al., ard et al., 2000), and microglial Fc receptor (i.e., microglial phagocytosis of A antibody-opsonized deposits) is not required for brain A clearance (Das et al., 2003). Thus, we suggest that the combination of blocking the microglial CD40 pathway and A immunotherapy further enhances microglial Abeta clearance. Previous clinical investigation has revealed that active immunization with A in humans confers the unacceptable risk of aseptic meningoencephalitis associated with T3.5 Discussion We have previously shown the CD40-CD40L interaction enhances pro-inflammatory microglial activation triggered by cerebral A deposits (Tan et al., 1This form of microglial activation is deleterious, as both genetic ablation of CD40L and CD40L neutralizing antibody reduce brain levels of several neurotoxic inflammatory cytokines and mitigate cerebral amyloidosis in AD mouse models (Tan et al., 2002a).establish a possible mechanism to explain these results, we previously quantified microglial phagocytic activity in CD40 deficient versus CD40 sufficient AD mice. Wobserved inhibition of microglial A phagocytosis upon CD40 ligation. This coincided with increased microglial co-localization of MHC class II with non-opsonized ApeptideMoreover, this APC phenotype was accompanied by upregulation of prinflammato Th1 cytokines such as TNF-, IL-1, IL-2, and IFN(Townsend e2005). These data suggest that CD40 pathway blockade induces switching of the microglial phenotype from a pro-inflammatory APC state to an anti-inflammatory, pro-phagocytic state (Townsend et al., 2005). Interestingly, brain A clearance in the Aimmunotherapy paradigm has previously been suggested to rely on microglial phagocytosis (B 129

PAGE 141

gliosis, and associated rise in CNS pro-inflammatory mediators (Schenk et al., 1ta and our 40 ever, 42G e -is M king the across the various groups cell infiltration, 999; Schenk and Yednock, 2002; Nicoll et al., 2003). Based on these dawork on the CD40-CD40L association with brain A levels, we investigated whether CD40 blockade could reduce cerebral A deposits without the undesirable inflammatory events in the CNS. First, we tested whether active A 1-42 vaccination of CD40 deficient mice could produce significant levels of A antibodies. Consistent with the requirement of CDsignaling for IgM to IgG class switching (Kawabe et al., 1994), homozygous CD40 deficient mice vaccinated with A 1-42 did not produce detectable A IgG antibodies, but had slightly increased levels of A IgM antibodies vs. wild-type controls. Howactive A 1vaccination of PSAPP/CD40 +/mice produced elevated plasma anti-A Igantibodies comparable to CD40 sufficient PSAPP mice which are consistent with a gendose-effect (Figure 6A). Interestingly, further reduction in cerebral amyloidosis in Avaccinated PSAPP/CD40 -/occurred essentially in the absence of A IgG antibodies. It well known that the CD40 pathway is essential for antibody isotype switching from Igto IgG, and this result suggests that the additional therapeutic benefit from blocCD40 pathway is independent of IgG in our system. However, given 1) the requirement of CD40 signaling for a diverse set of immunological responses and 2) that we did not use IgG deficient mice, this conclusion should be taken with caveats. Next, we quantified plasma levels of A 1-40 and A 1-42 species of A-immunized PSAPP mice in an attempt to determine the relationship between IgG, IgM, and efflux of A from the brain to the periphery. Similar elevations 130

PAGE 142

1-40, 42 species (Figure 6B). This lack of elevated plasma A1-40, 42 corretinct 1-42 resulted in reduced IFNand IL-2 production in the PSAPP/A1-42/CD40L antibody mouse group compared to PSAPP/A1-42/IgG mice (Figure 13A). Further, PSAPP/CD40/A1-42 mice of plasma A 1-40 42 species between CD40 heterozygous deficient or CD40 sufficient PSAPP mice were observed (Figure 6B). Thus, we suggest that A IgG-mediated brain to blood efflux was operating similarly in CD40 heterozygous and CD40 sufficient PSAPP mice. By contrast, A 1-42 vaccinated PSAPP/CD40 -/mice did not exhibit elevations in plasma A lated positively with the lack of A IgG antibody production in the homozygous CD40 deficient PSAPP mice. This is consistent with a lack of brain-to-blood efflux of Avia the peripheral sink hypothesis DeMattos et al., 2001), and suggests that a dismechanism is operating in these PSAPP/CD40 -/mice While CD40 deficient mice do produce normal levels of AIgM antibodies, no detectable elevation in peripheral Awas observed. This, too, is in accord with the notion that A IgG is required for brain to blood efflux of A (DeMattos et al., 2001). As pharmacotherapeutic proof of principle, we administered CD40L neutralizing antibody to A 1-42 vaccinated PSAPP mice, and confirmed greater reductions in cerebral amyloidosis compared to A 1-42 vaccinated PSAPP mice given control IgG. This experimental approach was performed to offset the possibility that genetic ablation, in and of itself, does not confer developmental changes that would result in modulation of A loads. We next went on to test T-cell specific immune responses against Aand foundthat recall stimulation of primary cultured splenocytes with Apeptide +/131

PAGE 143

pro-lenge. o al., l A) and d congo red staininPreviously, we showed that CD40 ligation shifts microglial response to A from an anti-inflammatory phagocytic phenotype to a pro-inflammatory APC response (Town et al., exhibited an increase in the anti-inflammatory Th2 cytokine, IL-10, and produced less circulating soluble CD40L (sCD40L) compared to PSAPP/CD40 +/+ /A 1-42 mice (Figure 9). Taken together, these findings suggest partial CD40 pathway inhibition reduces inflammatory but increases anti-inflammatory T-cell immune responses to AchalThis is particularly attractive as these results raise the possibility that 50% pharmacological inhibition of CD40 might be of benefit in the attenuation of the pro-inflammatory meningoencephalitis associated with active A vaccination in humans, while still allowing for production of A IgG antibodies. In addition to developing parenchymal A deposits and associated elevated inflammatory cytokines seen in the PSAPP model, the Tg2576 mouse model of AD alsdevelops cerebrovascular A deposits not unlike cerebral amyloid angiopathy (CAA), which is observed in the majority of AD patients (Nicoll et al., 2003; Wilcock et2004; Wilcock et al., 2007). To determine if reducing available CD40L might also mitigate CAA, we administered neutralizing CD40L antibody to A vaccinated Tg2576 mice. Indeed blockade of the CD40L with neutralizing antibody in combination with Avaccination produced the highest therapeutic effect (reduction of parenchymathe most minimal CAA-like pathology as measured by parenchymal an g respectively (Figs. 11D E). These results suggest CD40L antibody neutralization not only improves the cerebral amyloid reducing effect of A vaccination, but also confers a reduction in undesirable CAA-like pathology in Tg2657 mice. 132

PAGE 144

al ytokines can f ination paradigm, making this less liksults F-, but 2002; Townsend et al., 2005). In accord, here we observed downregulation of microgliAPC morphology in situ (Figs. 12A B), and reduction in CD45 and MHC II-positive microglia from A 1-42 vaccinated PSAPP mice treated with CD40L neutralizing antibody(Figure 12C). Indeed, reduction in MHC II expression, a measure of microglial APC phenotype, correlated with elevated brain levels of anti-inflammatory Th2 cTGF-1 and IL-10 (Figure 12D). Although NK cell-mediated activation of microgliabypass MHC and T cell receptors to produce a vaccine immune response, infiltration oNK cells into the CNS has not been reported in the A vacc ely. Another possibility is that so-called anti-ergotypic responses, defined as an immune response to the vaccine-activated host immune cells, could play a role in enhanced efficacy of A immunotherapy in conjunction with CD40 blockade. Short-term anti-ergotypic lines isolated from vaccinated Multiple Sclerosis (MS) patients demonstrate a mixed phenotype (both CD4 + and CD8 + cells). These cells secrete IFNand TNF-, but not TGF-1 (Correale et al., 1997; Hellings et al., 2004). Although we can not fully rule out this mechanism in our studies of CD40 blockade enhancement of reduced cerebral amyloidosis after A vaccination, it is interesting to note that our rein the CNS and in splenocytes show the converse: a reduction in IFNand TNan increase in TGF(Figure 12D and Figure 13A), suggesting the involvement of a different mechanism. Given our previous observation CD40 ligation induces switching of the microglial phenotype from a pro-phagocytic state endorsing Aphagocytosis to a pro133

PAGE 145

e s of auto-aggressive T-cells as was observed in the active A AN-1792 vaccineponse, r e s r hus, complete systemic blockade of CD40-CD40L would likely have dey inflammatory APC state (Townsend et al., 2005), we propose that combined CD40 blockade and A vaccination promotes microglial phagocytosis/clearance of A from thCNS. Additionally, T-cells derived from neutralizing CD40L antibody-treated A 1-42 vaccinated PSAPP were dramatically less neurotoxic to A producing neurons ex vivo(Figure 13B), suggesting further benefit afforded by combining these therapeutic approaches. However, it is worth noting that this latter result needs to be interpreted with the caveat that standard active A vaccination of AD mouse models (unless modified with the addition of pertussis toxin) (Furlan et al., 2003) does not produce appreciable brain infiltrate in AD patients (Nicoll et al., 2003; Town et al., 2005). CD40-CD154 interaction is essential for initiating the adaptive immune resas demonstrated by immune deficits observed in patients with mutations in the CD40 oCD40L genes. These patients develop type 1 hyper IgM immunodeficiency syndrom(HIGM1) that is characterized by recurrent bacterial and opportunistic infections (Durandy 2001; Fuleihan 2001; Levy et al., 1997a). Patients with homozygous mutation in the CD40 gene have a severe immunodeficiency termed HIGM3. Its clinical phenotype overlaps with that of HIGM1 and is characterized by defective generation of secondary antibodies and severe opportunistic infections, (Ferrari et al., 2001; Kutukculeet al., 2003) particularly Cryptosporidium enteritis and Pneumocystis carinii pneumonia (Levy et al., 1997b). T leterious immunosuppressive side effects. For this reason, pharmacotherapwould need to be titrated such that the CD40-CD154 pathway was partially inhibited. 134

PAGE 146

e, on by t is both safer and more effectiv Interestingly, as we have shown in our proof-of-concept paradigm in transgenic miceven a 50% blockade of CD40 is enough to increase the efficacy of the Abeta vaccine on reduction of cerebral amyloidosis. Our observations suggest that partial blockade of CD40 signaling, either by genetic or by pharmacologic means, increases the effectiveness of A 1-42 vaccinatifurther reducing cerebral amyloidosis and simultaneously promoting anti-inflammatory cellular immune processes in the brain and in the periphery. If the benefit afforded by CD40 pathway blockade to A 1-42 vaccinated AD mouse models can translate to the clinical syndrome, then pharmacotherapy aimed at reducing CD40 signaling in conjunction with A vaccination may represent an approach tha e in humans. Future studies will be required to isolate CD40-CD40L downstream signaling involved in reduced efficacy of A vaccination, as this may uncover additionaltargets for pharmacologic intervention. 135

PAGE 147

K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Lieberburg, I., ., Vasquez, N., Weiss, K., Welch, B., Seubert, P., Schenk, D., Yednock, T., 2000. Peripherally administered antibodies against amyloid beta-pel of Alzheimer disease. Nat Med. 6(8), 916-9. Bernhardi, R. V., Nicholls, J. G., 1999. Transformation of leech microglial cell tissue. J Exp Biol. 202 (Pt 6), 723-8. Bishop, G. A., Hostager, B. S., 2003. The CD40-CD154 interaction in B cell-T cell 368, 387-er's disease induce antibody responses against l responses. Vaccine. 24, 6321-31. hen, K., Iribarren, P., Hu, J., Chen, J., Gong, W., Cho, E. H., Lockett, S., Dunlop, N. M., Wag, J. M., 2006. Activation of Toll-like receptor 2 on microglia promotes cell uptake of Alzheimer disease-associated amyloid beta peptide. J Biol Chem. 281, 3651-9. Christie, R., Yamada, M., Moskowitz, M., Hyman, B., 2001. Structural and functional disruption of vascular smooth muscle cells in a transgenic mouse model of amyloid angiopathy. Am J Pathol. 158, 1065-71. Correale, J., McMillan, M., Li, S., McCarthy, K., Le, T., Weiner, L. P., 1997. Antigen presentation by autoreactive proteolipid protein peptide-specific T cell clones from chronic progressive multiple sclerosis patients: roles of co-stimulatory B7 molecules and IL-12. J Neuroimmunol. 72, 27-43. Das, P., Howard, V., Loosbrock, N., Dickson, D., Murphy, M. P., Golde, T. E., 2003. Amyloid-beta immunization effectively reduces amyloid deposition in FcRgamma-/knock-out mice. J Neurosci. 23(24), 8532-8. DeMattos, R. B., Bales, K. R., Cummins, D. J., Dodart, J. C., Paul, S. M., Holtzman, D. M., 2001. Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and 3.6 References Bard, F., Cannon, C., Barbour, R., Burke, R. L., Games, D., Grajeda, H., Guido, T., Hu, Motter, R., Nguyen, M., Soriano, F ptide enter the central nervous system and reduce pathology in a mouse mode morphology and properties following co-culture with injured central nervous system liaisons. Cytokine Growth Factor Rev. 14, 297-309. Blennow, K., de Leon, M. J., Zetterberg, H., 2006. Alzheimer's disease. Lancet. 403. Calingasan, N. Y., Erdely, H. A., Altar, C. A., 2002. Identification of CD40 ligand in Alzheimer's disease and in animal models of Alzheimer's disease and brain injury. Neurobiol Aging. 23, 31-9. Chackerian, B., Rangel, M., Hunter, Z., Peabody, D. S., 2006. Virus and virus-like particle-based immunogens for Alzheimamyloid-beta without concomitant T cel C n 136

PAGE 148

beta burden in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A. 98, 8850-5. ., Ugazio, A. G., Levy, Y., Catalan, N., urandy, A., Tbakhi, A., Notarangelo, L. D., Plebani, A., 2001. Mutations of CD40 gene Natl riedlich, A. L., Lee, J. Y., van Groen, T., Cherny, R. A., Volitakis, I., Cole, T. B., an animal model of Alzheimer's isease. J Neurosci. 24, 3453-9. an, R. L., 2001. The hyper IgM syndrome. Curr Allergy Asthma Rep. 1, 445-50. eview. mi, G., Martino, G., 2003. Vaccination with amyloid-beta eptide induces autoimmune encephalomyelitis in C57/BL6 mice. Brain. 126(Pt 2), 285-ouble-edged accine. Nat Med. 9, 389-90. ate a, A., Papassotiropoulos, A., Nitsch, R. M., 003. Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease. s of immunotherapeutic intervention by anti-CD40L CD154) antibody in an animal model of multiple sclerosis. J Clin Invest. 103, 281-90. therapeutic blockade of CD154-CD40 in xperimental autoimmune encephalomyelitis. J Clin Invest. 109, 233-41. decreases brain A Durandy, A., Honjo, T., 2001. Human genetic defects in class-switch recombination (hyper-IgM syndromes). Curr Opin Immunol. 13, 543-8. Review. Ferrari, S., Giliani, S., Insalaco, A., Al-Ghonaium, A., Soresina, A. R., Loubser, M., Avanzini, M. A., Marconi, M., Badolato, R D cause an autosomal recessive form of immunodeficiency with hyper IgM. Proc Acad Sci U S A. 2001 Oct 23;98(22):12614-9. F Palmiter, R. D., Koh, J. Y., Bush, A. I., 2004. Neuronal zinc exchange with the bloodvessel wall promotes cerebral amyloid angiopathy in d Fuleih R Furlan, R., Brambilla, E., Sanvito, F., Roccatagliata, L., Olivieri, S., Bergami, A., Pluchino, S., Uccelli, A., Co p 91. Greenberg, S. M., Bacskai, B. J., Hyman, B. T., 2003. Alzheimer disease's d v Hellings, N., Raus, J., Stinissen, P., 2004. T-cell vaccination in multiple sclerosis: updon clinical application and mode of action. Autoimmun Rev. 3(4), 267-75. Hock, C., Konietzko, U., Streffer, J. R., Tracy, J., Signorell, A., Muller-Tillmanns, B., Lemke, U., Henke, K., Moritz, E., Garcia, E., Wollmer, M. A., Umbricht, D., de Quervain, D. J., Hofmann, M., Maddalen 2 Neuron. 38, 547-54. Howard, L. M., Miga, A. J., Vanderlugt, C. L., Dal Canto, M. C., Laman, J. D., Noelle, R. J., Miller, S. D., 1999. Mechanism ( Howard, L. M., Ostrovidov, S., Smith, C. E., Dal Canto, M. C., Miller, S. D., 2002. Normal Th1 development following long-term e Jankowsky, J. L., Slunt, H. H., Ratovitski, T., Jenkins, N. A., Copeland, N. G., Borchelt 137

PAGE 149

17, 157-65. t, P., McConlogue, ., 1997. Amyloid precursor protein processing and A beta42 deposition in a transgenic erminal center formation. Immunity. 1, 67-78. ., ci. saccharide-induced inflammation exacerbates tau pathology by a cyclin-ependent kinase 5-mediated pathway in a transgenic model of Alzheimer's disease. J otarangelo, L. D., 2003. Disseminated cryptosporidium infection in an infant with yper-IgM syndrome caused by CD40 deficiency. emere, C. A., Spooner, E. T., LaFrancois, J., Malester, B., Mori, C., Leverone, J. F., protein llowing chronic, active Abeta immunization in PSAPP mice. Neurobiol Dis. 14, 10-8. nick, I., ., E., Sanal, O., Peitsch, M. C., Notarangelo, L. D., 1997. Clinical pectrum of X-linked hyper-IgM syndrome. J Pediatr. 131, 47-54. lzheimer's disease. Am J Pathol. 163, 2155-64. ryl lipid A MPL), cholera toxin B subunit (CTB) and E. coli enterotoxin LT(R192G). Vaccine. 23, D. R., 2001. Co-expression of multiple transgenes in mouse CNS: a comparison of strategies. Biomol Eng. Johnson-Wood, K., Lee, M., Motter, R., Hu, K., Gordon, G., Barbour, R., Khan, K., Gordon, M., Tan, H., Games, D., Lieberburg, I., Schenk, D., Seuber L mouse model of Alzheimer disease. Proc Natl Acad Sci U S A. 94, 1550-5. Kawabe, T., Naka, T., Yoshida, K., Tanaka, T., Fujiwara, H., Suematsu, S., Yoshida, N., Kishimoto, T., Kikutani, H., 1994. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and g 1 Kim, J., Onstead, L., Randle, S., Price, R., Smithson, L., Zwizinski, C., Dickson, D. WGolde, T., McGowan, E., 2007. Abeta40 inhibits amyloid deposition in vivo. J Neuros27, 627-33. Kitazawa, M., Oddo, S., Yamasaki, T. R., Green, K. N., LaFerla, F. M., 2005. Lipopoly d Neurosci. 25, 8843-53. Kutukculer, N., Moratto, D., Aydinok, Y., Lougaris, V., Aksoylar, S., Plebani, A., Genel,F., N h J Pediatr. 142, 194-6. L Matsuoka, Y., Taylor, J. W., DeMattos, R. B., Holtzman, D. M., Clements, J. D., Selkoe, D. J., Duff, K. E., 2003. Evidence for peripheral clearance of cerebral Abeta fo Levy, J., Espanol-Boren, T., Thomas, C., Fischer, A., Tovo, P., Bordigoni, P., ResFasth, A., Baer, M., Gomez, L., Sanders, E. A., Tabone, M. D., Plantaz, D., Etzioni, AMonafo, V., Abinun, M., Hammarstrom, L., Abrahamsen, T., Jones, A., Finn, A., Klemola, T., DeVries s Li, L., Cao, D., Garber, D. W., Kim, H., Fukuchi, K., 2003. Association of aortic atherosclerosis with cerebral beta-amyloidosis and learning deficits in a mouse model of A Maier, M., Seabrook, T. J., Lemere, C. A., 2005. Modulation of the humoral and cellular immune response in Abeta immunotherapy by the adjuvants monophospho ( 5149-59. 138

PAGE 150

D., Jiang, L., Das, P., Janus, C., Lemere, C. A., 006. Short amyloid-beta (Abeta) immunogens reduce cerebral Abeta load and learning K., arbinian, N., Darekar, P., Mihaly, L., Khalili, K., 2006. Beta-amyloid deposition and onsiveness to amyloid beta-peptide in amyloid precursor protein transgenic ice: implications for the pathogenesis and treatment of Alzheimer's disease. Proc Natl A., Imitola, J., Zota, V., Oida, T., Weiner, H. L., 2003. Microglia-mediated itric oxide cytotoxicity of T cells following amyloid beta-peptide presentation to Th1 f, G., Wilcock, D., Connor, K., Hatcher, J., Hope, C., Gordon, M., rendash, G. W., 2000. A beta peptide vaccination prevents memory loss in an animal Markham, H., Weller, R. O., 2003. an Alzheimer disease after immunization with amyloid-beta eptide: a case report. Nat Med. 9, 448-52. icrohemorrhage. Proc Natl Acad Sci U S A. 104, 2507-12. s kura, Y., Miyakoshi, A., Kohyama, K., Park, I. K., Staufenbiel, M., Matsumoto, Y., rgogozo, J. M., Gilman, S., Dartigues, J. F., Laurent, B., Puel, M., Kirby, L. C., Maier, M., Seabrook, T. J., Lazo, N. 2 deficits in an Alzheimer's disease mouse model in the absence of an Abeta-specific cellular immune response. J Neurosci. 26, 4717-28. Miklossy, J., Kis, A., Radenovic, A., Miller, L., Forro, L., Martins, R., Reiss D Alzheimer's type changes induced by Borrelia spirochetes. Neurobiol Aging. 27, 228-36. Monsonego, A., Maron, R., Zota, V., Selkoe, D. J., Weiner, H. L., 2001. Immune hyporesp m Acad Sci U S A. 98, 10273-8. Monsonego, n cells. J Immunol. 171, 2216-24. Morgan, D., Diamond, D. M., Gottschall, P. E., Ugen, K. E., Dickey, C., Hardy, J., DufK., Jantzen, P., DiCarlo, A model of Alzheimer's disease. Nature. 408, 982-5. Nicoll, J. A., Wilkinson, D., Holmes, C., Steart, P.,Neuropathology of hum p Nikolic, W. V., Bai, Y., Obregon, D., Hou, H., Mori, T., Zeng, J., Ehrhart, J., Shytle, R. D., Giunta, B., Morgan, D., Town, T., Tan, J., 2007. Transcutaneous beta-amyloid immunization reduces cerebral beta-amyloid deposits without T cell infiltration and m O'Keefe, G. M., Nguyen, V. T., Benveniste, E. N., 2002. Regulation and function of clasII major histocompatibility complex, CD40, and B7 expression in macrophages andmicroglia: Implications in neurological diseases. J Neurovirol. 8, 496-512. O 2006. Nonviral Abeta DNA vaccine therapy against Alzheimer's disease: long-termeffects and safety. Proc Natl Acad Sci U S A. 103, 9619-24. O Jouanny, P., Dubois, B., Eisner, L., Flitman, S., Michel, B. F., Boada, M., Frank, A., Hock, C., 2003. Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology. 61, 46-54. 139

PAGE 151

onomarev, E. D., Shriver, L. P., Dittel, B. N., 2006. CD40 expression by microglial ., Kulhanek, D., Eckenstein, F., 003. Inflammation and cerebral amyloidosis are disconnected in an animal model of Westendorp, R. G., 2001. Patients with Alzheimer's disease display a pro-n, D., Hardy, J., Town, T., Tan, J., 2005. Green tea pigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and ., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Liao, Z., Lieberburg, I., 3-r heng, J. G., Bora, S. H., Xu, G., Borchelt, D. R., Price, D. L., Koliatsos, V. E., 2003. igurdsson, E. M., Wisniewski, T., Frangione, B., 2002. A safer vaccine for Alzheimer's plays a entral role in regulating dendritic cell activation during infections that induce Th1 or attson, M. P., Flavell, R. ., Mullan, M., 1999. Microglial activation resulting from CD40-CD40L interaction after ation pathway. J Biol Chem. 275, 37224-31. P cells is required for their completion of a two-step activation process during central nervous system autoimmune inflammation. J Immunol. 176, 1402-10. Quinn, J., Montine, T., Morrow, J., Woodward, W. R 2 Alzheimer's disease. J Neuroimmunol. 137, 32-41. Remarque, E. J., Bollen, E. L., Weverling-Rijnsburger, A. W., Laterveer, J. C., Blauw, G. J. inflammatory phenotype. Exp Gerontol. 36, 171-6. Rezai-Zadeh, K., Shytle, D., Sun, N., Mori, T., Hou, H., Jeanniton, D., Ehrhart, J., Townsend, K., Zeng, J., Morga e reduces cerebral amyloidosis in Alzheimer transgenic mice. J Neurosci. 25, 8807-14. Schenk, D., Barbour, R., Dunn, W J. Motter, R., Mutter, L., Soriano, F., Shopp, G., Vasquez, N., Vandevert, C., Walker, S., Wogulis, M., Yednock, T., Games, D., Seubert, P., 1999. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature. 400, 177. Schenk, D. B., Yednock, T., 2002. The role of microglia in Alzheimer's disease: friend ofoe? Neurobiol Aging. 23, 677-9; discussion 683-4. S Lipopolysaccharide-induced-neuroinflammation increases intracellular accumulation of amyloid precursor protein and amyloid beta peptide in APPswe transgenic mice. Neurobiol Dis. 14, 133-45. S disease? Neurobiol Aging. 23, 1001-8. Straw, A. D., MacDonald, A. S., Denkers, E. Y., Pearce, E. J., 2003. CD154 c Th2 responses. J Immunol. 170, 727-34. Tan, J., Town, T., Paris, D., Mori, T., Suo, Z., Crawford, F., M A beta-amyloid stimulation. Science. 286, 2352-5. Tan, J., Town, T., Mullan, M., 2000. CD45 inhibits CD40L-induced microglial activvia negative regulation of the Src/p44/42 MAPK 140

PAGE 152

., ic e. ogo, T., Akiyama, H., Kondo, H., Ikeda, K., Kato, M., Iseki, E., Kosaka, K., 2000. own, T., Tan, J., Mullan, M., 2001a. CD40 signaling and Alzheimer's disease 1-2. Neurosci Lett. 307, 101-4. ., own, T., Tan, J., Flavell, R. A., Mullan, M., 2005. T-cells in Alzheimer's disease. ownsend, K. P., Town, T., Mori, T., Lue, L. F., Shytle, D., Sanberg, P. R., Morgan, D., ve J., 2000. CD40-CD40 ligand. J Leukoc Biol. 67, 2-17. kinase plasminogen-activator receptor in cultured uman central nervous system microglia. J Neurosci Res. 45, 392-9. N., y against Abeta in aged APP-transgenic mice verses cognitive deficits and depletes parenchymal amyloid deposits in spite of ., Gordon, M. N., 2007. Amyloid-beta accination, but not nitro-nonsteroidal anti-inflammatory drug treatment, increases Tan, J., Town, T., Crawford, F., Mori, T., DelleDonne, A., Crescentini, R., Obregon, DFlavell, R. A., Mullan, M. J., 2002a. Role of CD40 ligand in amyloidosis in transgenAlzheimer's mice. Nat Neurosci. 5, 1288-93. Tan, J., Town, T., Mullan, M., 2002b. CD40-CD40L interaction in Alzheimer's diseasCurr Opin Pharmacol. 2, 445-51. T Expression of CD40 in the brain of Alzheimer's disease and other neurological diseases. Brain Res. 885, 117-21. T pathogenesis. Neurochem Int. 39, 371-80. Town, T., Tan, J., Sansone, N., Obregon, D., Klein, T., Mullan, M., 2001b. Characterization of murine immunoglobulin G antibodies against human amyloid-beta 4 Town, T., Vendrame, M., Patel, A., Poetter, D., DelleDonne, A., Mori, T., Smeed, RCrawford, F., Klein, T., Tan, J., Mullan, M., 2002. Reduced Th1 and enhanced Th2 immunity after immunization with Alzheimer's beta-amyloid(1-42). J Neuroimmunol. 132, 49-59. T Neuromolecular Med. 7, 255-64. T Fernandez, F., Flavell, R. A., Tan, J., 2005. CD40 signaling regulates innate and adaptiactivation of microglia in response to amyloid beta-peptide. Eur J Immunol. 35, 901-10. van Kooten, C., Banchereau Washington, R. A., Becher, B., Balabanov, R., Antel, J., Dore-Duffy, P., 1996. Expression of the activation marker uro h Wilcock, D. M., Rojiani, A., Rosenthal, A., Subbarao, S., Freeman, M. J., Gordon, M. Morgan, D., 2004. Passive immunotherap re increased vascular amyloid and microhemorrhage. J Neuroinflammation. 1, 24. Wilcock, D. M., Jantzen, P. T., Li, Q., Morgan, D v vascular amyloid and microhemorrhage while both reduce parenchymal amyloid. Neuroscience. 144, 950-60. 141

PAGE 153

CHAPTER FOUR ZHEIMERS MICE RESULTS IN REDUCED CEREBRAL A DEPOSITS IN RATION AND MICROHEMORRHAGE ts are lacking. We have developed a novel e skin ion in TRANSCUTANEOUS A PEPTIDE IMMUNIZATION OF TRANSGENIC AL THE ABSENCE OF T-CELL INFILT 4.1 Abstract Alzheimers disease (AD) immunotherapy accomplished by vaccination with amyloid peptide (A has proved efficacious in AD mouse models. However, active A vaccination strategies for the treatment of cerebral amyloidosis without concurrent nduction of detrimental side effec i transcutaneous (t.c.) Avaccination approach and have evaluated efficacy and onitored for deleterious side effects, including meningoencephalitis and m m icrohemorrhage, in wild-type mice and in a transgenic mouse model of AD. We demonstrate that t.c. immunization of wild-type mice with aggregated A 1-42 plus the djuvant cholera toxin (CT) results in high-titer A antibodies (mainly of the a immunoglobulin G1 class) and A 1-42 -specific splenocyte immune responses. Confocal microscopy of the t.c. immunization site revealed Langerhans cells in areas of th containing the A 1-42 immunogen, suggesting that these unique innate immune cells participate in A 1-42 antigen processing. To evaluate the efficacy of t.c. immunizat 142

PAGE 154

reducing cerebral amyloidosis, transgenic PSAPP (APPsw, PSEN1dE9) mice were immunized with aggregated A1-42 peptide plus CT. Similar to wild-type mice, PSAPP mice showed high A antibody titers. Most importantly, t.c. immunization with A1-42 plus CT resulted in significant decr40, 42 levels coincident with incrbrt.c. immunization constitutes a novel, effectient strategy for ngles and s (Selkoe eal s this eases in cerebral A 1eased circulating levels of A 1-40, 42 suggesting brain-to-blood efflux of A. Reduction in cerebral amyloidosis was not associated with deleterious side-effects of ain T-cell infiltration or cerebral microhemorrhage. Together, these data suggest that ve and potentially safe treatm AD. 4.2 Introduction Alzheimers disease (AD) is the most common dementing illness and is pathologically characterized by the presence of intracellular neurofibrillary taextracellular senile plaques primarily composed of 40-42 amino acid A peptide2001). In a seminal report, Schenk and colleagues showed that intraperitonvaccination of the PDAPP transgenic mouse model of AD with A 1-42 plus Freundadjuvant resulted in dramatic reduction of cerebral amyloidosis (Schenk et al. 1999). This therapeutic approach is clearly highly efficacious; however, the safety of strategy has become an important concern. In a recent clinical trial, patients were administered a synthetic A peptide (AN-1792) plus adjuvant and approximately 6% ofthese patients developed aseptic meningoencephalitis, most likely mediated by brain-infiltrating activated T-cells (Hock et al. 2003; Bayer et al. 2005). This serious side143

PAGE 155

e t possible meningoencephalitis resulting from A vaccination, various strategies ave been attempted. Interestingly, recent works suggest that A-derived peptides l tissues (with adjuvant) results in effective clearanls of AD. t of A Such safe effect led to suspension of the clinical trial. Furthermore, passive transfer of A antibodies to transgenic AD mice results in cerebral microhemorrhage, a potentially adverse side-effect (Wilcock et al. 2004; Racke et al. 2005). Uncovering of these adversevents has re-directed A vaccination strategies towards the goal of developing an approach that is both safe and effective. Studies examining the brains of A vaccinated patients developing meningoencephalitis implicate A reactive T-cell subsets as major components of this deleterious response to active A vaccination (Nicoll et al. 2003; Ferrer et al. 2004). To subver h delivered intranasally to mucosal epithelia ce of A plaques and improvement of cognitive function in animal modeMoreover, T-cell reactivity appeared to be considerably reduced compared with other active immunization strategies (Maier et al. 2005; Maier et al. 2006). In other studies, differential T-cell responses were observed dependent upon the epitope/fragmenpeptide utilized for vaccination. Specifically, portions of the A peptide seemed to stimulate different T-cell responses, resulting in either pro-inflammatory T-helper cell type 1 (Th1) responses, or anti-inflammatory T-helper cell type 2 (Th2) responses. findings imply that A vaccination is not only efficacious, but may also prove to beand therefore a feasible strategy for AD therapy depending upon a number of factors including route of delivery, adjuvant choice, and A epitope administered. The skin is a well-established effective route for vaccination, including delivery 144

PAGE 156

. s al hans cell (LC) precursors, known as migratory CD14+rofessional Keratinocyte-derived IL-10 serves to buffer harmful pro-inflammatory immune activation and therby preserves skin barrier integrity (Niizeki; Streilein 1997). Taken together, these lines of evidence led us to hypothesize that targeting A immunotherapy to skin tissue may provide an immunotherapeutic approach that is both efficacious and safe. To evaluate this hypothesis, we tested a t.c. A immunization strategy using both wild-type and the transgenic PSAPP (APPsw, PSEN1dE9) mouse model of AD (Jankowsky et al. 2001). We found that t.c. immunization of non-transgenic C57BL/6 mice with aggregated A1-42 peptide plus the adjuvant cholera toxin (CT) resulted in high A antibody titers [mainly immunoglobulin (Ig) G1], and A-specific splenocyte immune responses after re-challenge with the peptide. Confocal microscopy of the t.c. immunization site revealed CD207 and CD11c double-positive Langerhans cells in areas of the skin containing the A immunogen, suggesting that these unique innate immune cells participate in A antigen processing. Further, of peptide-based vaccines (Beignon et al. 2005; Itoh; Celis 2005; Dell et al. 2006)Strong humoral and cellular immune responses have been elicited after transcutaneou(t.c.) vaccination (Giudice; Campbell 2006), largely owing to the diverse populations of resident antigen presenting cells (APCs) and other immune cells in the various dermlayers. Subsets of dermal-resident Langer LC precursors, are important immune regulators that demonstrate pAPC capability including reducing T-cell stimulatory function by producing anti-inflammatory cytokines (Larregina et al. 2001). Also, skin-resident keratinocytes releasethe anti-inflammatory cytokine interleukin (IL)-10 in response to certain stimuli. 1-42 1-42 1-42 145

PAGE 157

l. r ately obtained from R&D Systems (Minneapolis, MN). A1-40 1-42 ELISA kits were transgenic PSAPP mice t.c. immunized with aggregated A 1-42 peptide plus CT manifested effective immune responses against A in concert with reduced cerebral Apathology, demonstrating the effectiveness of this approach. These mice showed high A antibody titers and increased circulating Alevels, suggesting brain-to-blood effluxof A. Importantly, brain T-cell infiltration and cerebral microhemorrhage were not observed after t.c. immunization, indicating that this immunization strategy is potentially safe. 4.3 Materials and methods 4.3.1 Reagents. Lyophilized cholera toxin (CT), CT antibody, concanavalin (Con A) and mouseCD3 antibody were obtained from Sigma (St Louis, MO). A 1-42 peptide was purchasedfrom U.S. peptides (Rancho Cucamonga, CA). As previously described (Schenk et a1999), A 1-42 peptide was added to 0.9% saline (4 mg/mL), vortexed, and incubated fo24 h at 37 C. This solution was aliquoted, frozen and stored at 80 C. Immediprior to use, A aliquots were thawed and then mixed with CT (reconstituted with distilled water to 0.5 mg/mL). Antibody 4G8 directed against A (amino acids 17-26) was purchased from Chemicon/Millipore (Billerica, MA). Enzyme-linked immunoabsorbance assay (ELISA) kits for detection of IFN-, IL-2, and IL-4 were and Apurchased from IBL-American (Minneapolis, MN). Murine IgG and HRP-conjugated 146

PAGE 158

L). es nimals Wild-type C57BL/6 and PSAPP (APPsw, PSEN1dE9) mice were obtained from or, ME). Animals were housed and maintained in the ollege of Medicine Animal Facility at the University of South Florida, and all e in compliance with protocols approved by the University of South Florida rrg/m goat anti-mouse IgG were obtained from Pierce Biotechnology, Inc. (Rockford, IBiotinylated anti-mouse IgG1, IgG2a and IgG2b were purchased from Zymed Laboratories (South San Francisco, CA). Alexa-Fluor-conjugated secondary antibodi(including Alexa-Fluor 488 594 and 647 ) were purchased from Invitrogen (Carlsbad, CA). Lactate dehydrogenase (LDH) kit was obtained from Promega (Madison, WI). 4.3.2 A Jackson Laboratory (Bar Harb C experiments wer Institutional Animal Care and Use Committee. Brain sections from mice inducedwith experimental autoimmune encephalomyelitis were provided by Dr. Terrence Town and used as a positive control for CD3 staining. Brain sections for positive microhemohage staining from mice intraperitoneally passively given A antibodies were provided by Dr. Dave Morgan (Wilcock et al. 2006). 4.3.3 Transcutaneous (t.c.) immunization of mice To test whether t.c. delivery of A peptide could stimulate immune responses, we first t.c. immunized wild-type C57BL/6 mice. These mice (n = 10, 5 male/5 female) were t.c. immunized with human A 1-42 peptide (200 ouse) and CT (10 g/mouse) or CT alone (10 g/mouse) in 100 L of 0.9% saline on a weekly basis for the first 147

PAGE 159

e bi-ach munization, mice were anaesthetized. A small lower back section (1-2 cm2) was extra precaution not to damage the skin. The skin was then swabbed with acetonen. ed as withdrawn or PP mice (n = 9, 4 male/5 ale) at 4 months of age using the same procedure described above. No mortality was nd none of the animals exhibited sympto month. Thereafter, these mice were continually t.c. immunized with A 1-42 (100 g/mouse) and CT (5 g/mL) or CT alone (5 g/mouse) in 100 L of 0.9% salinweekly for the following 12 weeks. Transcutaneous immunization was performed according to a method described by Skelding and colleagues (Skelding et al. 2006) with minor changes. To ensure mice immobility for the duration of administration for e im shaved with an to remove surface oils and enhance penetration, allowed to air dry, and then re-hydrated by swabbing with 0.9% saline. The shaved edge was coated with a thin petroleum jelly layer to prevent unnecessary leakage of the immunization solutioLastly, 100 L of A 1-42 in combination with CT or CT alone in 0.9% saline was placon the shaved region and allowed to be absorbed for 2 h. At the end, the skin was washed with 0.9% saline and dried, so as to remove any remaining immunization solution. Mice were cleaned thereafter and returned to their cages. Blood won weeks 0 (immediately prior to the first immunization), 4, 8 and 16 (immediately prito the sacrifice of these mice). We then t.c. immunized PSA fem observed over the course of the t.c. immunization a ms causing their removal from the study. 4.3.4 Splenocyte cultures. Cell suspensions of splenocytes from individual mice were prepared as 148

PAGE 160

ll plates okine red nd 207 ti-mouse CD11c (1:50; Pierce iotechnology, Rockford, IL), and/or anti-human A antibody (clone 4G8; 1:500; ica, MA) or rat anti-mouse CD3 antibodies (1:200; eBioscinjugated previously described (Town et al. 2002) and passed in 0.5 mL aliquots into 24-weat 3 10 6 /mL. These cells were treated for 48 h with ConA (5 g/mL), anti-CD3 (1 g/mL) or A 1-42 (20 g/mL). Supernatants were then collected and assayed by cytELISA kits in strict accordance with the manufacturer's instruction (R&D Systems). TheBio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) was performed to measuretotal cellular protein from each of the cell groups under consideration just prior to quantification of cytokine release by ELISA, and cytokine secretion is expressed in pg/mg total cellular protein (mean SD). To verify whether stimulation of splenocytes produced any between-groups differences on cell death that might account for altecytokine profiles, LDH release assay was carried out as described (Tan et al. 2000) aLDH was not detected in any of the wells studied. 4.3.5 Immunofluorescence staining. Non-transgenic C57BL/6 mice were transcutaneouslly treated with A/CT, CT alone, or PBS for 18 h, as described above. The dorsal skin was removed by careful razor slicing around pre-labeled regions (1.5 cm in diameter) where the vaccine was applied. Skin or brain samples were routinely prepared for immunofluorescence staining. The staining was curried out using the following primary antibodies: anti-mouse CD(Langerin; 1:250; eBioscience San Diego, CA), an B Chemicon/Millipore, Biller ence) overnight at 4C, followed by appropriate secondary antibodies co 149

PAGE 161

ng .6 (coating buffer) in 96-well munoassay plates overnight at 4 C. The plates were washed with 0.05% Tween 20 in locked with blocking buffer (PBS with 1% BSA and 5%in 37 C. and 4) was with AlexaFluor 488 -594 and/or -647 (1:500; Invitrogen) for 45 min. Sections were then washed 3 times in PBS, and mounted with fluorescence mounting media containing DAPI (Vector Laboratories, Inc., CA) to counter-stain cell nuclei, and then viewed under an Olympus BX-51 microscope or visualized in independent channels using a Zeiss LSM510 META confocal microscope equipped with a 2-photon laser that was used for exciting DAPI. 4.3.6 A antibody ELISA. A antibodies in mouse plasma and brain homogenates were measured accordito previously described methods (Maier et al. 2005). Briefly, human A 1-40 peptide wascoated at 2 g/mL in 50 mM carbonate buffer, pH 9 im PBS (washing buffer) five times and b horse serum) for 2 h at room temperature. Murine IgG was serially diluted in coating buffer (1,000 0 ng/mL) to generate a standard curve. Mouse plasma and brahomogenate samples were diluted in blocking buffer at concentrations ranging from 1:400 to 1:102,400, added to the plates, and incubated for 2 h at room temperature. After 5 washes with washing buffer, a detection antibody (HRP-conjugated goat anti-mouse IgG, 1 mg/mL) was diluted (1:4,000), added to the plates and incubated for 1 h at Following 8 washes, tetramethylbenzidine (TMB) substrate was added to the platesincubated for 15 min at room temperature. Fifty L of stop solution (2 N N 2 SOadded to each well to terminate the reaction. The optical density of each well was 150

PAGE 162

de wells were blocked with blocking buffer for 2 h at room temperature. ouse plasma was serially diluted in blocking buffer, added to each well, and incubated ashes, the plates were incubated in secondary antibodies (biotinoom le immediately determined by a microplate reader at 450 nm. A antibodies were represented as ng per mL of plasma (mean SD). ELISAs for A antibody isotypes were carried out as previously described (Town et al. 2001; Lemere et al. 2002). Ninety-six well plates were coated with A 1-40 pepti(2 g/mL) in coating buffer and incubated for 2 h at 37 C. Following three washes in washing buffer, M for 1 h at 37 C. After five w ylated anti-mouse IgG1, IgG2a or IgG2b) diluted at 1:1,000 in blocking buffer for 1 h, followed by 30 min in streptevidin-HRP (1:200 in blocking buffer) at room temperature. TMB substrate was added to the plates and incubated for 15 min at rtemperature. Fifty L of stop solution was added to each well to terminate the reaction. The optical density of each well was immediately determined by a microplate reader at 450 nm. The ratios of IgG1 to IgG2a or IgG1 to IgG2b were calculated for each time point from each mouse individually using optical density values and then average ratio for each group (mean SD). 4.3.7 A ELISA. Mouse brains were isolated under sterile conditions on ice and placed in ice-cold lysis buffer as previously described (Rezai-Zadeh et al. 2005). Brains were then sonicated on ice for approximately 3 min, allowed to stand for 15 min at 4C, and centrifuged at 15,000 rpm for 15 min. This fraction represented the detergent-solub 151

PAGE 163

ogenates followed by a 1:10 dilution in lysis on with the VectaStain Elite ABC kit (Vector aboratories, Burlingame, CA) coupled with diaminobenzidine substrate. 4G8-positive examined under bright field using an Olympus (Tokyo, Japan) BX-51 micros diluted 70% fraction. A 1-40, 42 species were further subjected to acid extraction of brain homin 5 M guanidine buffer (Johnson-Wood et al. 1997) buffer. Soluble A 1-40, 42 were directly detected in plasma and brain homogenates prepared with lysis buffer described above at a 1:4 or 1:10 dilution, respectively. A 1-40,42 was quantified in these samples using the A 1-40, 42 ELISA kits (IBL-America, Minneapolis, Minnesota) in accordance with the manufacturers instructions, except that standards included 0.5 M guanidine buffer in some cases. A 1-40, 42 were represented as pg per mL of plasma and pg per mg of total protein (mean SD). 4.3.8 Immunohistochemistry and image analysis. As previously described (Tan et al. 2002), five coronal sections from each brain (5 m thickness) were cut with a 150 m interval. Sections were routinely deparaffinizedand hydrated in a graded series of ethanol before preblocking for 30 min at ambient temperature with serum-free protein block (Dako Cytomation, Carpinteria, CA). A immunohistochemical staining was performed using anti-human amyloidantibody (clone 4G8; 1:100) in conjuncti L A deposits were cope. For congo red histochemistry, sections were routinely deparaffinized and rinsed in 70% (v/v) ethanol before staining with fresh-filtered 1% (w/v) congo redin 70% ethanol for 5 min. These sections were rinsed three times for 5 min each inethanol, hydrated for 5 min in 0.9% saline, and mounted in Vectashield fluorescence 152

PAGE 164

ith an (Hain constant throughout the analysis session. We do not adjust thresholds for each to the estimation. Section-to-section variability in immunostaining is software then corrects for heterogeneity in background illumination (blank field correction) and calculates the measurement parameters for the entire field. For quantitative image analysis of immunohistochemistry, we combined unbiased sampling of sections with the videodensitometric procedures we have used extensively in the past (Wilcock et al., 2004). All procedures were performed by an individual blind to the experimental condition of each specimen. Sample numbers were randomized before the start of the tissue processing, and code was broken only after completion of the analysis. We sampled every fifth section from the mouse brain, starting with a section between 1 and 5, identified by a random number generator. Frontal cortical mounting media(Vector Laboratories). Congo red-positive -amyloid plaques were visualized using an Olympus BX-51 microscope. Quantitative image analysis was performed for both immunohistochemistry and Congo red-stained tissue sections wOncor V150 image analysis system. The software uses hue, saturation, and intensity SI) to segment objects in the image field. Operationally, thresholds for object segmentation are established by using a series of standard slides that have extremes of intensity for the stain being measured. Thresholds in HSI space are established that accurately identify objects on all standard slides, and these segmentation thresholds rem section to avoid the introduction of experimenter bias in minor, owing to rigid fixation and staining protocols. In most experiments using the same reaction product (e.g., DAB-peroxidase), the same thresholds settings can be used for different antibodies. After establishing the threshold parameters, the image field is digitized with a frame grabber. The computer 153

PAGE 165

uct. ea that e d by washed a) for 15 secondn VA) le measurements were performed on laminae II-VI of the most anterior portion of the cortex. Hippocampal measurements were performed by circumscribing the entire hippocampus or selected hippocampal subfields. Using the standard nomenclature, "A load" is defined as percent of area in the measurement field occupied by reaction prodSimilarly, "Congo red staining" (or "amyloid load") refers to the percent of the aris stained with Congo red. All values from a single mouse were averaged to represent thsingle value for that animal in statistical analyses. Statistical analyses were performeusing ANOVA followed by Fischer's LSD post hoc means comparison test (Statviewsoftware from SAS). The numbers of animals, not numbers of sections, have been usedfor statistical comparisons. 4.3.9 Perls Prussian blue reaction for ferric ion-hemosiderin. Sections were deparaffinized, hydrated through descending grades of ethanol, washed in distilled water, and incubated for 20 min in a solution containing 20% hydrochloric acid and 10% potassium ferrocyanide (VWR). These sections were3 X for 5 min with H 2 0 and counterstained with hematoxylin solution (Sigm s and then mounted (Wilcock et al. 2006). 4.3.10 Statistical analysis. Means and standard deviations were calculated according to standard practice. Iinstances of multiple comparisons of the means, one-way analysis of variance (ANOwas carried out with post-hoc comparison by Bonferronis method. In instances of singmean comparisons, a t-test for independent samples was used to assess significance. P 154

PAGE 166

sed c., tion. Twenty non-transgenic C57BL/6 mice at eight weeks f age were used for this experiment, and ten mice received aggregated A1-42 peptide ice were t.c. immunized over a 16-w antibody isotypes were determined by an IgG isotyping assay using an isotype-specific secondary antibody (Town et al. 2001). A antibodies were first detected at week 4 in all immunized mice and dramatically increased thereafter [(Figure 14A) P <0.001]. Consistent with our previous studies that utilized an intraperitoneal route of administration of A plus Fruends adjuvant (Town et al. 2002), A antibodies of the IgG1 isotype were produced values less than 0.05 were considered to be statistically significant. Data were analyusing the Statistical Package for the Social Sciences (SPSS), release 13.0 (SPSS InChicago, IL). 4.4 Results 4.4.1 Transcutaneous immunization of mice with A 1-42 peptide plus cholera toxin results in high A antibody titers. We first sought to determine whether t.c. administration of A 1-42 to mice could result in A antibody produc o with CT (A/CT), while the remaining ten received CT alone. M eek time course; weekly for the first 4 weeks and bi-weekly for the following 12 weeks. To characterize the kinetics of the humoral immune response following immunization, blood samples were taken at week 0 (baseline, immediately prior to the first immunization), 4, 8 and 16 (immediately prior to the sacrifice of these mice). Plasma A antibody titers were measured by ELISA. A 155

PAGE 167

re utaneous immunization was further evaluated by assaying CT antibody ters in plasma from these mice at weeks 0, 4, 8 and 16 following immunization. A n of results was observed, albeit CT antibody titers were higher in plasma om mice t.c. immunized with either A/CT compared with CT alone (data not shown). we were unable to detect A antibody titers in these animal at the highest level, while IgG2a antibodies directed against Awere present in significantly lesser quantity (P <0.001). A IgG2b antibody was least detectable (Figu14B, C). Transc ti similar patter fr As an additional control group, we injected non-transgenic C57BL/6 mice with PBS alone (n = 10) in parallel, and s, confirming the specificity of our titer assay (data not shown). 156

PAGE 168

157 Figure 14

PAGE 169

158

PAGE 170

159

PAGE 171

Figure 14. Generation of immune responses in wild-type C57BL/6 mice t.c. immunized with aggregated A1-42 peptide plus CT. (A) A antibody titers were measured by ELISA. Data are presented as mean SD (n = 10) of A antibodies (ng/mL plasma). One-way ANOVA followed by post hoc comparison revealed significant differences in anti-A titers when comparing week 4 to weeks 8, 12, or 16 (**P < 0.001). IgG isotypes were determined by an Ig isotyping assay and represented as ratios (mean SD; n = 10) of IgG1 to IgG2a (B) or IgG1 to IgG2b (C). One-way ANOVA followed by post hoc comparison revealed significant differences between the ratio of IgG1 and IgG2a versus IgG1 and IgG2b at each week shown (**P < 0.001). (D) Splenocytes were individually isolated and cultured from wild-type mice t.c. immunized with A1-42/CT, CT alone, or PBS (control). These cells were stimulated with Con A (5 g/mL) or A42 (20 L) for 48 h. Cultured supernatants were collected from these cells for IFN-, IL-2 and IL-4 cytokine analyses by ELISA. Data are presented as relative fold mean SD (n = 10) of each cytokine over PBS control. One-way ANOVA followed by post hoc comparison revealed significant differences between groups for levels of each of three cytokines [IFN-, IL-2 and IL-4 (**P <0.001)] following in vitro A1-42 challenge. As noted, there was also a significant difference in cytokine levels between IL-4 and either IFNor IL-2 following A1-42 challenge (##P <0.001). (E) To characterize the dermal immune responses to A/CT t.c. immunization, skin tissues were prepared from non-transgenic C57BL/6 mice t.c. immunized for 18 h with PBS (control, top panels), CT alone (middle panels) or with A/CT (bottom panels) as indicated and then analyzed by laser scanning confocal microscopy with the indicated antibodies (antibody 4G8 was used to reveal A). g/m 160

PAGE 172

161 Note the presence of CD207+CD11c+ LCs in A-positive regions in the A/CT t.c. immunized group.

PAGE 173

Figure 15 162

PAGE 174

163 Figure 15. A/CT t.c. immunization resulted in LC recruitment into dermal layers. To characterize the dermal immune responses to A/CT t.c. immunization, skin tissues were prepared from nontransgenic C57BL/6 mice treated with A/CT (Left), CT alone (Center), or PBS (Right) as indicated for 18 h and then analyzed. Skin frozen sections were stained with anti-mouse CD207 antibody/FITC-labeled anti-rat IgG2a, rabbit anti-human A antibody/Alexa Fluor-555-labeled anti-rabbit IgG, and DAPI. As noted, blue staining indicates all nucleated cells, ande green indicates CD207 positive cells (presumed LC, ).

PAGE 175

4.4.2 A-specific immune responses in from mice transcutaneously immunized with A plus cholera toxin. To further investigate splenocyte responses to A, primary cultures of splenocytes were established from individual mice (non-transgenic C57BL/6) t.c. immunized with A/CT, CT alone, or PBS only (control) and immune responses were compared. We quantified key cytokines produced by activated T-cells [interferon(IFN-, interleukin (IL)-2, IL-4] in splenocyte supernatants by ELISA as an indicator of immune responsiveness. Non-specific mitogenic stimulation of cultured splenocytes with concanavalin A (Con A) resulted in over two-fold increases in IFN-, IL-2, and IL-4 production in cells from mice immunized with A/CT or CT versus PBS-immunized controls (Figure 14D). No statistically significant difference was noted between A/CT and CT alone groups for each cytokine (P >0.05). Similar results were observed in cultured splenocytes stimulated with anti-CD3 (data not shown). On the other hand, specific recall stimulation with A1-42 peptide of primary cultured splenocytes from A/CT-t.c. immunized mice resulted in significantly increased production of IFN-, IL-2 and IL-4 compared to splenocytes cultured from mice immunized with CT alone (P <0.001, Figure 14D). Importantly, regarding the anti-inflammatory cytokine IL-4, an 8-fold increase in its secretion by splenocytes from A/CT-immunized mice following A1-42 recall stimulation was observed (P < 0.001) (Figure 14D). Taken together with the predominantly IgG1 A-specific humoral response in A/CT-t.c. immunized wild-type mice, this IL-4 result suggests an anti-inflammatory Th2-type immune response. splenocytes 164

PAGE 176

165 4.4.3 Transcutaneous immunization with A plus CT promotes recruitment of dermal Langerhans cells. Given that LCs often play a key role in skin immune responses, we asked whether LCs might be recruited to the site of t.c. A/CT immunization. Skin tissues and frozen sections were prepared from non-transgenic C57BL/6 mice 18 h after t.c. vaccination with A/CT or CT alone, and they were co-stained with antibodies against mouse CD207 antibody (Langerin, a pan-LC marker), mouse CD11c (a marker of an LC subset (Douillard et al. 2005) and/or rabbit anti-human A. A/CT t.c. immunization resulted in LC recruitment into dermal layers compared with CT alone or PBS-immunized controls (Figure 14E and Figure 15), where dermal LCs were much less frequently observed. Furthermore, these LCs tended to be found in regions of the skin that stained positive for A peptide by 4G8 A antibody (Figure 14E). These data show the migratory action of LCs in response to A/CT t.c. stimulation and suggest that this effect is important in mediating the initial immune response to A/CT t.c. immunization. 4.4.4 Transcutaneous immunization of PSAPP mice with A plus CT results in A-specific immune response and increased circulating A. Having shown high A antibody titers and cultured splenocyte immune responses to A in A/CT t.c. immunized non-transgenic C57BL/6 mice, we then asked whether this immunization strategy would reduce cerebral amyloidosis in a mouse model of AD. Eighteen double-transgenic (APPswe/PSEN1dE9) PSAPP mice, which overproduce human A and develop significant amyloid deposits by 8 months of age (Jankowsky et

PAGE 177

= 9) CT, while the remaining half received CT alone. The 16 ce, A PP t he 1-42 rly al. 2001), were immunized at four months of age in this study. Half of them (nreceived aggregated A 1-42 peptide with -week procedure that we employed was identical to that used above for non-transgenic C57BL/6 mice. Blood samples were taken at weeks 0, 4, 8 and 16 followingimmunization. Plasma A antibody titers from these mice were measured by ELISA. Significant increases in A antibody titers were observed in PSAPP mice t.c. immunized with A/CT (P < 0.001) (Figure 16A). Similar to non-transgenic C57BL/6 miantibodies were first detected at week four in plasma from A/CT-immunized PSAmice, and dramatically increased thereafter. By contrast, these Aantibodies were nodetected in plasma from CT-vaccinated control mice (Figure 16A). Two weeks after tfinal immunization, primary splenocytes were isolated and cultured from individual miceRecall stimulation of splenocytes from A/CT-t.c. immunized PSAPP mice with A peptide resulted in significantly increased production of IFNand IL-2, and particulaIL-4 (data not shown), similar to results from A/CT-t.c. immunized non-transgenic C57BL/6 mice. 166

PAGE 178

Figure 16 167

PAGE 179

were t #P Figure 16. Increased systemic Aafter A 1-42 /CT t.c. immunization of PSAPP mice. For A analysis, blood samples were individually collected from A/CT or CT alone t.c.-immunized PSAPP mice at the time points indicated. (A) Plasma A antibody titersmeasured by ELISA. Data are presented as mean SD (n = 9) of A antibodies (pg/mL plasma). ANOVA followed by post hoc comparison revealed significant differences between A/CT and CT t.c.-immunized PSAPP mice for plasma A antibody levels at each time point as indicated (**P <0.001). Moreover, this analysis revealed significandifferences between time points within the A/CT t.c. immunized group as indicated ( #<0.001). (B and C) Plasma A peptides were measured separately by A ELISA. Data are presented as mean SD (n = 9) of A or A (pg/mL plasma). One-way ANOVA followed by post hoc comparison revealed significant differences between A/CT and CT alone t.c.-immunized PSAPP mice for plasma A levels at each time point as indicated (*P <0.05; **P <0.001). Arrows below the panels show each t.c. immunization with respect to time of blood sample collection. 1-40, 42 1-40 1-42 168

PAGE 180

169 It has been hypothesized that passively-administered A antibodies create a peripheral sink; in essence, shifting the equilibrium of A levels from the brain to the blood (DeMattos et al. 2001; Matsuoka et al. 2003). To determine whether this phenomenon was occurring in the A/CT-t.c. immunization paradigm, we quantified A levels in the blood by ELISA. In support of this hypothesis, we found significantly increased circulating A1-40, 42 in PSAPP mice t.c. immunized with A/CT as early as 4 weeks after immunization (Figure 16B and C). Importantly, plasma A levels increased rapidly to the highest values of 781 118 pg/mL and 129 46 pg/mL, respectively, by week 8 (two weeks after the third booster t.c. immunization). Thereafter, plasma A levels remained relatively constant through to the time of sacrifice following 16 weeks of immunization. 4.4.5 PSAPP mice transcutaneously immunized with A plus cholera toxin show reduced cerebral amyloidosis in the absence of T-cell infiltrates or cerebral microhemorrhage. Having shown significantly increased circulating A levels in PSAPP mice t.c. immunized with A/CT, we sought to evaluate A/-amyloid pathology in these mice. Using a sandwich ELISA-based method, detergent-soluble A1-40, 42 levels were reduced by approximately 53 and 48%, respectively (P <0.001) (Figure 17A). Insoluble A1-40, 42 (prepared by acid extraction of detergent-insoluble material in 5 M guanidine and subsequent ELISA) levels were reduced by 50 and 54%, respectively, in A/CT t.c.-immunized PSAPP mice (P <0.001) (Figure 17B). We further analyzed -amyloid plaques in brains of mice that received t.c. immunization with A/CT or CT alone by

PAGE 181

sis antibodies in brain homogenates from A/CT-t.c. immunized PSAPP mice [18.87 6.25 (mean ng/mg total protein SD)], while A antibodies were undetectable in PSAPP mice t.c. immunized with CT-alone (data not shown). Given the presence of these A antibodies in the brain, it is possible that additional A clearance mechanisms (i.e., mediated by the Fc receptor on phagocytic microglia) are operating. 4G8 immnunohistochemistry and congo red histochemistry (Figure 17D and F, respectively). At 10 months of age, A/CT-t.c. immunized PSAPP mice showed 42-58% (Figure 17E) and 61-65% (Figure 17G) reductions in 4G8 immunoreactive and congo red-positive A deposits, respectively, across hippocampal and cortical brain regions examined. Together, these results demonstrate that t.c. immunization with A/CT is effective in reducing cerebral amyloidosis in PSAPP mice. As shown above, we found that peripheral levels of human A 1-40, 42 were increased in mice t.c. immunized with A/CT. To determine this systemic increase in human A 1-40, 42 was associated with a reduction in cerebral A levels, we performed correlation analysis and noted an inverse correlation between plasma and brain soluble A (Figure 17C and Figure 18), suggesting that circulating A antibodies play an important role in clearance of A from brain to blood via the peripheral sink hypothe(DeMattos et al. 2001; Matsuoka et al. 2003). This effect is likely not solely responsible for reduced cerebral amyloidosis, as, interestingly, we detect A 170

PAGE 182

Figure 17 171

PAGE 183

172

PAGE 184

173

PAGE 185

174

PAGE 186

175 175 Figure 17. Reduction of cerebral A/-amyloid pathology in PSAPP mice t.c. immunized with A1-42/CT. Detergent-soluble A1-40, 42 peptides (A) and insoluble A1-40, 42 prepared from 5 M guanidine extraction (B) in brain homogenates were measured separately by ELISA. Data are presented as mean SD (n = 9) of A1-40 or A1-42 (pg/mg protein), and reductions for each group are indicated. (A and B), A t test revealed a significant between-group difference for either soluble or insoluble A1-40, 42 (P < 0.001). (C) A significant inverse correlation (P < 0.001) between plasma and brain soluble A levels was revealed. Plasma A levels are presented as percentage mean SD (n = 9) of soluble circulating Aat 16 weeks following t.c. immunization of PSAPP mice with A/CT over CT control mice. (D) Mouse brain coronal paraffin sections were stained with monoclonal anti-human A antibody 4G8. Left, A1-42/CT t.c. immunized PSAPP mice; right, CT t.c. immunized PSAPP mice. The top panels are from the cingulate cortex (CC), the middle panels are from the hippocampus (H), and the bottom panels are from the entorhinal cortex (EC). (E) Percentages (plaque burden, area plaque/total area) of A antibody-immunoreactive A plaques (mean SD; n = 9) were calculated by quantitative image analysis, and reductions for each mouse brain area analyzed are indicated. (F) Mouse brain sections from the indicated regions were stained with congo red. Left, A1-42/CT t.c. immunized PSAPP mice; right, CT t.c. immunized PSAPP mice. (G) Percentages of congo red-stained plaques (mean SD; n = 9) were quantified by image analysis, and reductions for each brain region are indicated. (E and G) a t test for independent samples revealed significant differences (P < 0.001) between-groups for each brain region examined.

PAGE 187

176 Figure 18

PAGE 188

byFigure 18. Simultaneous analysis of plasma and brain soluble A levels on a mouse-mouse basis. Data are presented as percentage of circulating or brain soluble A at 16 weeks after A/CT t.c. immunization of individual PSAPP mice (n = 9) relative to average A levels of control mice t.c. immunized with CT alone. Each color bar represents plasma A levels and brain-soluble A levels from each individual mouse as indicated. 177

PAGE 189

In a recent clinical trial, patients peptide (AN-1792) plus adjuvant and approximately 6% of these patients developed aseptic meningoencephalitis, most likely mediated by brain-infiltrating activated T-cells (Hock et al. 2003; Bayer et al. 2005). This serious complication led to suspension of the clinical trial. We sought to investigate whether A/CT t.c. immunization might induce T-cell infiltration into the brain. We immunostained brain sections from mice immunized with A/CT, CT alone, or, as a positive control, non-transgenic C57BL/6 mice subcutaneously injected with myelin oligodendrocyte protein emulsified in complete Freunds adjuvant (brains were isolated 20 days after immunization, when copious amounts of T-cells have infiltrated the brain). As shown in Figure 19A-C, we did not detect CD3-positive T-cells in brains from mice t.c. immunized with CT or A/CT; however, this was not due to a technical issue as T-cells were detected in the positive control tissue (Figure 19A). Further, we also immunostained brain sections from the above mice with CD4 or CD8 antibodies (which stain different subsets of T-cells) and did not detect T-cell infiltration in brains of mice t.c. immunized with CT or A/CT, while such cells were detected in our positive control tissue (data not shown). It has been reported that mice receiving passive transfer of A antibodies manifest cerebral microhemorrhage (Wilcock et al. 2004; Racke et al. 2005). To investigate this potentially adverse side-effect of A/CT t.c. immunization, we carried out microhemorrhage analysis via Perls Prussian blue stain (which can detect even minute amounts of deposited iron derived from blood (Wilcock et al. 2004; Racke et al. 2005). We did not detect positive staining using this method in either t.c. immunized group, but were administered a synthetic A 178

PAGE 190

tissues that both the Prussian blue stain and apolipoprotein B analyses were negative in t.c. immunized PSAPP mice, suggesting that detection of A antibodies in the brains of these mice (as mentioned above) was not due to poor perfusion efficiency or detectable breakdown of the blood-brain-barrier, but rather was likely due to physiological entry of A antibodies into the brain parenchyma. did observe staining in our positive control tissue [in this case, from mice intraperitoneally passively given A antibodies (Wilcock et al. 2006)] (Figure 19D-F). As an additional indicator of possible blood-brain-barrier breakdown, we analyzed apolipoprotein B (present in blood but not normally in brain) levels in these brainby Western blot and it was undetectable in the t.c. immunized groups (Figure 20). It is noteworthy 179

PAGE 191

Figure 19 180

PAGE 192

/CT t.c. was panel is the neocortex. Magnification for CD3 staining is 10 X while it is 20 X for microhemorrhage. Figure 19. Absence of T-cell infiltration or brain microhemorrhage in Aimmunized mice. To characterize the safety of the vaccine, brain sections were stained for CD3 as an indicator of T-cell infiltration (top panels). Staining for hemosiderin also performed to identify microhemorrhage (bottom panels) in mice immunized with A/CT (B and E), or CT alone (C and F). These stained sections were compared to positive controls. For CD3 staining, the positive control consisted of CD3-positive brain sections from EAE mice (A). For microhemorrhage, experimental sections were compared to sections from mouse brains suffering microhemorrhage (D). Each panel is representative of staining repeated in triplicate for each brain section for either CD3 or hemosiderin. The brain region shown for each 181

PAGE 193

182 Figure 20

PAGE 194

nd n Figure 20. Apolipoprotein B protein was undetectable in brain tissue homogenatesderived from both A/CT and CT t.c.-immunized mice. Brain homogenates were prepared from PSAPP mice t.c.-immunized with A/CT or CT, and an aliquot corresponding to 100 g of total protein was electrophoretically separated by using 4%SDS-polyacrylamide gels. In addition, a series of mouse plasma dilutions (100, 50, a25 g per lane) was loaded as positive controls. Western blot analysis by apolipoprotein B antibody (ab20737, Abcam, Inc, Cambridge, MA) shows apolipoprotein B100 iplasma but not in brain homogenates as indicated. 183

PAGE 195

184 4.5 Discussion To translate animal A immunization approaches into successful clinical AD therapies, such strategies should not only be efficacious, but also be safe, including avoiding meningoencephalitic reactions to A immunization previously observed in humans (Janus 2003; Monsonego et al. 2003). Experimental and postmortem evidence suggests that such aseptic meningoencephalitis observed in AD patients after A vaccination resulted from CNS invasion by A reactive T-cells (Monsonego et al. 2003; Maier et al. 2006). The requirement of A-reactive T-cells for cerebral amyloid plaque clearance mediated by active A vaccination strategies still remains unclear (Monsonego et al. 2003; Orgogozo et al. 2003; Ferrer et al. 2004). A previous study utilizing intranasal delivery of short A-derived peptides lacking T-cell reactive epitopes with a specific immune-modulating adjuvant (mutant Escherichia coli heat-labile enterotoxin; LT R192G), demonstrated the possibility of potentiating an effective humoral anti-A response while minimizing A reactive T-cells (Maier et al. 2006), suggesting that A-reactive T-cells are not necessary for an effective A antibody response. If this proves to be true in humans, such an approach may represent reasonable therapeutic potential; however, it should be noted that anosmia/hyposmia may limit the usefulness of intranasal A immunization. Further evidence that A-reactive T-cells are likely not required for Aimmunotherapy efficacy comes from passive immunization studies, which have shown that humoral responses alone may be sufficient to effectively reduce cerebral amyloid burden, and thereby mitigate neurodegeneration (Dickstein et al. 2006; Ma et al. 2006).

PAGE 196

of an y ositive for A, showing that these LCs migrate to the t.c. immunization site and likely participate in antigen processing. The skin immune environment has likely evolved over millennia, owing to constant bombardment of the skin with various antigenic stimuli, resulting in a delicate balance between immunogenic and tolerogenic responses. It is noteworthy that transcutaneous/epicutaneous immunization has been successful in mitigating neurodegenerative disease in both induced and spontaneous forms of experimental autoimmune encephalomyelitis (EAE), mouse models of the demyelinating disease multiple sclerosis (Bynoe et al. 2003; Bynoe et al. 2005). To determine the ability of A t.c. immunization to effectively produce A antibodies, we began our investigation in non-transgenic C57BL/6 mice, where we measured the kinetics of A-specific antibody titers. Remarkably, the A antibody response was observed as early as week four in all immunized mice and dramatically increased thereafter, remaining elevated through 16 weeks post-initial vaccination. Antibody isotype characterization demonstrated a predominantly IgG1 Here, we investigated the potential of t.c. Aimmunization for the treatmentAD-like cerebral amyloidosis in transgenic mice. Transcutaneous immunization isattractive route of delivery, as it is convenient, relatively painless, and minimallinvasive. This strategy is also appealing because the epidermal and dermal immune systems provide a unique environment for immune stimulation due to LC antigen presentation (Rozis et al. 2005; Renn et al. 2006; Schiller et al. 2006; Strid et al. 2006). Indeed, following A/CT t.c. immunization, we observed cells double-positive for CD207 and CD11c in dermal regions that stained p 185

PAGE 197

gG2b) response in line with our previous report utilizing an intrapeas ly ion Notwithstanding the need for these critical studies, A immunization appears to modulate immune responses based on three major criteria: 1) tissue route of delivery, 2) antigen epitope utilized for immunization, and 3) properties of the co-administered adjuvant. Whether Th2 polarization in this study occurred due to route of delivery, CT adjuvant choice, or the genetic background of the C57BL/6 strain (Rosas et al. 2005; Fukushima et al. 2006) remains to be fully determined in future studies. It has been reported that CT promotes an anti-inflammatory Th2 immune response (Eriksson et al. 2003), and our data demonstrating IgG1 subtype antibodies produced in the greatest (IgG1>IgG2a>I ritoneal route of A vaccination plus Freunds adjuvant (Town et al. 2002). Production of IgG1 and IgG2b are typically due to anti-inflammatory Th2 cytokine signaling, whereas IgG2a typically results from pro-inflammatory Th1 signaling (Abbet al. 1996). The Th2 cytokine profile is likely favorable for inducing antibody production and thus A clearance without the overt pro-inflammatory (i.e., possibcontributing to auto-immune responses) Th1-type activation that typifies cellular immuneresponses (Romagnani 2000; Schwarz et al. 2001; Town et al. 2002). Accordingly, to circumvent meningoencephalitic reactions, many studies investigating vaccinatmethods for reducing cerebral amyloidosis in AD have attempted to bias Th cell responses towards Th2 profiles utilizing various strategies (Kim et al. 2005; Dasilva et al. 2006; Ghochikyan et al. 2006). The effectiveness and potential safety of these strategies seems promising, but further investigation is needed to confirm whether the link between Th cell responses and meningoencephalitis in AD patients is causitive. 186

PAGE 198

proport e c ll nd ccination matory Th2-type he A ; /CT t.c. immunization of non-transgenic C57BL/6 mice produces both A-specific local LC immune response and systemic immune response characterized by high A antibody titers that are sustained throughout the immunization protocol. To determine the potential therapeutic efficacy of A t.c. immunization, 6 month-old double-transgenic (APPsw, PSEN1dE9) PSAPP mice [which develop robust amyloid pathology at 8 months of age (Jankowsky et al. 2001)], were immunized against A/CT ion (compared to IgG2a or IgG2b antibodies) supports this notion. Of note, CT antibody titers were observed (data not shown), indicating an immunogenic response tothis adjuvant. To confirm specific systemic versus local immune cell activation, wanalyzed primary cultures of isolated splenocytes from t.c. immunized non-transgeniC57BL/6 mice and found that A/CT t.c. immunization conferred A-specific T-ceresponse as measured by secretion of cytokines IFN-, IL-2 and IL-4 upon aggregated A 1-42 peptide recall challenge. Importantly, there was a marked increase in IL-4 secretion compared to IFNor IL-2, further suggesting Th2 immune responses after A/CT t.c. immunization. This is in agreement with our previous study, where we fouTh2-type cytokine responses both in vivo and ex vivo after intraperitoneal A vawith Freunds adjuvant (Town et al. 2002). Further, the Th2-type response that we observed following A/CT t.c. immunization is important as anti-inflamimmune responses are likely preferred to pro-inflammatory Th1-responses in tvaccination paradigm, given that pro-inflammatory Th1 cells likely contributed to the aseptic meningoencephalitis in the human clinical trial of AN-1792 (Nicoll et al. 2003Town et al. 2005). When taken together, these findings show that A 187

PAGE 199

antibody oup. only nation, that A/CT t.c. immunization is effective at mitigating cerebral amyloidosis and suggest activation of A brain-to-blood clearance. This peripheral sink mechanism has been reported by others following passive transfer of A antibodies to AD transgenic mice (DeMattos et al. 2001; Matsuoka et al. 2003). In order for AD immunotherapy approaches to be useful, they must not only be efficacious, but such approaches must also be safe and well-tolerated. Importantly, while we did observe peripheral A-specific T-cell responses consistent with an anti-ex vivo by IL-4 secretion after A recall stimulation of splenocytes) after A/CT t.c. or CT alone for 16 weeks. Results showed consistently high and sustained Atiters throughout the 16 week immunization period only in the A/CT immunized grInterestingly, the magnitude of A antibody response in A/CT t.c. immunized was about half of that in wild-type mice (compare Figure 14A with Figure 15A), supporting the notion that transgenic mouse models of AD are hypo-responsive to A vacciprobably owing to overexpression of the human APP transgene throughout their lives(Monsonego et al. 2001). This humoral response correlated with high plasma levels of A 1-40, 42 peptides which peaked around eight weeks and remained relatively constant through to 16 weeks. Immunohistological and histochemical analyses of A immunoreactive plaques and congophilic plaques, respectively, in cortical and hippocampal brain sections showed reductions by approximately 50% compared to CT t.c. immunized PSAPP mice, and a negative correlation existed between brain A and blood A levels following A/CT t.c. immunization. Taken together, these results show inflammatory Th2 response (characterized in vivo by IgG1 A antibody production and 188

PAGE 200

A ort trace ct in immunization, no signs of aseptic meningoencephalitis and/or cell-mediated immunity were observed in brains as evidenced by lack of CD3 positive T-cell infiltrates. However, we did observe evidence of humoral immunity in brain as demonstrated byantibody titers in brain homogenates, similar to data from previous reports using other modes of A immunotherapy (Bard et al. 2000; Bacskai et al. 2001), suggesting transpof A antibodies across the blood-brain-barrier. This observation was not due to poor PBS perfusion at the time of sacrifice, as Perls stain (which normally detects evenamounts of iron which could be present due poor perfusion) results were consistently negative. Finally, other investigators have reported that passive transfer of A antibodies to transgenic AD mice results in cerebral microhemorrhage (Wilcock et al. 2004; Racke et al. 2005). Importantly, Perls stain did not show this potentially adverse side-effemice t.c. immunized with A/CT. Thus, when taken together, t.c. immunization holds potential as a novel, effective, and safe potential treatment strategy for AD. 189

PAGE 201

lymphocytes. Nature. 383, 787-793. ts hethetic Abeta42 (AN1792) in patients with AD. Neurology. 64, 94-101. Beignon, A.S., Brown, F., Eftekhari, P., Kramer, E., Briand, J.P., Muller, S., Partidos, elicits potent neutralising anti-FMDV antibody responses. Vet Immunol Immunopathol. with autoantigenic peptides induces T suppressor cells that prevent experimental allergic cLaurin, J., 2006. Immunization with amyloid burden and alters plaque morpho vels. ad Sci U S A. 98, 8850-8855. 4.6 References Abbas, A.K., Murphy, K.M., Sher, A., 1996. Functional diversity of helper T Bacskai, B.J., Kajdasz, S.T., Christie, R.H., Carter, C., Games, D., Seubert, P., Schenk, D., Hyman, B.T., 2001. Imaging of amyloid-beta deposits in brains of living mice permidirect observation of clearance of plaques with immunotherapy. Nat Med. 7, 369-372. Bard, F., Cannon, C., Barbour, R., Burke, R.L., Games, D., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Lieberburg, I., Motter, R., Nguyen, M., Soriano, F., Vasquez, N., Weiss, K., Welch, B., Seubert, P., Schenk, D., Yednock, T., 2000. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzimer disease. Nat Med. 6, 916-919. Bayer, A.J., Bullock, R., Jones, R.W., Wilkinson, D., Paterson, K.R., Jenkins, L., Millais, S.B., Donoghue, S., 2005. Evaluation of the safety and immunogenicity of syn C.D., 2005. A peptide vaccine administered transcutaneously together with cholera toxin104, 273-280. Bynoe, M.S., Evans, J.T., Viret, C., Janeway, C.A., Jr., 2003. Epicutaneous immunizationencephalomyelitis. Immunity. 19, 317-328. Bynoe, M.S., Viret, C., Flavell, R.A., Janeway, C.A., Jr., 2005. T cells from epicutaneously immunized mice are prone to T cell receptor revision. Proc Natl Acad Sci U S A. 102, 2898-2903. Dasilva, K.A., Brown, M.E., Westaway, D., Mamyloid-beta using GM-CSF and IL-4 reduces logy. Neurobiol Dis. Dell, K., Koesters, R., Linnebacher, M., Klein, C., Gissmann, L., 2006. Intranasal immunization with human papillomavirus type 16 capsomeres in the presence of non-toxic cholera toxin-based adjuvants elicits increased vaginal immunoglobulin leVaccine. 24, 2238-2247. DeMattos, R.B., Bales, K.R., Cummins, D.J., Dodart, J.C., Paul, S.M., Holtzman, D.M., 2001. Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer's disease. Proc Natl Ac 190

PAGE 202

Dickstein, D.L., Biron, K.E., Ujiie, M., Pfeifer, C.G., Jeffries, A.R., Jefferies, W.A., imer disease. Faseb J. 20, 426-433. Douillard, P., Stoitzner, P., Tripp, C.H., Clair-Moninot, V., Ait-Yahia, S., McLellan, ct subsets of langerin/CD207 dendritic cells, only one of which represents epidermalB n 2275-2282. Giudice, E.L., Campbell, J.D., 2006. Needle-free vaccine delivery. Adv Drug Deliv Rev. 58, 68-89. Hock, C., Konietzko, U., Streffer, J.R., Tracy, J., Signorell, A., Muller-Tillmanns, B., Lemke, U., Henke, K., Moritz, E., Garcia, E., Wollmer, M.A., Umbricht, D., de Quervain, D.J., Hofmann, M., Maddalena, A., Papassotiropoulos, A., Nitsch, R.M., 2003. Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease. Neuron. 38, 547-554. Itoh, T., Celis, E., 2005. Transcutaneous immunization with cytotoxic T-cell peptide epitopes provides effective antitumor immunity in mice. J Immunother. 28, 430-437. Jankowsky, J.L., Slunt, H.H., Ratovitski, T., Jenkins, N.A., Copeland, N.G., Borchelt, D.R., 2001. Co-expression of multiple transgenes in mouse CNS: a comparison of strategies. Biomol Eng. 17, 157-165. Janus, C., 2003. Vaccines for Alzheimer's disease: how close are we? CNS Drugs. 17, 457-474. 2006. Abeta peptide immunization restores blood-brain barrier integrity in Alzhe A.D., Eggert, A., Romani, N., Saeland, S., 2005. Mouse lymphoid tissue contains distinderived Langerhans cells. J Invest Dermatol. 125, 983-994. Eriksson, K., Fredriksson, M., Nordstrom, I., Holmgren, J., 2003. Cholera toxin and its subunit promote dendritic cell vaccination with different influences on Th1 and Th2 development. Infect Immun. 71, 1740-1747. Ferrer, I., Boada Rovira, M., Sanchez Guerra, M.L., Rey, M.J., Costa-Jussa, F., 2004. Neuropathology and pathogenesis of encephalitis following amyloid-beta immunizatioin Alzheimer's disease. Brain Pathol. 14, 11-20. Fukushima, A., Yamaguchi, T., Ishida, W., Fukata, K., Taniguchi, T., Liu, F.T., Ueno, H., 2006. Genetic background determines susceptibility to experimental immune-mediated blepharoconjunctivitis: comparison of Balb/c and C57BL/6 mice. Exp Eye Res.82, 210-218. Ghochikyan, A., Mkrtichyan, M., Petrushina, I., Movsesyan, N., Karapetyan, A., CribbsD.H., Agadjanyan, M.G., 2006. Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch. Vaccine. 24, 191

PAGE 203

Johnson-Wood, K., Lee, M., Motter, R., Hu, K., Gordon, G., Barbour, R., Khan, K., onlogue, processing and A beta42 deposition in a transgenic ouse model of Alzheimer disease. Proc Natl Acad Sci U S A. 94, 1550-1555. combinant adenovirus vectors encoding amyloid beta-protein and GM-CSF. Vaccine. emere, C.A., Spooner, E.T., Leverone, J.F., Mori, C., Clements, J.D., 2002. Intranasal a, Q.L., Lim, G.P., Harris-White, M.E., Yang, F., Ambegaokar, S.S., Ubeda, O.J., aier, M., Seabrook, T.J., Lemere, C.A., 2005. Modulation of the humoral and cellular C.A., 2006. mouse model in the absence of an Abeta-specific cellular mune response. J Neurosci. 26, 4717-4728. ., Lu, uff, K., 2003. Novel therapeutic approach for the eatment of Alzheimer's disease by peripheral administration of agents with an affinity to yloid beta-peptide in amyloid precursor protein transgenic ice: implications for the pathogenesis and treatment of Alzheimer's disease. Proc Natl einer, H.L., 2003. Increased T cell reactivity to amyloid beta Gordon, M., Tan, H., Games, D., Lieberburg, I., Schenk, D., Seubert, P., McCL., 1997. Amyloid precursor protein m Kim, H.D., Cao, Y., Kong, F.K., Van Kampen, K.R., Lewis, T.L., Ma, Z., Tang, D.C., Fukuchi, K., 2005. Induction of a Th2 immune response by co-administration of re 23, 2977-2986. Larregina, A.T., Morelli, A.E., Spencer, L.A., Logar, A.J., Watkins, S.C., Thomson, A.W., Falo, L.D., Jr., 2001. Dermal-resident CD14+ cells differentiate into Langerhanscells. Nat Immunol. 2, 1151-1158. L immunotherapy for the treatment of Alzheimer's disease: Escherichia coli LT and LT(R192G) as mucosal adjuvants. Neurobiol Aging. 23, 991-1000. M Glabe, C.G., Teter, B., Frautschy, S.A., Cole, G.M., 2006. Antibodies against beta-amyloid reduce Abeta oligomers, glycogen synthase kinase-3beta activation and tau phosphorylation in vivo and in vitro. J Neurosci Res. 83, 374-384. M immune response in Abeta immunotherapy by the adjuvants monophosphoryl lipid A (MPL), cholera toxin B subunit (CTB) and E. coli enterotoxin LT(R192G). Vaccine. 23, 5149-5159. Maier, M., Seabrook, T.J., Lazo, N.D., Jiang, L., Das, P., Janus, C., Lemere, Short amyloid-beta (Abeta) immunogens reduce cerebral Abeta load and learning deficits in an Alzheimer's disease im Matsuoka, Y., Saito, M., LaFrancois, J., Gaynor, K., Olm, V., Wang, L., Casey, EY., Shiratori, C., Lemere, C., D tr beta-amyloid. J Neurosci. 23, 29-33. Monsonego, A., Maron, R., Zota, V., Selkoe, D.J., Weiner, H.L., 2001. Immune hyporesponsiveness to am m Acad Sci U S A. 98, 10273-10278. Monsonego, A., Zota, V., Karni, A., Krieger, J.I., Bar-Or, A., Bitan, G., Budson, A.E., Sperling, R., Selkoe, D.J., W 192

PAGE 204

rotein in older humans and patients with Alzheimer disease. J Clin Invest. 112, 415-422. eimer disease after immunization with amyloid-beta eptide: a case report. Nat Med. 9, 448-452. 25-et of patients with AD after Abeta42 munization. Neurology. 61, 46-54. an, W.P., Holtzman, D.M., Bales, K.R., itter, B.D., May, P.C., Paul, S.M., DeMattos, R.B., 2005. Exacerbation of cerebral ic of enn, C.N., Sanchez, D.J., Ochoa, M.T., Legaspi, A.J., Oh, C.K., Liu, P.T., Krutzik, ., Sun, N., Mori, T., Hou, H., Jeanniton, D., Ehrhart, J., ownsend, K., Zeng, J., Morgan, D., Hardy, J., Town, T., Tan, J., 2005. Green tea ebral amyloidosis in Alzheimer transgenic mice. J Neurosci. 25, 8807-8814. Satoskar, A.R., 2005. Genetic background influences immune responses nd disease outcome of cutaneous L. mexicana infection in mice. Int Immunol. 17, 1347-ozis, G., de Silva, S., Benlahrech, A., Papagatsias, T., Harris, J., Gotch, F., Dickson, G., tein: r J Immunol. 35, 2617-2626. -Wood, K., Khan, K., Kholodenko, D., Lee, M., Liao, Z., Lieberburg, I., p Nicoll, J.A., Wilkinson, D., Holmes, C., Steart, P., Markham, H., Weller, R.O., 2003. Neuropathology of human Alzh p Niizeki, H., Streilein, J.W., 1997. Hapten-specific tolerance induced by acute, low-dose ultraviolet B radiation of skin is mediated via interleukin-10. J Invest Dermatol. 10930. Orgogozo, J.M., Gilman, S., Dartigues, J.F., Laurent, B., Puel, M., Kirby, L.C., Jouanny, P., Dubois, B., Eisner, L., Flitman, S., Michel, B.F., Boada, M., Frank, A., Hock, C., 2003. Subacute meningoencephalitis in a subs im Racke, M.M., Boone, L.I., Hepburn, D.L., Parsadainian, M., Bryan, M.T., Ness, D.K., Piroozi, K.S., Jordan, W.H., Brown, D.D., Hoffm G amyloid angiopathy-associated microhemorrhage in amyloid precursor protein transgenmice by immunotherapy is dependent on antibody recognition of deposited forms amyloid beta. J Neurosci. 25, 629-636. R S.R., Sieling, P.A., Cheng, G., Modlin, R.L., 2006. TLR Activation of Langerhans Cell-Like Dendritic Cells Triggers an Antiviral Immune Response. J Immunol. 177, 298-305. Rezai-Zadeh, K., Shytle, D T epigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and reduces cer Romagnani, S., 2000. T-cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol.85, 9-18; quiz 18, 21. Rosas, L.E., Keiser, T., Barbi, J., Satoskar, A.A., Septer, A., Kaczmarek, J., Lezama-Davila, C.M., a 1357. R Patterson, S., 2005. Langerhans cells are more efficiently transduced than dermal dendritic cells by adenovirus vectors expressing either group C or group B fibre proimplications for mucosal vaccines. Eu Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., HuangJ., Johnson 193

PAGE 205

otter, R., Mutter, L., Soriano, F., Shopp, G., Vasquez, N., Vandevert, C., Walker, S., -3-chiller, M., Metze, D., Luger, T.A., Grabbe, S., Gunzer, M., 2006. Immune response chiatric disorders. Brain Behav Immun. 15, 340-370. kelding, K.A., Hickey, D.K., Horvat, J.C., Bao, S., Roberts, K.G., Finnie, J.M., nt an, J., Town, T., Mori, T., Wu, Y., Saxe, M., Crawford, F., Mullan, M., 2000. CD45 n, T., Crawford, F., Mori, T., DelleDonne, A., Crescentini, R., Obregon, D., lavell, R.A., Mullan, M.J., 2002. Role of CD40 ligand in amyloidosis in transgenic antibodies against human amyloid-beta1-2. Neurosci Lett. 307, 101-104. R., zheimer's beta-amyloid(1-42). J Neuroimmunol. 32, 49-59. ilcock, D.M., Rojiani, A., Rosenthal, A., Subbarao, S., Freeman, M.J., Gordon, M.N., M Wogulis, M., Yednock, T., Games, D., Seubert, P., 1999. Immunization with amyloidbeta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature. 400, 17177. S modifiers--mode of action. Exp Dermatol. 15, 331-341. Schwarz, M.J., Chiang, S., Muller, N., Ackenheil, M., 2001. T-helper-1 and T-helper-2 responses in psy Selkoe, D.J., 2001. Alzheimer's disease: genes, proteins, and therapy. Physiol Rev. 81741-766. S Hansbro, P.M., Beagley, K.W., 2006. Comparison of intranasal and transcutaneous immunization for induction of protective immunity against Chlamydia muridarum respiratory tract infection. Vaccine. 24, 355-366. Strid, J., Callard, R., Strobel, S., 2006. Epicutaneous immunization converts subsequeand established antigen-specific T helper type 1 (Th1) to Th2-type responses. Immunology. T opposes beta-amyloid peptide-induced microglial activation via inhibition of p44/42 mitogen-activated protein kinase. J Neurosci. 20, 7587-7594. Tan, J., Tow F Alzheimer's mice. Nat Neurosci. 5, 1288-1293. Town, T., Tan, J., Sansone, N., Obregon, D., Klein, T., Mullan, M., 2001. Characterization of murine immunoglobulin G 4 Town, T., Vendrame, M., Patel, A., Poetter, D., DelleDonne, A., Mori, T., SmeedCrawford, F., Klein, T., Tan, J., Mullan, M., 2002. Reduced Th1 and enhanced Th2 immunity after immunization with Al 1 Town, T., Tan, J., Flavell, R.A., Mullan, M., 2005. T-cells in Alzheimer's disease. Neuromolecular Med. 7, 255-264. W Morgan, D., 2004. Passive immunotherapy against Abeta in aged APP-transgenic mice reverses cognitive deficits and depletes parenchymal amyloid deposits in spite of 194

PAGE 206

increased vascular amyloid and microhemorrhage. J Neuroinflammation. 1, 24. eta uce parenchymal amyloid with minimal ascular consequences in aged amyloid precursor protein transgenic mice. J Neurosci. 26, Wilcock, D.M., Alamed, J., Gottschall, P.E., Grimm, J., Rosenthal, A., Pons, J., RonanV., Symmonds, K., Gordon, M.N., Morgan, D., 2006. Deglycosylated anti-amyloid-bantibodies eliminate cognitive deficits and red v 5340-5346. 195

PAGE 207

zheimers disease is the most common progressive neurodegenerative disorder. mal deposits, deposition of A in the cerebral vasculature (known as CAA) is another lis et al., f the neuroinflammatory response can be regarded as a central feature of AD CHAPTER FIVE DISCUSSION Al Based on genetic, biochemical, and post-mortem evidence, A peptides are key etiological contributors to AD pathogenesis (Selkoe, 2001). In addition to parenchy A pathological feature of AD, and occurs with 83% frequency in AD patients (El 1 996; Selkoe, 2001; Jellinger, 2002; Green et al., 2005). It is well know that there is a chronic activation of inflammatory pathways in AD brain, including the production o p roinflammatory cytokines and acute-phase reactants in and around A plaques. In addition, a number of other molecules important in the proinflammatory acute-phase re sponse are also found at high levels in AD brain, including the activated glial product S100 beta, the protease -1-antichymotripsin and -2-macroglobulin, the prostaglandin g enerating enzymes cyclooxygenases 1 and 2, and components of the classical protein complement cascade such as C1q and C3 (Akiyama et al., 2000; McGeer and McGeer,2001). Thus, brain. 196

PAGE 208

5.1 Microglia and central nervous system like cells, it is becoming increasingly clear that reactive microglia play more verse roles in the CNS. Microglial activation is often used to refer to a single phenotype; however, a continuum of microglial activation exists, with phagocytic response (innate activation) at one end and antigen presenting cell function (adaptive activation) at the other. Where activated microglia fall in this spectrum seems to be highly dependent on the type of stimulation provided. Here, we suggest a model wherein microglial cells exist in at least two functionally discernable states once activated, namely a phagocytic phenotype (innate activation) or an antigen presenting phenotype (adaptive activation), as governed by their stimulatory environment. When challenged with certain PAMPs (particularly CpG-DNA), murine microglia seem to activate a mixed response characterized by enhanced phagocytosis and pro-inflammatory cytokine production as well as adaptive activation of T cells. In the EAE model, murine microglia seem to largely support adaptive activation of encephalitogenic T cells in the presence of the CD40-CD40 ligand interaction. In the context of A challenge, CD40 ligation is able to shift activated microglia from innate to adaptive activation. Further, it seems that the cytokine milieu that microglia are exposed to biases these cells to innate activation (i.e., anti-inflammatory Th2-associated cytokines such as IL-4, IL-10, TGF-1) or an adaptive form of activation (i.e., pro-inflammatory Th1-associated cytokines such as IFN-, IL-6, and TNF-; summarized in Fig. 21). Microglia are innate immune cells of myeloid origin that take up residence in the central nervous system (CNS) during embryogenesis. While classically regarded as macrophagedi 197

PAGE 209

Figure 21 198

PAGE 210

microglial activation responses. In the contextnting y roglia. ll; kin; IFN, interferon, TNF, tumor necrosi Figure 21. Model for innate versus adaptive of -amyloid challenge, microglia activate a phagocytic response. If co-stimulated with CD40 ligand, a shift from innate activation to adaptive antigen-presecell response ensues. Additionally, certain anti-inflammatory Th2-type cytokines shift this balance back towards innate phagocytic response, while some pro-inflammatorTh1-associated cytokines tip the balance further towards adaptive activation of micSee the text and Table for references. Abbreviations used: APC, antigen presenting ceCD40L, CD40 ligand; Th1, CD4+ T helper cell type I response; Th2, Th type II response; TGF, transforming growth factor; IL, interleu s factor. 199

PAGE 211

Not all forms of microglial activation as, as activated microglia may serve a protective role as was shown in A1-42-immunized mouse models of AD. It seems that enhanced microglial phagocytosis of -amyloid plaques is at least partly responsible for the therapeutic benefit in these animals, so perhaps stimulation of innate microglial activation contributes to these reported benefits. In conclusion for microglial activation, if we can learn how to better harness microglia in order to produce specific forms of microglial activation, this could be key to turn a pathogenic cell into a therapeutic modality. 5.2 Immunotherapy for Alzheimer disease In a seminal report, Schenk and colleagues showed that peripheral immunization of the PDAPP mouse model of AD with A1-42 peptide resulted in high antibody titers, a small fraction of which (0.1%, (Bard et al., 2000) crossed the blood-brain-barrier and entered the brain parenchyma. Most importantly, these authors found that A1-42 vaccination markedly diminished -amyloid plaque burden (Schenk et al., 1999). These authors also found evidence of cells in the brains of the A1-42 immunization animals that contained A. Many of these cells stained for the activated microglia marker MHC II and phenotypically resembled activated microglia, suggesting that these cells were able to phagocytose A deposits. In a follow-up report, Bard and colleagues supported this hypothesis by showing ex vivo that certain antibodies against A peptides could trigger microglial phagocytosis and subsequent clearance of A through the Fc receptor (Bard et al., 2000) (Bard et al., 2003). Clearance of brain -amyloid deposits was beneficial, as re deleteriou 200

PAGE 212

mouse d st-mortem brain of one patient who died as a consequence of this side-effect of treatment, there was significant clearance of A plaques in parts of the neocortex and, in other areas where plaques remained, A-immunoreactivity was associated with microglia (Nicoll et al., 2003). It is not yet clear whether this fulminate infiltration of T cells in AD patients who developed aseptic T cell meningoencephalitis was due to adaptive activation of microglia, but this is a distinct possibility given that microglia did seem to recognize antibody-opsonized A (Nicoll et al., 2003) (Monsonego and Weiner, 2003). These results indicate the potentially damaging and overwhelming effects of a full-blown T cell autoimmune response, which does not normally occur in AD, and which may have been mediated by adaptively activated microglia. Thus, to translate animal A immunization approaches into successful clinical AD therapies, such strategies should not only be efficacious, but also be safe, including avoiding meningoencephalitic reactions to A immunization previously observed in humans A 1-42 -vaccinated mice had markedly reduced cognitive impairment as assayed by behavioral testing in AD mice (Janus et al., 2000) (Morgan et al., 2000). Thus, inmodels of AD, innate (phagocytic) microglial activation mediated by the Fc receptor in the presence of antibody-opsonized A appears beneficial rather than deleterious. Based on the above-mentioned data, a human clinical trial was begun to peripherally administer a synthetic A 12 peptide (AN-1792) with an adjuvant to AD patients. Unfortunately, the trial was halted when a small percentage of patients developed aseptic T cell meningoencephalitis. This response most likely occurrebecause of an immune reaction to A mediated by infiltrating T cells (Pfeifer et al., 2002). In the po 201

PAGE 213

et une ave been elicited after transcutaneous (t.c.) vaccination (Giudice and ampbell, 2006). Taken together, these lines of evidence led us to hypothesize that y provide an immunotherapeutic approach that is b d o (Janus, 2003; Monsonego et al., 2003b). 5.2.1. Immunotherapy: transcutaneous vaccination Transcutaneous (t.c.) vaccination is an attractive route of delivery, as it is convenient, relatively painless, and minimally invasive. The skin is a well-established effective route for vaccination, including delivery of peptide-based vaccines (Masliahal., 1991; Tan et al., 2002a; Beignon et al., 2005). Strong humoral and cellular immresponses h C targeting A immunotherapy to skin tissue ma oth efficacious and safe. Here, we first found that t.c. immunization of non-transgenic C57BL/6 mice with aggregated A 1-42 peptide plus the adjuvant cholera toxin (CT) resulted in high A antibody titers [mainly immunoglobulin (Ig) G1], and A 1-42 -specific splenocyte immune responses after re-challenge with the peptide. Similarresponse was observed in transgenic PSAPP mice and resulted in effective immune responses against A in concert with reduced cerebral A pathology, demonstrating the effectiveness of this approach. These mice showed high A antibody titers and increasecirculating Alevels, suggesting brain-to-blood efflux of A. Importantly, brain T-cell infiltration and cerebral microhemorrhage were not observed after t.c. immunization, indicating that this immunization strategy is potentially safe. A immunization appears tmodulate immune responses based on three major criteria: 1) tissue route of delivery, 2) antigen epitope utilized for immunization, and 3) properties of the co-administered 202

PAGE 214

CT t al., 2003), test T-cell ted e found ination l., adjuvant. Whether Th2 polarization in our study occurred due to route of delivery, adjuvant choice, or the genetic background of the C57BL/6 strain (Rosas et al., 2005; Fukushima et al., 2006) remains to be fully determined in future studies. It has been reported that CT promotes an anti-inflammatory Th2 immune response (Eriksson e and our data demonstrating IgG1 subtype antibodies produced in the greaproportion (compared to IgG2a or IgG2b antibodies) supports this notion. Further we showed that splenocytes isolated from t.c. immunized mice and found A-specificresponses as measured by secretion of cytokines IFN-, IL-2 and IL-4 upon aggregaA 1-42 peptide recall challenge. Importantly, there was a marked increase in IL-4 secretion compared to IFNor IL-2, further suggesting Th2 immune responses after A/CT t.c. immunization. This is in agreement with our previous study, where wTh2-type cytokine responses both in vivo and ex vivo after intraperitoneal A vaccwith Freunds adjuvant (Town et al., 2002). Further, the Th2-type response that we observed following A/CT t.c. immunization is important as anti-inflammatory Th2-typeimmune responses are likely preferred to pro-inflammatory Th1-responses in the A vaccination paradigm, given that pro-inflammatory Th1 cells likely contributed to the aseptic meningoencephalitis in the human clinical trial of AN-1792 (Nicoll et al., 2003; Town et al., 2005). Finally, other investigators have reported that passive transfer of Aantibodies to transgenic AD mice results in cerebral microhemorrhage (Arendash et a2001; Racke et al., 2005). Importantly, Perls stain did not show this potentially adverse side-effect in mice t.c. immunized with A/CT. Thus, when taken together, t.cimmunization holds potential as a novel, effective, and safe prospective treatment 203

PAGE 215

strategy for AD. e have previously shown the CD40-CD40L interaction enhances pro-bral A deposits (Tan et al., 1999a). This foL and utilized 40gic hese W inflammatory microglial activation triggered by cere rm of microglial activation is deleterious, as both genetic ablation of CD40CD40L neutralizing antibody reduce brain levels of several neurotoxic inflammatory cytokines and mitigate cerebral amyloidosis in AD mouse models (Tan et al., 2002a). Blocking the CD40-CD40L interaction mitigates A-induced inflammatory responses and enhances Aclearance (Tan et al., 2002b; Townsend et al., 2005). Here, we genetic and pharmacologic approaches to test whether CD40-CD40L blockade couldenhance the efficacy of A 1-42 immunization, while limiting potentially damaging inflammatory responses. We show that genetic or pharmacologic interruption of CDCD40L interaction enhanced A 1-42 immunization efficacy to reduce cerebral amyloidosisin the PSAPP and Tg2576 mouse models of AD. Potentially deleterious pro-inflammatory immune responses, cerebral amyloid angiopathy (CAA) and cerebralmicrohemorrhage were reduced or absent in these combined approaches. Pharmacoloblockade of CD40L decreased T-cell neurotoxicity to A-producing neurons. Further reduction of cerebral amyloidosis in A-immunized PSAPP mice completely deficient for CD40 occurred in the absence of A immunoglobulin G (IgG) antibodies or efflux ofA from brain to blood, but was rather correlated with anti-inflammatory cytokine profiles and reduced plasma soluble CD40L. Recently soluble CD40L was reported to be significantly increased in AD patients vs. healthy elderly controls, further supporting a role for this receptor/ligand dyad in the pathogenesis of AD (Desideri et al., 2006). T 204

PAGE 216

it fer ach be r, as results suggest CD40-CD40L blockade promotes anti-inflammatory cellular immune responses, likely resulting in promotion of microglial phagocytic activity and A clearance while precluding generation of neurotoxic A-reactive T-cells. If the benefafforded by CD40 pathway blockade to A 1-42 vaccinated AD mouse models can translate to the clinical syndrome, then pharmacotherapy aimed at reducing CD40 signaling in conjunction with A vaccination may represent an approach that is both saand more effective in humans. Future studies will be required to isolate CD40-CD40L downstream signaling involved in reduced efficacy of A vaccination, as this may uncover additional targets for pharmacologic intervention. 5.2.2. Immunotherapy: human umbilical cord blood cells Considering that inflammation plays a crucial role in propagation of Alzheimers disease, a second or parallel therapy would be desired. This alternative therapy approwould mainly immunomodulate peripheral immunity such that desired anti-inflammatory Th2 responses would predominate, in other words an increased Th2/Th1 ration shouldobserved. A possible strategy would involve a partial blockade of CD40-40L. Howeveit is rather challenging to partially block CD40-40L interaction with the use of CD40 agonists or CD40L antagonists since it could lead to immunodeficiencies and hyper IgM syndrome. Human cord blood cells (HUCBC) have unique immunomodulatory potentialHUCBC have been shown to oppose the pro-inflammatory Th1 response, as demonstrated in an animal model of stroke where HUCBC infusion promoted a strong anti-inflammatory Th2 response (Vendrame et al., 2004). Importantly, this effect w 205

PAGE 217

duced infarct volume and rescue of behavioral deficit (Vendrame et al., 2004). mmatory o ly, reduced s the a ant associated with re HUCBC infusion has also shown therapeutic benefit in other neuroinflaconditions including multiple sclerosis, amyotrophic lateral sclerosis, age-related neuromacular degeneration, and Parkinsons disease (El-Badri et al., 2006; Garbuzova-Davis et al., 2006; Henning et al., 2006). In AD preclinical models, administration of these cells to the PSAPP mouse model of AD was associated with extension of lifespanalthough high doses were administered in this paradigm (Ghorpade et al., 2001). Here, we showed a marked reduction of A levels/-amyloid plaques and associated astrocytosis following multiple low dose infusions of HUCBC. HUCBC infusions alsreduced cerebral vascular A deposits in the Tg2576 AD mouse model. Interestingthese effects were associated with suppression of the CD40-CD40L interaction as evidenced by decreased circulating and brain soluble CD40L (sCD40L) and elevated systemic IgM levels, attenuated CD40L-induced inflammatory responses, andsurface expression of CD40 on microglia. Importantly, deficiency of CD40 abolisheeffect of HUCBC on elevated plasma A levels. Moreover, microglia isolated from HUCBC-infused PSAPP mice demonstrated increased phagocytosis of A. Further, serfrom HUCBC-infused PSAPP mice significantly increased microglial phagocytosis of A 1-42 peptide while inhibiting IFN--induced microglial CD40 expression. Increased microglial phagocytic activity in this scenario was inhibited by addition of recombinCD40L protein. Thus, the data suggests that HUCBC infusion confers mitigation of AD-like pathology by disrupting CD40L activity. When taken together, our results provide the basis for a novel immunomodulatory strategy for AD using HUCBC. 206

PAGE 218

es as uch as ed to les the vaccine as they provide the host with required tools for needed immunomodulation. nd they are readily available. B In conclusion, there is no current treatment for Alzheimers disease. Immunotherapy is emerging as a potential therapy not just for AD but other diseaswell. Previous vaccines were very effective yet came with undesired side effects for a small population of patients. Since then, the accent have been given to safety as mto efficacy of any treatment. Alternative routes, adjuvants, and antigens could be usbypass this problem. Here, we showed that transcutaneous immunization of a whole A peptide with a cholera toxin adjuvant could be the answer. This vaccine could further beenhanced with inhibitors of yet to be identified specific CD40-40L signaling molecuinvolved in inflammation. Lastly, human cord blood cells could be used in parallel with In addition, these cells do not represent any ethical concerns a ased on these finding and current trends in research it is highly likely that future AD treatments will incorporate both the vaccineand the cell-based therapies. Future studies are needed to further examine the safety of these approaches since both have shown very effective. 207

PAGE 219

of bovine bone marrow-derived macrophages. Vet Immunol Immunopathol. 41, 211-27. Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G. M., Cooper, N. R., S., Hampel, H., Hull, M., Landreth, G., Lue, L., Mrak, R., Mackenzie, I. R., McGeer, P. del, R., Shen, Y., Streit, W., Strohmeyer, R., Tooyoma, I., Van Muiswinkel, F. L., Veerhuis, R., Inflammation and Alzheimer's disease. Neurobiol Aging. 21, 383-421. Alexopoulou, L., Holt, A. C., Medzhitov, R., Flavell, R. A., 2001. Recognition of double-32-8. from the yolk sac, and which proliferate in the brain. Brain Res Dev Brain Res. 117, 145-ns dnock, T., 2003. Epitope and isotype specificities of antibodies to beta -amyloid peptide for protection against Alzheimer's disease-like neuropathology. Proc Natl Acad Sci U S A. REFERENCE Adler, H., Peterhans, E., Jungi, T. W., 1994. Generation and functional characterization Eikelenboom, P., Emmerling, M., Fiebich, B. L., Finch, C. E., Frautschy, S., Griffin, WL., O'Banion, M. K., Pachter, J., Pasinetti, G., Plata-Salaman, C., Rogers, J., RyWalker, D., Webster, S., Wegrzyniak, B., Wenk, G., Wyss-Coray, T., 2000. stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature. 413, 7 Alliot, F., Godin, I., Pessac, B., 1999. Microglia derive from progenitors, originating 52. Arendash, G. W., Gordon, M. N., Diamond, D. M., Austin, L. A., Hatcher, J. M., Jantzen, P., DiCarlo, G., Wilcock, D., Morgan, D., 2001. Behavioral assessment of Alzheimer's transgenic mice following long-term Abeta vaccination: task specificity and correlatiobetween Abeta deposition and spatial memory. DNA Cell Biol. 20, 737-44. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B., Palucka, K., 2000. Immunobiology of dendritic cells. Annu Rev Immunol. 18, 767-811. Bard, F., Cannon, C., Barbour, R., Burke, R. L., Games, D., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Lieberburg, I., Motter, R., Nguyen, M., Soriano, F., Vasquez, N., Weiss, K., Welch, B., Seubert, P., Schenk, D., Yednock, T., 2000. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med. 6, 916-9. Bard, F., Barbour, R., Cannon, C., Carretto, R., Fox, M., Games, D., Guido, T., HoenowK., Hu, K., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, C., Lee, M., Motter, R., Nguyen, M., Reed, A., Schenk, D., Tang, P., Vasquez, N., Seubert, P., Ye 208

PAGE 220

100, 20 precursor protein and modulation by apolipoprotein E. Nature. 388, 878-81. Becher, B., Durell, B. G., Miga, A. V., Hickey, W. F., Noelle, R. J., 2001. The clinical the expression of CD40 within the central nervous system. J Exp Med. 193, 967-74. Beignon, A. S., Brown, F., Eftekhari, P., Kramer, E., Briand, J. P., Muller, S., Partidos, elicits potent neutralising anti-FMDV antibody responses. Vet Immunol Immunopathol. from toll-like receptors. Science. 304, 1014-8. Blom, A. B., Radstake, T. R., Holthuysen, A. E., Sloetjes, A. W., Pesman, G. J., Sweep, 2003. Increased expression of Fcgamma receptors II and III on macrophages of lpha and matrix metalloproteinase. Arthritis Rheum. 48, 1002-14. Brazelton, T. R., Rossi, F. M., Keshet, G. I., Blau, H. M., 2000. From marrow to brain: receptors in the human central nervous system. J Neuropathol Exp Neurol. 61, 1013-21. Calingasan, N. Y., Erdely, H. A., Altar, C. A., 2002. Identification of CD40 ligand in Alzheimer's disease and in animal models of Alzheimer's disease and brain injury. Neurobiol Aging. 23, 31-9. Carson, M. J., Reilly, C. R., Sutcliffe, J. G., Lo, D., 1998. Mature microglia resemble immature antigen-presenting cells. Glia. 22, 72-85. Chackerian, B., Rangel, M., Hunter, Z., Peabody, D. S., 2006. Virus and virus-like particle-based immunogens for Alzheimer's disease induce antibody responses against amyloid-beta without concomitant T cell responses. Vaccine. 24, 6321-31. Chung, H., Brazil, M. I., Soe, T. T., Maxfield, F. R., 1999. Uptake, degradation, and release of fibrillar and soluble forms of Alzheimer's amyloid beta-peptide by microglial cells. J Biol Chem. 274, 32301-8. Cornet, A., Vizler, C., Liblau, R., 1998. [Experimental autoimmune encephalomyelitis]. Rev Neurol (Paris). 154, 586-91. 23-8. Barger, S. W., Harmon, A. D., 1997. Microglial activation by Alzheimer amyloid course of experimental autoimmune encephalomyelitis and inflammation is controlled by C. D., 2005. A peptide vaccine administered transcutaneously together with cholera toxin104, 273-80. Blander, J. M., Medzhitov, R., 2004. Regulation of phagosome maturation by signals F. G., van de Loo, F. A., Joosten, L. A., Barrera, P., van Lent, P. L., van den Berg, W. B.rheumatoid arthritis patients results in higher production of tumor necrosis factor a expression of neuronal phenotypes in adult mice. Science. 290, 1775-9. Bsibsi, M., Ravid, R., Gveric, D., van Noort, J. M., 2002. Broad expression of Toll-like 209

PAGE 221

Dalpke, A. H., Schafer, M. K., Frey, M., Zimmermann, S., Tebbe, J., Weihe, E., Heeg, K., 2002. Immunostimulatory CpG-DNA activates murine microglia. J Immunol. 168, 4854-63. Desideri, G., Cipollone, F., Necozione, S., Marini, C., Lechiara, M. C., Taglieri, G., Zuliani, G., Fellin, R., Mezzetti, A., d C., 2006. Enhanced soluble CD40 gand and Alzheimer's disease: Evidence of a possible pathogenetic role. Neurobiol glitis, M. A., Mezey, E., 1997. Hematopoietic cells differentiate into both microglia and ly, D., Heyman, ., 1996. Cerebral amyloid angiopathy in the brains of patients with Alzheimer's disease: riksson, K., Fredriksson, M., Nordstrom, I., Holmgren, J., 2003. Cholera toxin and its B ischer, H. G., Reichmann, G., 2001. Brain dendritic cells and macrophages/microglia in es. berg, C. D., Willing, A. E., Saporta, S., ameron, D. F., Desjarlais, T., Daily, J., Kuzmin-Nichols, N., Chamizo, W., Klasko, S. J., i Orio, F., Ferri, li Aging. E macroglia in the brains of adult mice. Proc Natl Acad Sci U S A. 94, 4080-5. El-Badri, N. S., Hakki, A., Saporta, S., Liang, X., Madhusodanan, S., Willing, A. E., Sanberg, C. D., Sanberg, P. R., 2006. Cord blood mesenchymal stem cells: Potential usein neurological disorders. Stem Cells Dev. 15, 497-506. Ellis, R. J., Olichney, J. M., Thal, L. J., Mirra, S. S., Morris, J. C., Beek A the CERAD experience, Part XV. Neurology. 46, 1592-6. E subunit promote dendritic cell vaccination with different influences on Th1 and Th2 development. Infect Immun. 71, 1740-7. F central nervous system inflammation. J Immunol. 166, 2717-26. Forman, H. J., Torres, M., 2001. Redox signaling in macrophages. Mol Aspects Med. 22, 189-216. Fujiwara, N., Kobayashi, K., 2005. Macrophages in inflammation. Curr Drug Targets Inflamm Allergy. 4, 281-6. Fukushima, A., Yamaguchi, T., Ishida, W., Fukata, K., Taniguchi, T., Liu, F. T., Ueno, H., 2006. Genetic background determines susceptibility to experimental immune-mediated blepharoconjunctivitis: comparison of Balb/c and C57BL/6 mice. Exp Eye R82, 210-8. Garbuzova-Davis, S., Gografe, S. J., San C K., Sanberg, P. R., 2006. Maternal transplantation of human umbilical cord blood cells provides prenatal therapy in Sanfilippo type B mouse model. Faseb J. 20, 485-7. Gerritse, K., Laman, J. D., Noelle, R. J., Aruffo, A., Ledbetter, J. A., Boersma, WClaassen, E., 1996. CD40-CD40 ligand interactions in experimental allergic 210

PAGE 222

sis. Proc Natl Acad Sci U S A. 93, 2499-504. and viral fection regulate matrix metalloproteinase expression: implications for human ev. 58, 68-89. reen, D. A., Masliah, E., Vinters, H. V., Beizai, P., Moore, D. J., Achim, C. L., 2005. 1-14. n costimulation duction, T cell activation, and experimental allergic encephalomyelitis. Science. 273, rewal, I. S., Flavell, R. A., 1998. CD40 and CD154 in cell-mediated immunity. Annu The amyloid hypothesis of Alzheimer's disease: progress nd problems on the road to therapeutics. Science. 297, 353-6. ouse resident microglia: isolation nd characterization of immunoregulatory properties with naive CD4+ and CD8+ T-cells. eceptor 9, yD88, and DNA-dependent protein kinase catalytic subunit in the effects of two ignalling and the function of dendritic cells. Chem munol Allergy. 86, 120-35. rovic, L., Sanberg, P., Balis, J., Morgan, M. B., 2006. encephalomyelitis and multiple sclero Ghorpade, A., Persidskaia, R., Suryadevara, R., Che, M., Liu, X. J., Persidsky, Y., Gendelman, H. E., 2001. Mononuclear phagocyte differentiation, activation, in immunodeficiency virus type 1-associated dementia. J Virol. 75, 6572-83. Giudice, E. L., Campbell, J. D., 2006. Needle-free vaccine delivery. Adv Drug Deliv R Goldsby, R., Kindt, T., Osborne, B., Kuby, J., Mononuclear Phagocytes. In: R. Goldsby, (Ed.), Immunology. Freeman and Co., New York, 2002, pp. 38-19. G Brain deposition of beta-amyloid is a common pathologic feature in HIV positive patients. Aids. 19, 407-11. Greenberg, S. M., Bacskai, B. J., Hyman, B. T., 2003. Alzheimer disease's double-edged vaccine. Nat Med. 9, 389-90. Gregory, C. D., Devitt, A., 2004. The macrophage and the apoptotic cell: an innate immune interaction viewed simplistically? Immunology. 113, Grewal, I. S., Foellmer, H. G., Grewal, K. D., Xu, J., Hardardottir, F., Baron, J. L., Janeway, C. A., Jr., Flavell, R. A., 1996. Requirement for CD40 ligand i in 1864-7. G Rev Immunol. 16, 111-35. Hardy, J., Selkoe, D. J., 2002 a Havenith, C. E., Askew, D., Walker, W. S., 1998. M a Glia. 22, 348-59. Hemmi, H., Kaisho, T., Takeda, K., Akira, S., 2003. The roles of Toll-like r M distinct CpG DNAs on dendritic cell subsets. J Immunol. 170, 3059-64. Hemmi, H., Akira, S., 2005. TLR s Im Henning, R. J., Burgos, J. D., Ond 211

PAGE 223

uman umbilical cord blood progenitor cells are attracted to infarcted myocardium and Konietzko, U., Streffer, J. R., Tracy, J., Signorell, A., Muller-Tillmanns, B., emke, U., Henke, K., Moritz, E., Garcia, E., Wollmer, M. A., Umbricht, D., de ornung, V., Rothenfusser, S., Britsch, S., Krug, A., Jahrsdorfer, B., Giese, T., Endres, o CpG ligodeoxynucleotides. J Immunol. 168, 4531-7. utic intervention by anti-CD40L D154) antibody in an animal model of multiple sclerosis. J Clin Invest. 103, 281-90. blockade of CD154-CD40 in xperimental autoimmune encephalomyelitis. J Clin Invest. 109, 233-41. via -9 (TLR9). Faseb J. 18, 412-4. tory drugs and the risk f Alzheimer's disease. N Engl J Med. 345, 1515-21. akawa, R., Kaisho, T., Hemmi, H., Tajima, K., Uehira, K., Ozaki, Y., omizawa, H., Akira, S., Fukuhara, S., 2002. Interferon-alpha and interleukin-12 are 07-12. Arbour, N., Manusow, J., Montgrain, V., Blain, M., McCrea, E., Shapiro, A., ntel, J. P., 2005. TLR Signaling Tailors Innate Immune Responses in Human Microglia i, M., H significantly reduce myocardial infarction size. Cell Transplant. 15, 647-58. Hock, C., L Quervain, D. J., Hofmann, M., Maddalena, A., Papassotiropoulos, A., Nitsch, R. M., 2003. Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease. Neuron. 38, 547-54. H S., Hartmann, G., 2002. Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity t o Howard, L. M., Miga, A. J., Vanderlugt, C. L., Dal Canto, M. C., Laman, J. D., Noelle, R. J., Miller, S. D., 1999. Mechanisms of immunotherape (C Howard, L. M., Ostrovidov, S., Smith, C. E., Dal Canto, M. C., Miller, S. D., 2002. Normal Th1 development following long-term therapeutic e Iliev, A. I., Stringaris, A. K., Nau, R., Neumann, H., 2004. Neuronal injury mediated stimulation of microglial toll-like receptor in t' Veld, B. A., Ruitenberg, A., Hofman, A., Launer, L. J., van Duijn, C. M., Stijnen, T., Breteler, M. M., Stricker, B. H., 2001. Nonsteroidal antiinflamma o Ito, T., Am T induced differentially by Toll-like receptor 7 ligands in human blood dendritic cell subsets. J Exp Med. 195, 15 Iwasaki, A., Medzhitov, R., 2004. Toll-like receptor control of the adaptive immune responses. Nat Immunol. 5, 987-95. Jack, C. S., A and Astrocytes. J Immunol. 175, 4320-30. Janeway, C. A., Jr., Medzhitov, R., 2002. Innate immune recognition. Annu Rev Immunol. 20, 197-216. Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., ChishtM. A., Horne, P., Heslin, D., French, J., Mount, H. T., Nixon, R. A., Mercken 212

PAGE 224

e munization reduces behavioural impairment and plaques in a model of Alzheimer's s. 17, rrossay, D., Napolitani, G., Colonna, M., Sallusto, F., Lanzavecchia, A., 2001. loid dendritic cells. Eur J Immunol. 31, 3388-93. ptors obial antigens. J Exp Med. 194, 863-9. on cytokine, costimulatory molecule, and Toll-like ceptor expression. J Neuroimmunol. 130, 86-99. 2) is pivotal for cognition of S. aureus peptidoglycan but not intact bacteria by microglia. Glia. 49, 567-g, P. A., Volpe, J. J., Vartanian, T., 2002. The toll-like receptor TLR4 is ecessary for lipopolysaccharide-induced oligodendrocyte injury in the CNS. J Neurosci. ehnardt, S., Massillon, L., Follett, P., Jensen, F. E., Ratan, R., Rosenberg, P. A., Volpe, c Natl Acad Sci S A. 100, 8514-9. in reduces oxidative damage and amyloid pathology in an Alzheimer ansgenic mouse. Journal of Neuroscience. 21, 8370-8377. d and learning eficits in an Alzheimer's disease mouse model in the absence of an Abeta-specific asliah, E., Mallory, M., Hansen, L., Alford, M., Albright, T., Terry, R., Shapiro, P., Bergeron, C., Fraser, P. E., St George-Hyslop, P., Westaway, D., 2000. A beta peptid im disease. Nature. 408, 979-82. Janus, C., 2003. Vaccines for Alzheimer's disease: how close are we? CNS Drug457-74. Ja Specialization and complementarity in microbial molecule recognition by human myeand plasmacytoid Jellinger, K. A., 2002. Alzheimer disease and cerebrovascular pathology: an update. J Neural Transm. 109, 813-36. Kadowaki, N., Ho, S., Antonenko, S., Malefyt, R. W., Kastelein, R. A., Bazan, F., Liu, YJ., 2001. Subsets of human dendritic cell precursors express different toll-like receand respond to different micr Kielian, T., Mayes, P., Kielian, M., 2002. Characterization of microglial responses to Staphylococcus aureus: effects re Kielian, T., Esen, N., Bearden, E. D., 2005. Toll-like receptor 2 (TLR re 76. Lehnardt, S., Lachance, C., Patrizi, S., Lefebvre, S., Follett, P. L., Jensen, F. E., Rosenber n 22, 2478-86. L J. J., Vartanian, T., 2003. Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway. Pro U Lim, G. P., Chu, T., Yang, F. S., Beech, W., Frautschy, S. A., Cole, G. M., 2001. The curry spice curcum tr Maier, M., Seabrook, T. J., Lazo, N. D., Jiang, L., Das, P., Janus, C., Lemere, C. A.2006. Short amyloid-beta (Abeta) immunogens reduce cerebral Abeta loa d cellular immune response. J Neurosci. 26, 4717-28. M Sundsmo, M., Saitoh, T., 1991. Immunoreactivity of CD45, a protein phosphotyrosine 213

PAGE 225

atsushima, G. K., Taniike, M., Glimcher, L. H., Grusby, M. J., Frelinger, J. A., Suzuki, trophy. er, P. L., 1998. The importance of inflammatory mechanisms in lzheimer disease. Exp Gerontol. 33, 371-8. se. cMahon, E. J., Bailey, S. L., Castenada, C. V., Waldner, H., Miller, S. D., 2005. eda, L., Cassatella, M. A., Szendrei, G. I., Otvos, L., Jr., Baron, P., Villalba, M., d edzhitov, R., Janeway, C., Jr., 2000a. The Toll receptor family and microbial edzhitov, R., Janeway, C. A., Jr., 2000b. How does the immune system distinguish self e patterns of self and nonself by the nate immune system. Science. 296, 298-300. w. degenerative diseases: roles of apoptotic neurons nd chronic stimulation. Brain Res Brain Res Rev. 48, 251-6. e in amyloid precursor protein transgenic ice: implications for the pathogenesis and treatment of Alzheimer's disease. Proc Natl onsonego, A., Imitola, J., Zota, V., Oida, T., Weiner, H. L., 2003a. Microglia-mediated phosphatase, in Alzheimer's disease. Acta Neuropathol (Berl). 83, 12-20. M K., Ting, J. P., 1994. Absence of MHC class II molecules reduces CNS demyelination, microglial/macrophage infiltration, and twitching in murine globoid cell leukodysCell. 78, 645-56. McGeer, E. G., McGe A McGeer, P. L., McGeer, E. G., 2001. Inflammation, autotoxicity and Alzheimer diseaNeurobiol Aging. 22, 799-809. M Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis. Nat Med. 11, 335-9. M Ferrari, D., Rossi, F., 1995. Activation of microglial cells by beta-amyloid protein aninterferon-gamma. Nature. 374, 647-50. M recognition. Trends Microbiol. 8, 452-6. M from nonself? Semin Immunol. 12, 185-8; discussion 257-344. Medzhitov, R., Janeway, C. A., Jr., 2002. Decoding th in Mezey, E., Chandross, K. J., Harta, G., Maki, R. A., McKercher, S. R., 2000. Turningblood into brain: cells bearing neuronal antigens generated in vivo from bone marroScience. 290, 1779-82. Minghetti, L., Ajmone-Cat, M. A., De Berardinis, M. A., De Simone, R., 2005. Microglial activation in chronic neuro a Monsonego, A., Maron, R., Zota, V., Selkoe, D. J., Weiner, H. L., 2001. Immune hyporesponsiveness to amyloid beta-peptid m Acad Sci U S A. 98, 10273-8. M nitric oxide cytotoxicity of T cells following amyloid beta-peptide presentation to Th1 cells. J Immunol. 171, 2216-24. 214

PAGE 226

Monsonego, A., Weiner, H. L., 2003. Immunotherapeutic approaches to Alzheimer's disease. Science. 302, 834-8. R., Selkoe, D. J., Weiner, H. L., 2003b. Increased T cell reactivity to amyloid 12, organ, D., Diamond, D. M., Gottschall, P. E., Ugen, K. E., Dickey, C., Hardy, J., Duff, ., peptide vaccination prevents memory loss in an animal odel of Alzheimer's disease. Nature. 408, 982-5. h amyloid-beta eptide: a case report. Nat Med. 9, 448-52. e, R. Transcutaneous beta-amyloid munization reduces cerebral beta-amyloid deposits without T cell infiltration and efe, G. M., Nguyen, V. T., Benveniste, E. N., 2002. Regulation and function of class major histocompatibility complex, CD40, and B7 expression in macrophages and l Abeta DNA vaccine therapy against Alzheimer's disease: long-term ffects and safety. Proc Natl Acad Sci U S A. 103, 9619-24. ate and lsson, T., 1995. Cytokine-producing cells in experimental autoimmune el, M., Kirby, L. C., uanny, P., Dubois, B., Eisner, L., Flitman, S., Michel, B. F., Boada, M., Frank, A., Microglial cells internalize ggregates of the Alzheimer's disease amyloid beta-protein via a scavenger receptor. Monsonego, A., Zota, V., Karni, A., Krieger, J. I., Bar-Or, A., Bitan, G., Budson, A. E.Sperling, beta protein in older humans and patients with Alzheimer disease. J Clin Invest. 1415-22. M K., Jantzen, P., DiCarlo, G., Wilcock, D., Connor, K., Hatcher, J., Hope, C., Gordon, MArendash, G. W., 2000. A beta m Nicoll, J. A., Wilkinson, D., Holmes, C., Steart, P., Markham, H., Weller, R. O., 2003. Neuropathology of human Alzheimer disease after immunization wit p Nikolic, W. V., Bai, Y., Obregon, D., Hou, H., Mori, T., Zeng, J., Ehrhart, J., ShytlD., Giunta, B., Morgan, D., Town, T., Tan, J., 2007 im microhemorrhage. Proc Natl Acad Sci U S A. 104, 2507-12. O'Ke II microglia: Implications in neurological diseases. J Neurovirol. 8, 496-512. Okura, Y., Miyakoshi, A., Kohyama, K., Park, I. K., Staufenbiel, M., Matsumoto, Y., 2006. Nonvira e Olson, J. K., Miller, S. D., 2004. Microglia initiate central nervous system innadaptive immune responses through multiple TLRs. J Immunol. 173, 3916-24. O encephalomyelitis and multiple sclerosis. Neurology. 45, S11-5. Orgogozo, J. M., Gilman, S., Dartigues, J. F., Laurent, B., Pu Jo Hock, C., 2003. Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology. 61, 46-54. Paresce, D. M., Ghosh, R. N., Maxfield, F. R., 1996. a Neuron. 17, 553-65. 215

PAGE 227

regates of the lzheimer's disease amyloid beta-protein by microglial cells. J Biol Chem. 272, 29390-7. feifer, M., Boncristiano, S., Bondolfi, L., Stalder, A., Deller, T., Staufenbiel, M., tion of a two-step activation process during central ervous system autoimmune inflammation. J Immunol. 176, 1402-10. t, hmann, I., de Boer, B. A., Frotscher, M., Kreutzberg, G. W., Persons, D. ., Dirnagl, U., 2001. Targeting gene-modified hematopoietic cells to the central nervous 7, riller J, Prinz M, Heikenwalder M, Zeller N, Schwarz P, Heppner FL, Aguzzi A.Priller, ie. J. ffman, W. P., Holtzman, D. M., Bales, K. ., Gitter, B. D., May, P. C., Paul, S. M., DeMattos, R. B., 2005. Exacerbation of Neurosci. 25, 629-36. : emington, LT., Babcock, AA., Zehntner, SP., Owens, T., 2007. Microglial recruitment, 70, J., Satoskar, A. A., Septer, A., Kaczmarek, J., Lezama-avila, C. M., Satoskar, A. R., 2005. Genetic background influences immune responses Paresce, D. M., Chung, H., Maxfield, F. R., 1997. Slow degradation of agg A Pessac, B., Godin, I., Alliot, F., 2001. [Microglia: origin and development]. Bull Acad Natl Med. 185, 337-46; discussion 346-7. P Mathews, P. M., Jucker, M., 2002. Cerebral hemorrhage after passive anti-Abeta immunotherapy. Science. 298, 1379. Ponomarev, E. D., Shriver, L. P., Dittel, B. N., 2006. CD40 expression by microglial cells is required for their comple n Priller, J., Flugel, A., Wehner, T., Boentert, M., Haas, C. A., Prinz, M., Fernandez-KletF., Prass, K., Bec A system: use of green fluorescent protein uncovers microglial engraftment. Nat Med.1356-61. P J., 2006. Early and rapid engraftment of bone marrowderived microglia in scrapNeurosci. 26, 11753. Qureshi, S. T., Medzhitov, R., 2003. Toll-like receptors and their role in experimental models of microbial infection. Genes Immun. 4, 87-94. Racke, M. M., Boone, L. I., Hepburn, D. L., Parsadainian, M., Bryan, M. T., Ness, D. K., Piroozi, K. S., Jordan, W. H., Brown, D. D., Ho R cerebral amyloid angiopathy-associated microhemorrhage in amyloid precursor proteintransgenic mice by immunotherapy is dependent on antibody recognition of deposited forms of amyloid beta. J Ransohoff RM, Liu L, Cardona AE. 2007, Chemokines and chemokine receptorsmultipurpose players in neuroinflammation. Int Rev Neurobiol. 82:187-204. R activation and proliferation in response to primary demyelination. Am. J. Pathol. 11713. Rosas, L. E., Keiser, T., Barbi, D and disease outcome of cutaneous L. mexicana infection in mice. Int Immunol. 17, 1347-57. 216

PAGE 228

chenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, F., Shopp, G., Vasquez, N., Vandevert, C., Walker, S., ogulis, M., Yednock, T., Games, D., Seubert, P., 1999. Immunization with amyloid--. B., Yednock, T., 2002. The role of microglia in Alzheimer's disease: friend or e? Neurobiol Aging. 23, 677-9; discussion 683-4. hapshak, P., Duncan, R., Minagar, A., Rodriguez de la Vega, P., Stewart, R. V., hortman, K., Liu, Y. J., 2002. Mouse and human dendritic cell subtypes. Nat Rev tions that induce Th1 or h2 responses. J Immunol. 170, 727-34. n, L., e ceptors 9 and 3 as essential components of innate immune defense against mouse an, J., Town, T., Paris, D., Mori, T., Suo, Z., Crawford, F., Mattson, M. P., Flavell, R. ion ., Crawford, F., Mattson, M. P., Flavell, A., Mullan, M., 1999b. Microglial activation resulting from CD40-CD40L interaction is, D., Placzek, A., Parker, T., Crawford, F., Yu, H., Humphrey, J., ullan, M., 1999c. Activation of microglial cells by the CD40 pathway: relevance to S J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Liao, Z., Lieberburg, I., Motter, R., Mutter, L., Soriano W beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature. 400, 1737. Schenk, D fo Selkoe, D. J., 2001. Alzheimer's disease: genes, proteins, and therapy. Physiol Rev. 81, 741-66. S Goodkin, K., 2004. Elevated expression of IFN-gamma in the HIV-1 infected brain. Front Biosci. 9, 1073-81. S Immunol. 2, 151-61. Straw, A. D., MacDonald, A. S., Denkers, E. Y., Pearce, E. J., 2003. CD154 plays a central role in regulating dendritic cell activation during infec T Swanborg, R. H., 1995. Experimental autoimmune encephalomyelitis in rodents as a model for human demyelinating disease. Clin Immunol Immunopathol. 77, 4-13. Szekely, C. A., Thorne, J. E., Zandi, P. P., Ek, M., Messias, E., Breitner, J. C., GoodmaS. N., 2004. Nonsteroidal anti-inflammatory drugs for the prevention of Alzheimer's disease: a systematic review. Neuroepidemiology. 23, 159-69 Tabeta, K., Georgel, P., Janssen, E., Du, X., Hoebe, K., Crozat, K., Mudd, S., Shamel,Sovath, S., Goode, J., Alexopoulou, L., Flavell, R. A., Beutler, B., 2004. Toll-lik re cytomegalovirus infection. Proc Natl Acad Sci U S A. 101, 3516-21. T A., Mullan, M., 1999a. Microglial activation resulting from CD40-CD40L interactafter beta-amyloid stimulation. Science. 286, 2352-5. Tan, J., Town, T., Paris, D., Mori, T., Suo, Z. M R after beta-amyloid stimulation. Science. 286, 2352-2355. Tan, J., Town, T., Par M 217

PAGE 229

an, J., Town, T., Crawford, F., Mori, T., DelleDonne, A., Crescentini, R., Obregon, D., 3. ase. ogo, T., Akiyama, H., Kondo, H., Ikeda, K., Kato, M., Iseki, E., Kosaka, K., 2000. s. own, T., Tan, J., Mullan, M., 2001. CD40 signaling and Alzheimer's disease after immunization with Alzheimer's beta-amyloid(1-42). J Neuroimmunol. 32, 49-59. 64. ., ing regulates innate and adaptive ctivation of microglia in response to amyloid beta-peptide. Eur J Immunol. 35, 901-10. ltiple 40 ligand. J Leukoc Biol. 67, 2-17. ., lical cord lood cells in a rat model of stroke dose-dependently rescues behavioral deficits and Fitzgerald, K. A., Fenton, M. J., 2003. TLRs: differential adapter utilization y toll-like receptors mediates TLR-specific patterns of gene expression. Mol Interv. 3, -like receptor 3 mediates West Nile virus entry into the brain causing lethal ncephalitis. Nat Med. 10, 1366-73. multiple sclerosis. Journal of Neuroimmunology. 97, 77-85. T Flavell, R. A., Mullan, M. J., 2002a. Role of CD40 ligand in amyloidosis in transgenic Alzheimer's mice. Nat Neurosci. 5, 1288-9 Tan, J., Town, T., Mullan, M., 2002b. CD40-CD40L interaction in Alzheimer's diseCurr Opin Pharmacol. 2, 445-51. T Expression of CD40 in the brain of Alzheimer's disease and other neurological diseaseBrain Res. 885, 117-21. T pathogenesis. Neurochem Int. 39, 371-80. Town, T., Vendrame, M., Patel, A., Poetter, D., DelleDonne, A., Mori, T., Smeed, R., Crawford, F., Klein, T., Tan, J., Mullan, M., 2002. Reduced Th1 and enhanced Th2 immunity 1 Town, T., Tan, J., Flavell, R. A., Mullan, M., 2005. T-cells in Alzheimer's disease. Neuromolecular Med. 7, 255Townsend, K. P., Town, T., Mori, T., Lue, L. F., Shytle, D., Sanberg, P. R., Morgan, DFernandez, F., Flavell, R. A., Tan, J., 2005. CD40 signal a Tsukada, N., Miyagi, K., Matsuda, M., Yanagisawa, N., 1994. Expression of Fc epsilon R2/CD23 and p55 IL-2R/CD25 on peripheral blood macrophages/monocytes in musclerosis. J Neuroimmunol. 55, 127-33. van Kooten, C., Banchereau, J., 2000. CD40-CD Vendrame, M., Cassady, J., Newcomb, J., Butler, T., Pennypacker, K. R., Zigova, TSanberg, C. D., Sanberg, P. R., Willing, A. E., 2004. Infusion of human umbi b reduces infarct volume. Stroke. 35, 2390-5. Vogel, S. N., b 466-77. Wang, T., Town, T., Alexopoulou, L., Anderson, J. F., Fikrig, E., Flavell, R. A., 2004. Toll e 218

PAGE 230

the uced incidence of AD with NSAID but not H2 receptor antagonists: the Cache ounty Study. Neurology. 59, 880-6. n, A., 2002. CD40-mediated p38 itogen-activated protein kinase activation is required for immunoglobulin class switch Yamamoto, M., Takeda, K., Akira, S., 2004. TIR domain-containing adaptors definespecificity of TLR signaling. Mol Immunol. 40, 861-8. Zandi, P. P., Anthony, J. C., Hayden, K. M., Mehta, K., Mayer, L., Breitner, J. C., 2002. Red C Zhang, K., Zhang, L., Zhu, D., Bae, D., Nel, A., Saxo m recombination to IgE. J Allergy Clin Immunol. 110, 421-8. 219

PAGE 231

OUT THE AUTHOR tlantic University in 2002 and his Masters degree in Biology uring his Masters program he worked on f e laboratory of Dr. Jun Tan where he worked on immunotherapy for Alzheimers as in the department of Molecular Medicine within AB William Veljko Nikolic received his Bachelors degree in Biology from Florida A microbiology/immunology track in 2004. D systemic lupus erythematosus in the laboratory of Dr. James X. Hartmann. In the fall o2004 he joined the Medical Science Ph.D. program at the University of South Florida in th disease. His formal appointment w college of Medicine. He successfully defended his doctoral dissertation on June 13, 2008 at the University of South Florida. 220