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Short Communications |
From the Departments of Neurology,*
Pathology,
and
Psychiatry,
New York University School of
Medicine, New York; and the New York State Institute for Basic Research
in Developmental Disabilities,
Staten Island,
New York
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date there is no cure or effective therapy for reducing a patient’s amyloid burden or preventing amyloid deposition in AD. Amyloid-related therapeutic strategies include the use of compounds that affect processing of the amyloid-ß precursor protein (APP),7 interfere with fibril formation, or that promote fibril disassembly.8-10 In vitro studies have shown that monoclonal antibodies raised against the N-terminal region of Aß can disaggregate Aß fibrils, maintain Aß solubility, and prevent Aß toxicity in cell culture.11,12 Schenk and colleagues13 extended these studies to an in vivo situation by vaccinating transgenic (Tg) APP mice (PDAPP) with aged, fibrillar Aß1-42 in which the treatment resulted in a reduction of amyloid burden and associated pathology. This effect is thought to be mediated by antibodies, because peripherally administered antibodies against Aß have been shown to reduce brain parenchymal amyloid burden.14 In addition, intranasal immunization with freshly solubilized Aß1-40 reduces cerebral amyloid burden.15 Recently, two studies demonstrated that a vaccination-induced reduction in brain amyloid deposits resulted in cognitive improvements.16,17 Both groups used the same protocol as Schenk and colleagues,13 which is injection of fibrillar Aß1-42 peptide mixed with Freund’s adjuvant. Although these results provide promise for using immunomodulation as a general approach to treat AD, immunization with intact Aß may not be feasible for humans because of potential toxicity. First, Aß crosses the blood brain barrier in experimental animals,18 forms toxic fibrils,4 and further it can seed fibril formation.19 Therefore, in humans, it is possible that Aß1-42 can co-deposit on existing amyloid plaques leading to increased toxicity, and may actually promote plaque formation. Secondly, although autoimmunity has not been observed in the mice vaccinated with human Aß, it is more likely to occur in humans vaccinated with the human form of Aß. Overall, the use of peptides that are not fibrillogenic/toxic and not identical to endogenous Aß should reduce the likelihood of these serious side effects.
We have in earlier reports demonstrated that LPFFD, a nonamyloidogenic peptide with sequence homology to Aß, blocks fibril formation,8 and induces in vivo disassembly of fibrillar Aß deposits.9 Extension of peptides with these properties to include the major immunogenic sites of Aß takes advantage of their lack of amyloidogenicity/toxicity while resulting in antibody generation against Aß, that leads to clearance of Aß plaques.
We have now designed a highly soluble nonamyloidogenic peptide homologous to Aß that has a reduced ability to adopt a ß-sheet conformation and, therefore, a much lower risk of toxicity in humans. This peptide was designed to have reduced fibrillogenic potential and enhanced immunogenicity while maintaining the major immunogenic sites of Aß peptides. Accordingly, the peptide contains the first 30 amino acid residues of Aß with six lysine residues at the N-terminus. Poly-L-lysine enhances immunogenicity,20 and the coupling of lysine residues to the C-terminus of short Aß sequences within the 15 to 25 domain of Aß has recently been proposed by Pallitto and colleagues21 in the design of anti-ß-sheet peptides or Aß fibrillogenesis inhibitors. The use of potential peptide inhibitors of fibril formation as immunogens has never been previously proposed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The peptides used (Aß1-40, Aß1-42, Aß1-30-NH2, and K6Aß1-30-NH2) were synthesized at the Keck Foundation (Yale University, New Haven, CT), as described previously.9 The peptide used for the immunizations, K6Aß1-30-NH2, maintains the two major immunogenic sites of Aß peptides, which are residues 1 to 11 and 22 to 28 of Aß1-42 based on the antigenic index of Jameson and Wolf,22 and on preliminary results obtained in our laboratory. All other chemicals were from Sigma, St. Louis, MO, unless otherwise stated.
Secondary Structure Studies
Secondary structure (
-helix, ß-sheet, and random coil) of the
peptides was evaluated by circular dichroism as described
previously.8,23
Results are expressed as molar ellipticity
in units of degree cm2
dmol-1, and
the data were analyzed by the Lincomb and convex constraint
algorithms24
to obtain the percentages of different types
of secondary structure.
Study of Amyloid Fibril Formation in Vitro
Aliquots of the peptides prepared in 0.1 mol/L Tris, pH 7.4, were incubated for different times, and their fibril formation compared to that of Aß1-30-NH2 and Aß1-42. In vitro fibrillogenesis was evaluated by a fluorometric assay based on the fluorescence emission by thioflavin T, as we have previously described.8,25 Thioflavin T binds specifically to amyloid and this binding produces a shift in its emission spectrum and a fluorescent enhancement proportional to the amount of amyloid formed.26
Neurotoxicity
The potential neurotoxicity of K6Aß1-30-NH2 (1 to 100 µmol/L) was evaluated at 2 and 6 days in a human neuroblastoma cell line (SK-N-SH) using the standard MTT assay as described by the manufacturer (Roche Molecular Biochemicals, Indianapolis, IN). Aß1-30-NH2, Aß1-40, and Aß1-42 were used as control peptides. Briefly, cells were plated at 10,000 cells/100 µl culture medium per well in flat-bottom, 96-well microtiter plates. The cells were allowed to attach to the plate overnight in an incubator (37°C, 5.0% CO2), and then 10 µl of freshly prepared peptide solution (in nanopure H2O) was added. Aß1-42 was only partially soluble at 100 µmol/L and was, therefore, added as a suspension at that concentration. Subsequent steps were as described in the assay protocol.
Animals
The vaccination was performed in the Tg2576 APP mouse model developed by Karen Hsiao and colleagues.27 These mice develop Aß plaques as early as at 11 to 13 months of age. We chose this model over the double Tg APP/PS1 model,28 because the age of onset and progression of Aß deposition in the single Tg APP mice more closely resembles that of AD. Age-matched vehicle-treated Tg mice and non-Tg littermates receiving K6Aß1-30-NH2 were used as controls, and the animals received their first injection at 11 to 13 months, at which time few plaques should already be present. Four mice were in each group. The animals were maintained on a 12-hour light-dark cycle, and had access to food and water ad libitum. The animal care was in accordance with institutional guidelines.
Vaccine Administration
K6Aß1-30-NH2 was supplied as trifluoroacetic acid salt. The immunization procedure was performed as previously described by Schenk and colleagues13 except that the peptide was not incubated overnight at 37°C before injection. Briefly, the peptide was dissolved in phosphate-buffered saline (PBS) at a concentration of 2 mg/ml and then mixed 1:1 (v/v) with the adjuvant or PBS. Complete Freund’s adjuvant was used for the first injection, incomplete Freund’s adjuvant for the next three injections, and PBS from the fifth injection forward. The mice received a subcutaneous injection of 100 µl of the mixture (ie, 100 µg/100 µl) followed by a second injection 2 weeks later, and then monthly thereafter.
Antibody Titers
Antibody titers were determined by serial dilutions of sera using an enzyme-linked immunosorbent assay (ELISA) as described previously,29 in which Aß or its derivative is coated onto microtiter wells. The titer, defined as the dilution yielding 50% of the maximum signal, was detected by a goat anti-mouse IgG linked to a horseradish peroxidase (Amersham Pharmacia Biotech, Piscataway, NJ), and tetramethyl benzidine (Pierce, Rockford, IL) was the substrate.
Histology
Mice were anesthetized with sodium pentobarbital (150 mg/kg, intraperitoneally), perfused transaortically with phosphate buffer and the brains processed as previously described.5 The right hemisphere was immersion-fixed in periodate-lysine-paraformaldehyde, whereas the left hemisphere was snap-frozen for measurements of Aß levels using established ELISA methods.30,31 Serial coronal sections (40 µm) were cut and five series of sections at 0.2-mm intervals were saved for histological analysis of 1) 6E10-, 2) Congo Red-, 3) interleukin-1ß (IL-1ß-OX42/tomato lectin-, 4) GFAP-, and 5) cresyl violet-stained sections. 6E10 recognizes Aß and stains both pre-amyloid and Aß plaques.32 Congo Red staining was performed to identify amyloid lesions in these animals. GFAP is a component of the glial intermediate filaments that form part of the cytoskeleton and is found predominantly in astrocytes. Microglia seem to be the major source of IL-1 within the central nervous system,33 and OX-42 recognizes CD11b on microglia, a rat equivalent of the human C3bi receptor.34 Tomato lectin binds to poly-N-acetyl lactosamine residues and has in neural tissue specific affinity for microglial cells.35 Both astrocytes and microglia are associated with Aß deposits. Staining with cresyl violet was performed to determine whether the immunization was causing neuronal shrinkage and/or cell loss in these animals. After sectioning, the series were placed in ethylene glycol cryoprotectant and stored at -20°C until used.
Cresyl Violet and Congo Red: Mounted sections were defatted in xylene and hydrated in a gradient of ethyl alcohol and water series. Staining was performed as previously described.5,6,8
6E10, GFAP, IL-1ß, and OX-42: Staining was performed as previously described.5,8 Briefly, sections were incubated in 6E10 (kindly provided by Richard Kascsak, Institute for Basic Research) primary antibody that selectively binds to human Aß at a 1:1000 dilution. A mouse-on-mouse immunodetection kit (Vector Laboratories, Burlingame, CA) was used in which the anti-mouse IgG secondary antibody was used at a 1:2000 dilution. GFAP (1:500; DAKO, Glostrup, Denmark), IL-1ß (1:250; Endogen, Rockford, IL), and OX-42 (1:250; Biosource Int., Camarillo, CA) staining was performed the same way as the 6E10 staining, except the secondary antibody was diluted 1:1300. The sections were reacted in 3,3'-diaminobenzidine tetrahydrochloride (DAB) with or without nickel ammonium sulfate (Ni; Mallinckrodt, Paris, KY) intensification. For double-labeling of IL-1ß and Aß plaques, sections were first stained for IL-1ß (DAB/Ni; black) in which peroxidase was the enzyme. The plaques (6E10) were then stained using the Vector Red alkaline-phosphatase substrate kit I (Vector).
Tomato Lectin: Sections removed from the cryoprotectant were washed in PBS, 0.3% Triton-X-100 in PBS (PBS-Tx), and then incubated for 30 minutes in 0.3% hydrogen peroxide in PBS to quench endogenous peroxidase activity. After 2 hours of incubation with tomato lectin (10 µg/ml PBS; Vector), sections were washed in PBS-Tx and then reacted with avidin-horseradish peroxidase (Vector) for 1 hour. Subsequent steps were as those used for the antibody staining.
Image Analysis
Immunohistochemistry of tissue sections was quantified with a Bioquant image analysis system, and unbiased sampling was used.36 All procedures were performed by an individual blind to the experimental condition of the study. Cortical area analyzed was dorsomedially from the cingulate cortex and extended ventrolaterally to the rhinal fissure within the right hemisphere. The area of the grid was 800 x 800 µm2 and amyloid load was measured in 10 frames per mouse (each: 640 x 480 µm2), chosen randomly. Hippocampal measurements were performed on the entire hippocampus in a similar manner as the cortical analysis. The Aß burden is defined as the percentage of area in the measurement field occupied by reaction product.
Sandwich ELISA Assay for Soluble Aß Levels
Before extraction of Aß from brain tissue, 10% (w/v) homogenates were prepared in tissue homogenization buffer (20 mmol/L Tris, pH 7.4, 250 mmol/L sucrose, 1 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L EGTA). Immediately before use, 1/100 volume of 100 mmol/L phenylmethylsulfonyl fluoride stock solution (in ethanol) and 1/1000 volume of LAP (5 mg each of leupeptin, antipain, and pepstatin A per ml of N-N-dimethylformamide) were added to the homogenization buffer. The homogenate was then thoroughly mixed with an equal volume of 0.4% diethylamine/100 mmol/L NaCl, then spun at 135,000 x g for 1 hour at 4°C, and subsequently neutralized with 1/10 volume 0.5 mol/L Tris, pH 6.8. The samples were then aliquoted, flash-frozen on dry ice, and stored at -80°C until loaded onto plates. Soluble Aß levels were measured in the left hemisphere using monoclonal antibody 6E10 (specific to an epitope present on 1 to 16 amino acid residues of Aß), rabbit antiserum R162 (specific for Aß40), and rabbit antiserum 165 (specific for Aß42) in a double-antibody sandwich ELISA as described previously.30,31 The optical density (OD) was measured at 450 nm in a micro-ELISA reader. The relationship between OD and Aß40 or Aß42 concentrations was determined by a four-parameter logistic log function. Nonlinear curve fitting was performed with KlinetiCalc program (Biotek Instruments, Inc., Winooski, VT) to convert OD of plasma to estimated concentrations. All samples were coded, and the investigators were blinded to group assignment until levels were measured and recorded. The detection limit of the assay is 10 pg/ml for Aß40 and Aß42. The percent coefficient of variation normally ranges from 8 to 14% (interassay) and 10 to 18% (intraassay).
Data Analysis
The cell culture data were analyzed by one-way analysis of variance, followed by a Dunnett’s test for post hoc analysis (GraphPad Prism 3.0). An unbiased stereological image analysis system (Bioquant; R&M Biometrics Inc., Nashville, TN) was used to determine the amyloid burden in 6E10-stained brain sections. The data for the amyloid burden and the levels of soluble Aß within the brain were analyzed by a Student’s t-test, two-tailed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Circular Dichroism Assay
Compounds with high ß-sheet content are more toxic and more
likely to form fibrils than compounds with low ß-sheet
content.37
The peptide with the polylysine at the
N-terminus had much less ß-sheet content that the amidated Aß1-30
or Aß1-42 (Table 1)
.
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Aß1-42 was already fibrillar at t = 0, whereas
Aß1-30-NH2 gradually formed fibrils throughout
time (Figure 1A)
. The relatively high
degree of thioflavin T staining of the
Aß1-30-NH2 versus Aß1-42 after 6
days reflects the known batch-to-batch variability of Aß peptide
fibril formation,38
as well as some degree of pellet
formation by the Aß1-42 with prolonged incubation.
K6Aß1-30-NH2 did not form fibrils after
incubation at 37°C for at least 15 days.
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To further assess the safety of this vaccination approach the
neurotoxicity of K6Aß1-30-NH2 was determined.
K6Aß1-30-NH2 had no effect on cell viability at
2 days and was slightly trophic at 6 days (P <
0.05), whereas Aß1-40 and Aß1-42 were toxic
(P < 0.05 to 0.001) to the human
neuroblastoma cells (SK-N-SH), compared to vehicle group, as determined
by the MTT assay (Figure 1, B and C)
. Aß1-30-NH2 had
no effect. During the incubation period, aggregates were visible under
the microscope only in culture wells containing Aß1-42 (10 to 100
µmol/L).
Antibody Titer
Tg2576 and their non-Tg littermates were vaccinated with K6Aß1-30-NH2 or vehicle. Almost all of the mice developed antibodies against the immunogen (K6Aß1-30-NH2), that cross-reacted with Aß1-40 and Aß1-42. The titer, defined as the dilution yielding 50% of the maximum signal, ranged from a few hundreds to several thousands (data not shown). Vehicle-treated animals injected with the adjuvant and PBS did not develop antibodies against these three peptides (data not shown). Nontransgenic mice had generally higher titer against all three peptides, and the polyclonal antibodies had higher avidity for the immunogen compared to Aß1-40 and Aß1-42. These findings are as expected because the immunogen is based on the human sequence of Aß that differs in three amino acids from the mouse Aß,39 and K6Aß1-30-NH2 that elicited the immune response should have more binding motifs for antibodies than the intact Aß peptides.
Amyloid Burden and Associated Histopathology
The mice were killed at 18 to 20 months of age after 7 months of
treatment, and their right hemisphere was processed for histology as
described.5
The brain sections were stained with cresyl
violet, Congo Red, tomato lectin, and with antibodies against: 1) human
Aß (6E10), microglia (OX-42, IL-1ß), and GFAP (anti-GFAP). After
K6Aß1-30-NH2 vaccination, cortical and
hippocampal amyloid burden in the Tg mice was reduced by 89 and
81%, respectively (Figure 2, A and B
, and Figure 3, A and B
), as
determined by stereological techniques. The total number of Congo
Red-positive amyloid deposits was reduced in the immunized animals;
however, the percentage of Aß-immunoreactive lesions that were Congo
Red-positive appeared to remain the same as in the nonimmunized Tg
mice. The clearance of the amyloid deposits seemed to be similar in
other brain regions. Selected brain sections from a control mouse with
high amyloid burden and an immunized mouse with reduced amyloid burden
were stained with sera from several immunized and control mice, whose
antibody titer ranged from 0 to 3000. As expected, with increasing
titer more plaques were stained and the pattern was similar in both
mice (data not shown). There was no obvious difference between the Tg
treatment groups in cresyl violet staining. Reactive astrocytes were
observed associated with all amyloid plaques. Because the
vehicle-treated Tg mice had a higher plaque burden, they had more
clusters of astrocytes than immunized Tg mice. OX-42 staining of
ramified rather than phagocytic (ameboid) microglia was predominantly
observed associated with plaques. To verify that this lack of
microglial phagocytes was not because of down-regulation of the CD11b
receptor, the binding motif of OX-42,34
sections from all
treatment groups were stained with tomato lectin. This particular
lectin binds to poly-N-acetyl lactosamine residues found
predominantly in ramified and phagocytic microglial cells, in addition
to endothelial and ependymal cells.35
These two latter
cell types were stained in all of the mice. The microglial lectin
staining resembled the OX-42 staining. In other words, in both
immunized and control Tg groups, the microglia did not have phagocytic
morphology and number of ramified microglial processes per plaque
seemed to be similar between immunized and nonimmunized mice (data not
shown). On the other hand, IL-1ß staining of ramified microglial
cells was prominent surrounding the Aß plaques in the control Tg mice
(Figure 2C)
, whereas virtually no IL-1ß staining was observed in the
immunized mice (Figure 2D)
. Significantly, there was no indication of
glomerulonephritis in hematoxylin and eosin-stained kidney
sections from the K6Aß1-30-NH2-treated mice,
suggesting that the mice had not developed an autoimmune disorder at
the time of sacrifice.
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Measurements of soluble Aß levels were performed on the left
hemisphere of the mice whose right hemisphere was used for histology.
Soluble Aß1-42 was reduced by 57% after vaccination with
K6Aß1-30-NH2 for 7 months
(P = 0.0019), compared to control group (Figure 3C)
. Although there was a trend for reduced levels of soluble total
Aß and Aß1-40 in the K6Aß1-30-treated group, the values were not
significantly different from the vehicle group.
Overall, immunization in Tg APP mice with nonamyloidogenic/non toxic (low ß-sheet content) Aß homologous peptide results in a similar reduction of amyloid burden as observed by Schenk and colleagues13 in which they used a fibrillar/toxic (high ß-sheet content) Aß1-42.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism of the vaccination-induced reduction in cerebral amyloid burden is not fully understood. However, based on the passive vaccination study by Bard and colleagues14 it is likely that antibodies have a pivotal role. Interestingly, they demonstrated that there was no correlation between antibody efficacy and affinity for soluble Aß or binding to aggregated synthetic Aß peptide. Effective antibodies were, however, able to bind to plaques in unfixed brain sections. Janus and colleagues,17 using the same protocol as Schenk and colleagues13 observed that the sera from Aß-immunized mice preferentially stained dense core plaques rather than diffuse Aß deposits suggesting that the antibodies may have a higher affinity for ß-sheet Aß. Based on these somewhat contradictory findings more studies are needed on Aß-antibody interactions that may give insight into the mechanism of antibody-mediated Aß clearance. It is unlikely that these antibodies are affecting the production of Aß because they do not recognize APP.15 It is more probable that the antibodies enhance clearance of Aß through microglial activation after antibody binding to Aß plaques.13,14 Their effect may also in part be because of binding to soluble Aß within the brain, that alters the equilibrium between deposited Aß versus soluble Aß. Given the numerous reports that show that Aß can bi-directionally cross the blood brain barrier,40-47 the vaccination effect may be in part mediated through binding of the antibodies to soluble Aß in peripheral fluids. Subsequent reduction in peripheral Aß levels may alter the equilibrium between Aß found within and outside the central nervous system that may result in efflux of Aß out of this compartment. This is particularly likely since most of the antibodies are circulating in peripheral tissues and the tg mice have high levels of Aß not only within the central nervous system but also in the periphery. A recent report shows that in the Tg2576 mice, plasma levels of Aß decrease as cerebral plaque burden increases.48 This suggests an interaction between these two compartments that can be manipulated.
Interestingly, in the behavioral vaccination study by Morgan and colleagues,16 they observed a partial reversal in cognitive deficits in APP/PS1 mice although cerebral amyloid burden as measured by immunohistochemistry was not significantly reduced. As pointed out by Morgan and colleagues,16 soluble Aß has been proposed to cause synapse loss in APP Tg mice, as some Tg lines have reduced synaptophysin staining in the dentate gyrus without Aß deposits.49 Therefore, a possible explanation for the cognitive improvement in the immunized mice in the absence of reduced plaque burden, was a decrease in soluble Aß, although this potential connection was not measured in their study.16 Our results show that after 7 months of treatment, the 81 to 89% reduction in amyloid plaque burden is associated with a 57% reduction in soluble Aß1-42 within the brain, whereas the reduction in soluble total Aß and Aß1-40 was not significantly different from the control group. In other words, soluble Aß is reduced less than plaque Aß. However, detailed time course studies must be performed to determine further any changes in the equilibrium between plaque Aß and soluble Aß both within and outside the brain. Our findings indirectly demonstrate the importance of Aß1-42 for plaque maintenance. Overall, it is likely that several different mechanisms may result in reduction of cerebral amyloid burden, depending on the animal model and the properties of the peptide used for immunization.
Numerous studies have suggested that amyloid deposition can activate inflammatory cascades in the brain, such as increased IL-1ß production associated with neuronal injury and death.6,50 It is possible that our immunization with Aß homologous peptides could also stimulate such negative inflammatory pathways, along with amyloid reduction. However, we observed few phagocytic microglia in our immunized animals, as identified by OX-42 immunoreactivity or tomato lectin binding. This is not surprising because after 7 months of treatment most of the plaques have been cleared. Furthermore, in the immunized group of mice microglial IL-1ß staining was virtually absent, whereas numerous ramified IL-1ß-positive microglia were associated with the plaques in the control Tg group. We have previously reported a similar lack of IL-1ß staining in a rat model of cerebral amyloidosis after treatment with a ß-sheet breaker peptide.9 However, in that acute study (16 days) this effect was associated with an extensive increase in phagocytic OX-42 staining, indicating that phagocytes do not express IL-1ß. Our current observations may suggest that an important effect of the immunization is reduced inflammation within the brain.
We are currently further exploring the therapeutic potential of K6Aß1-30-NH2 and related peptides by using aluminum-based adjuvants that are approved for use in humans, and by assessing the behavioral status of the mice before and during the treatment period. Overall, our approach has a much lower risk of leading to toxic effects in humans, than the use of Aß1-40/42, while maintaining the therapeutic potential of immunization for AD.
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Footnotes |
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Supported by Metlife Award (to B. F.) and National Institutes of Health grants AG15408, AR02594, AG17617, and AG05891.
Accepted for publication May 4, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secretase inhibitors reduce ß-amyloid peptide levels in brain. J Neurochem 2001, 76:173-181[Medline]
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J.-M. Orgogozo, S. Gilman, J.-F. Dartigues, B. Laurent, M. Puel, L. C. Kirby, P. Jouanny, B. Dubois, L. Eisner, S. Flitman, et al. Subacute meningoencephalitis in a subset of patients with AD after A{beta}42 immunization Neurology, July 8, 2003; 61(1): 46 - 54. [Abstract] [Full Text] [PDF]
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F. Bard, R. Barbour, C. Cannon, R. Carretto, M. Fox, D. Games, T. Guido, K. Hoenow, K. Hu, K. Johnson-Wood, et al. Epitope and isotype specificities of antibodies to beta -amyloid peptide for protection against Alzheimer's disease-like neuropathology PNAS, February 18, 2003; 100(4): 2023 - 2028. [Abstract] [Full Text] [PDF]
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E. M. Sigurdsson, D. R. Brown, M. Daniels, R. J. Kascsak, R. Kascsak, R. Carp, H. C. Meeker, B. Frangione, and T. Wisniewski Immunization Delays the Onset of Prion Disease in Mice Am. J. Pathol., July 1, 2002; 161(1): 13 - 17. [Abstract] [Full Text] [PDF]
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M. T. Heneka, E. Galea, V. Gavriluyk, L. Dumitrescu-Ozimek, J. Daeschner, M. K. O'Banion, G. Weinberg, T. Klockgether, and D. L. Feinstein Noradrenergic Depletion Potentiates beta -Amyloid-Induced Cortical Inflammation: Implications for Alzheimer's Disease J. Neurosci., April 1, 2002; 22(7): 2434 - 2442. [Abstract] [Full Text] [PDF]
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