David A. Loeffler

A Quantitative Analysis of Isoferritins in Select Regions of Aged, Parkinsonian, and Alzheimer's Diseased Brains

Abstract: The brain requires a ready supply of iron for normal neurological function, but free iron is toxic. Consequently, iron bioavailability must be stringently regulated. Recent evidence has suggested that the brain iron regulatory system is dysfunctional in neurological disorders such as Alzheimers and Parkinsons diseases (AD and PD, respectively). A key component of the iron regulatory system in the brain is ferritin. Ferritin consists of 24 subunits, which are distinguished as either a heavy‐chain (H) or light‐chain (L) isoform. These peptide subunits are genetically and functionally distinct. Thus, the ability to investigate separately the types of ferritin in brain should provide insight into iron management at both the cellular and the molecular level. In this study, the ratio of isoferritins was determined in select regions of adult elderly AD and PD human brains. The H‐rich ferritin was more abundant in the young brain, except in the globus pallidus where the ratio of H/L ferritin was 1:1. The balance of H/L isoferritins was influenced by age, brain region, and disease state. With normal aging, both H and L ferritin increased; however, the age‐associated increase in isoferritins generally failed to occur in AD and PD brain tissue. The imbalance in H/L isoferritins was disease and region specific. For example, in frontal cortex, there was a dramatic (fivefold) increase in the ratio of H/L ferritin in AD brains but not in PD brains. In PD, caudate and putamen H/L ratios were higher than in AD and the elderly control group. The analysis of isoferritin expression in brain provides insight into regional iron regulation under normal conditions and suggests a loss of ability to maintain iron homeostasis in the two disease states. This latter observation provides further evidence of dysfunction of iron homeostatic mechanisms in AD and PD and may contribute significantly to understanding the underlying pathogenesis of each, particularly in relation to iron‐induced oxidative damage.

Cellular immune response to intrastriatally implanted allogeneic bone marrow stromal cells in a rat model of Parkinson's disease

BackgroundMarrow stromal cells (MSC), the non-hematopoietic precursor cells in bone marrow, are being investigated for therapeutic potential in CNS disorders. Although in vitro studies have suggested that MSC may be immunologically inert, their immunogenicity following transplantation into allogeneic recipients is unclear. The primary objective of this study was to investigate the cellular immune response to MSC injected into the striatum of allogeneic recipients (6-hydroxydopamine [6-OHDA]-hemilesioned rats, an animal model of Parkinsons disease [PD]), and the secondary objective was to determine the ability of these cells to prevent nigrostriatal dopamine depletion and associated motor deficits in these animals.Methods5-Bromo-2-deoxyuridine (BrdU) – labeled MSC from two allogeneic sources (Wistar and ACI rats) were implanted into the striatum of adult Wistar rats at the same time as 6-OHDA was administered into the substantia nigra. Behavioral tests were administered one to two weeks before and 16–20 days after 6-OHDA lesioning and MSC transplantation. Immunocytochemical staining for T helper and T cytotoxic lymphocytes, microglia/macrophages, and major histocompatibility class I and II antigens was performed on post-transplantation days 22–24. MSC were detected with an anti-BrdU antibody.ResultsTissue injury due to the transplantation procedure produced a localized cellular immune response. Unexpectedly, both sources of allogeneic MSC generated robust cellular immune responses in the host striatum; the extent of this response was similar in the two allograft systems. Despite these immune responses, BrdU+ cells (presumptive MSC) remained in the striatum of all animals that received MSC. The numbers of remaining MSC tended to be increased (p = 0.055) in rats receiving Wistar MSC versus those receiving ACI MSC. MSC administration did not prevent behavioral deficits or dopamine depletion in the 6-OHDA-lesioned animals.ConclusionMSC, when implanted into the striatum of allogeneic animals, provoke a marked immune response which is not sufficient to clear these cells by 22–24 days post-transplantation. In the experimental paradigm in this study, MSC did not prevent nigrostriatal dopamine depletion and its associated behavioral deficits. Additional studies are indicated to clarify the effects of this immune response on MSC survival and function before initiating trials with these cells in patients with PD or other neurodegenerative disorders.

Complement activation in the Parkinson's disease substantia nigra: an immunocytochemical study.

BackgroundInflammatory processes are increased in the Parkinsons disease (PD) brain. The long-term use of nonsteroidal anti-inflammatory drugs has been associated, in retrospective studies, with decreased risk for PD, suggesting that inflammation may contribute to development of this disorder. The objective of this study was to determine the extent of complement activation, a major inflammatory mechanism, in PD.MethodsSubstantia nigra specimens from young normal subjects (n = 11–13), aged normal subjects (n = 24–28), and subjects with PD (n = 19–20), Alzheimers disease (AD; n = 12–13), and dementia with Lewy bodies (DLB; n = 9) were stained for iC3b and C9, representing early- and late-stage complement activation, respectively. Numbers of iC3b+, C9+, and total melanized neurons in each section were counted in a blinded fashion. Nonparametric analyses were used to evaluate differences between groups and to evaluate correlations between complement staining, numbers of melanized neurons, and the duration of PD.ResultsLewy bodies in both PD and DLB specimens stained for iC3b and C9. Staining was also prominent on melanized neurons. The percentage of iC3b+ neurons was significantly increased in PD vs. aged normal and AD specimens, and in young normal vs. aged normal specimens. C9 immunoreactivity was significantly increased in PD vs. AD specimens, but unlike iC3b, the increased C9 staining in PD and young normal specimens did not achieve statistical significance vs. aged normal specimens. iC3b and C9 staining in PD specimens was not correlated with the numbers of remaining melanized neurons, nor with the duration of PD.ConclusionComplement activation occurs on Lewy bodies and melanized neurons in the PD substantia nigra. Early complement activation (iC3b) is increased on melanized neurons in PD vs. aged normal specimens, and late-stage complement activation (C9) also tends to increase. This latter finding suggests that complement activation may contribute to loss of dopaminergic neurons in some individuals with PD. Complement activation on melanized neurons appears to decrease with normal aging, suggesting a possible neuroprotective role for this process in the normal substantia nigra.

Intravenous immunoglobulin and Alzheimer's disease: what now?

Intravenous immunoglobulin (IVIG) products are prepared from purified plasma immunoglobulins from large numbers of healthy donors. Pilot studies with the IVIG preparations Octagam and Gammagard in individuals with mild-to-moderate Alzheimer’s disease (AD) suggested stabilization of cognitive functioning in these patients, and a phase II trial with Gammagard reported similar findings. However, subsequent reports from Octagam’s phase II trial and Gammagard’s phase III trial found no evidence for slowing of AD progression. Although these recent disappointing results have reduced enthusiasm for IVIG as a possible treatment for AD, it is premature to draw final conclusions; a phase III AD trial with the IVIG product Flebogamma is still in progress. IVIG was the first attempt to use multiple antibodies to treat AD. This approach should be preferable to administration of single monoclonal antibodies in view of the multiple processes that are thought to contribute to AD neuropathology. Development of “AD-specific” preparations with higher concentrations of selected human antibodies and perhaps modified in other ways (such as increasing their anti-inflammatory effects and/or ability to cross the blood–brain barrier) should be considered. Such preparations, if generated with recombinant technology, could overcome the problems of high cost and limited supplies, which have been major concerns relating to the possible widespread use of IVIG in AD patients. This review summarizes the recent AD IVIG trials and discusses the major issues relating to possible use of IVIG for treating AD, as well as the critical questions which remain.

Antibody concentrations to Aβ1–42 monomer and soluble oligomers in untreated and antibody–antigen-dissociated intravenous immunoglobulin preparations

Cognitive improvement in Alzheimers disease (AD) patients treated with intravenous immunoglobulin (IvIg) has been attributed to its antibodies to amyloid beta (Abeta). We compared the concentrations of specific antibodies to soluble Abeta1-42 conformations, namely Abeta1-42 monomer and Abeta1-42 soluble oligomers, between three IvIg preparations, Gamunex, Gammagard, and Flebogamma. To determine specific antibody concentrations to these Abeta1-42 conformations, nonspecific binding of the IvIg preparations to the Abeta reverse sequence, Abeta42-1, was subtracted. These antibodies were measured in untreated IvIg preparations and also after they were treated to dissociate antibody-antigen complexes, because this procedure has been reported to increase the detectable levels of serum anti-Abeta antibodies. Antibody levels to Abeta1-42 monomer were significantly higher in untreated Gamunex than in the other two IvIg preparations, and antibody-antigen dissociation increased the measured anti-Abeta monomer concentrations in Gamunex and Gammagard. Dissociated Gamunex and Gammagard had higher anti-Abeta monomer levels than Flebogamma. Generally similar results were found for antibodies to soluble Abeta1-42 oligomers, with the exception that after antibody-antigen dissociation, only Gammagard had significantly higher antibody levels than Flebogamma. These differences in antibody concentrations to Abeta1-42 conformations (particularly to Abeta1-42 soluble oligomers, thought to be the most neurotoxic conformation of soluble Abeta) and the increased availability of these antibodies after antibody-antigen complex dissociation have important implications for IvIg treatment of AD patients.

α-Synuclein and Anti-α-Synuclein Antibodies in Parkinson’s Disease, Atypical Parkinson Syndromes, REM Sleep Behavior Disorder, and Healthy Controls

α-synuclein is thought to play a key role in Parkinson’s disease (PD) because it is the major protein in Lewy bodies, and because its gene mutations, duplication, and triplication are associated with early-onset PD. There are conflicting reports as to whether serum and plasma concentrations of α-synuclein and anti-α-synuclein antibodies differ between PD and control subjects. The objectives of this study were to compare the levels of α-synuclein and its antibodies between individuals with typical PD (n = 14), atypical Parkinson syndromes (n = 11), idiopathic rapid eye movement sleep behavior disorder (n = 10), and healthy controls (n = 9), to assess the strength of association between these serum proteins, and to determine group sizes needed for a high probability (80% power) of detecting statistical significance for 25% or 50% differences between typical PD and control subjects for these measurements. Analysis of log-transformed data found no statistically significant differences between groups for either α-synuclein or its antibodies. The concentrations of these proteins were weakly correlated (Spearman rho = 0.16). In subjects with typical PD and atypical Parkinson syndromes, anti-α-synuclein antibody levels above 1.5 µg/ml were detected only in subjects with no more than four years of clinical disease. Power analysis indicated that 236 and 73 samples per group would be required for an 80% probability that 25% and 50% differences, respectively, in mean α-synuclein levels between typical PD and control subjects would be statistically significant; for anti-α-synuclein antibodies, 283 and 87 samples per group would be required. Our findings are consistent with those previous studies which suggested that serum concentrations of α-synuclein and its antibodies are not significantly altered in PD.

In situ hybridization for detection of nocardial 16S rRNA: reactivity within intracellular inclusions in experimentally infected cynomolgus monkeys—and in Lewy body-containing human brain specimens

Our previous studies found that experimental infection of BALB/c mice with the Gram-positive bacterium Nocardia asteroides induced a parkinsonian-type syndrome with levodopa-responsive movement abnormalities, loss of nigrostriatal dopaminergic neurons, depletion of striatal dopamine, and intraneuronal inclusions in the substantia nigra (SN) with an appearance similar to Lewy bodies. In the present study, an in situ hybridization technique was developed to detect nocardial 16S ribosomal RNA (rRNA), using a Nocardia-specific probe (B77). Cerebral cortical specimens from cynomolgus monkeys were examined for the presence of nocardial RNA 48 h, 3.5 months, and 1 year after experimental infection with N. asteroides. Hybridization reactions were detected within Nocardia-like structures 48 h after infection and within intracellular inclusion bodies (immunoreactive for alpha-synuclein and ubiquitin) in one of two 3.5-month-infected monkeys. The in situ hybridization procedure was then applied in a blinded fashion to 24 human SN specimens with Lewy bodies and 11 human SN specimens without Lewy bodies (including five normal controls). Hybridization reactions were detected in nine Lewy body-containing specimens and none of the others. Reactivity was limited to inclusions with the appearance of Lewy bodies, with the exception of one specimen in which intracellular reactivity was also observed in Nocardia-like structures. These results suggest a possible association between Nocardia and neurodegenerative disorders in which Lewy bodies are present.

Specificity and Sensitivity of the Abeta Oligomer ELISA

Abeta soluble oligomers are believed to play a key role in the development of Alzheimers disease (AD). An enzyme-linked immunosorbent assay (ELISA) commonly used to measure these proteins uses the same monoclonal antibody as both capture and reporter antibody. The objective of this study was to examine the specificity and sensitivity of this procedure, using monoclonal anti-Abeta antibody 6E10 as capture antibody and biotinylated 6E10 as reporter antibody. At comparable concentrations of Abeta soluble oligomers and low molecular weight (LMW) Abeta peptides, optical density (OD) values were four- to five-fold higher for the oligomer preparation than for the LMW Abeta. The LMW Abeta preparation, when evaluated by western blots of gels run under native conditions, showed only one band even after storage at 4 °C for more than two months, suggesting that the ELISA was detecting Abeta monomer as well as Abeta oligomers. Possible explanations for these results are that (1) the LMW Abeta preparation may contain Abeta oligomer species below the limit of detection of western blot, but still detectable by ELISA, or (2) some nonspecific binding of the LMW Abeta to the ELISA plate may have occurred, allowing its relevant epitope to remain available for binding by the reporter antibody. Because of the possibility that this ELISA may not be oligomer-specific, it seems prudent to suggest that it should be used in combination with other methods, rather than as the sole technique, for measuring Abeta oligomers in biological specimens.

Intravenous immunoglobulin products contain specific antibodies to recombinant human tau protein.

Intravenous immunoglobulin (IVIG) products are prepared from plasma immunoglobulins from healthy donors. Pilot studies suggest that IVIG may stabilize cognitive functioning in patients with mild-to-moderate Alzheimers disease. This study measured antibodies to recombinant human tau protein in the IVIG products Gammagard (Baxter), Gamunex (Talecris), and Flebogamma (Grifols). Anti-tau antibodies were measured by ELISA, subtracting IVIGs polyvalent binding from its binding to tau-coated wells to calculate specific anti-tau antibody levels. Because polyvalent binding of IVIG products may interfere with ELISA measurement of their specific antibody levels, the percentage of binding of each IVIG product to tau-coated wells that was specific for tau was also determined. Specific anti-tau antibodies were detected in all three IVIG products, with significant differences between these products (p<0.001) even when Flebogammas anti-tau antibodies were doubled to account for its preparation as a 5% solution vs. 10% solutions for Gammagard and Gamunex (means: Gammagard, 3.1 μg/ml; Gamunex, 2.5 μg/ml; Flebogamma, 1.2 μg/ml). The percentages of each IVIG products specific binding to tau-coated wells also varied between the various products (p<0.001) and between all pairs of IVIG products (means: Gammagard, 73.1%; Flebogamma, 54.5%; Gamunex, 37.4%; p<0.01 for all pairwise comparisons). These findings indicate that IVIG products contain specific anti-tau antibodies. The concentrations of these antibodies and the percentages of specific binding of IVIG to tau-coated wells vary between IVIG products. Further studies are indicated to determine if IVIG also contains antibodies to pathologic forms of tau.

Early complement activation increases in the brain in some aged normal subjects

Complement activation is increased in Alzheimers disease (AD) and may contribute to the development and progression of this disorder. To compare early complement activation between normal and AD brain specimens, C4d and iC3b concentrations were measured in hippocampus, entorhinal cortex, temporal cortex, parietal cortex, and cerebellum from aged normal and AD subjects n=10-14 for both), and in hippocampus and entorhinal cortex from younger normal subjects (n=5-6). C4d and iC3b levels increased 2.3- to 4.6-fold in AD versus aged normal specimens (all P <0.05), with lowest concentrations of these activation proteins generally in cerebellum. No significant differences were present between aged and younger normal C4d and iC3b levels in hippocampus or entorhinal cortex. However, the concentrations of these proteins were markedly increased in several aged normal specimens. Normal subject age was moderately associated with both C4d (r=0.49) and iC3b (r=0.53) concentrations in the hippocampus. Increased brain complement activation in some elderly individuals may promote the subsequent development of AD.