The Relationship Between Plasma Aß Levels, Cognitive Function and Brain Volumetrics: Sydney Memory and Ageing Study.

OBJECTIVES: Determine whether (1) a relationship exists between plasma amyloid-ß (Aß)1- 40 and 1-42 peptide levels, brain volumetrics and cognitive performance in elderly individuals with and without amnestic mild cognitive impairment (aMCI), (2) plasma Aß peptide levels differ between apolipoprotein E (APOE) ε4 carriers and non-carriers and (3) longitudinal changes in cognition and brain volume relate to Aß levels. METHODS: Subjects with aMCI (n = 89) and normal cognition (n = 126) were drawn from the Sydney Memory and Aging Study (Sydney MAS), a population based study of non-demented 70-90 year old individuals; 39 Alzheimer's disease (AD) patients were recruited from a specialty clinic. Sydney MAS participants underwent brain MRI scans and were assessed on 19 cognitive measures and were APOE ε4 genotyped. Plasma levels of Aß1-40 and 1-42 were quantified using ELISA. RESULTS: Wave1 plasma levels of Aß peptides and Aß1-42/1-40 ratio were lower in aMCI and AD, and Aß1-42 was positively associated with global cognition and hippocampal volume and negatively with white matter hyperintensities. The relationships of Aß1-40 and Aß1-42 were predominantly observed in ε4 allele carriers and non-carriers respectively. Longitudinal analysis revealed greater decline in global cognition and memory for the highest quintiles of Aß1-42 and the ratio measure. CONCLUSION: Plasma Aß levels and the Aß1-42/1-40 ratio are related to cognition and hippocampal volumes, with differential associations of Aß1-40 and Aß1-42 in ε4 carriers and non-carriers. These data support the Aß sink model of AD pathology, and suggest that plasma Aß measures may serve as biomarkers of AD.

Plasma protein profiling of Mild Cognitive Impairment and Alzheimer's disease using iTRAQ quantitative proteomics.

BACKGROUND: With the promise of disease modifying treatments, there is a need for more specific diagnosis and prognosis of Alzheimer's disease (AD) and mild cognitive impairment (MCI). Plasma biomarkers are likely to be utilised to increase diagnostic accuracy and specificity of AD and cognitive decline. METHODS: Isobaric tags (iTRAQ) and proteomic methods were used to identify potential plasma biomarkers of MCI and AD. Relative protein expression level changes were quantified in plasma of 411 cognitively normal subjects, 19 AD patients and 261 MCI patients. Plasma was pooled into 4 groups including normal control, AD, amnestic single and multiple domain MCI (aMCI), and nonamnestic single and multiple domain MCI (nMCI). Western-blotting was used to validate iTRAQ data. Integrated function and protein interactions were explored using WEB based bioinformatics tools (DAVID v6.7 and STRING v9.0). RESULTS: In at least two iTRAQ replicate experiments, 30 proteins were significantly dysregulated in MCI and AD plasma, relative to controls. These proteins included ApoA1, ApoB100, complement C3, C4b-binding protein, afamin, vitamin D-binding protein precursor, isoform 1 of Gelsolin actin regulator, Ig mµ chain C region (IGHM), histidine-rich glycoprotein and fibrinogen ß and γ chains. Western-blotting confirmed that afamin was decreased and IGHM was increased in MCI and AD groups. Bioinformatics results indicated that these dysregulated proteins represented a diversity of biological processes, including acute inflammatory response, cholesterol transport and blood coagulation. CONCLUSION: These findings demonstrate that expression level changes in multiple proteins are observed in MCI and AD plasma. Some of these, such as afamin and IGHM, may be candidate biomarkers for AD and the predementia condition of MCI.

Plasma apolipoprotein levels are associated with cognitive status and decline in a community cohort of older individuals.

OBJECTIVES: Apolipoproteins have recently been implicated in the etiology of Alzheimer's disease (AD). In particular, Apolipoprotein J (ApoJ or clusterin) has been proposed as a biomarker of the disease at the pre-dementia stage. We examined a group of apolipoproteins, including ApoA1, ApoA2, ApoB, ApoC3, ApoE, ApoH and ApoJ, in the plasma of a longitudinal community based cohort. METHODS: 664 subjects (257 with Mild Cognitive Impairment [MCI] and 407 with normal cognition), mean age 78 years, from the Sydney Memory and Aging Study (MAS) were followed up over two years. Plasma apolipoprotein levels at baseline (Wave 1) were measured using a multiplex bead fluorescence immunoassay technique. RESULTS: At Wave 1, MCI subjects had lower levels of ApoA1, ApoA2 and ApoH, and higher levels of ApoE and ApoJ, and a higher ApoB/ApoA1 ratio. Carriers of the apolipoprotein E ε4 allele had significantly lower levels of plasma ApoE, ApoC3 and ApoH and a significantly higher level of ApoB. Global cognitive scores were correlated positively with ApoH and negatively with ApoJ levels. ApoJ and ApoE levels were correlated negatively with grey matter volume and positively with cerebrospinal fluid (CSF) volume on MRI. Lower ApoA1, ApoA2 and ApoH levels, and higher ApoB/ApoA1 ratio, increased the risk of cognitive decline over two years in cognitively normal individuals. ApoA1 was the most significant predictor of decline. These associations remained after statistically controlling for lipid profile. Higher ApoJ levels predicted white matter atrophy over two years. CONCLUSIONS: Elderly individuals with MCI have abnormal apolipoprotein levels, which are related to cognitive function and volumetric MRI measures cross-sectionally and are predictive of cognitive impairment in cognitively normal subjects. ApoA1, ApoH and ApoJ are potential plasma biomarkers of cognitive decline in non-demented elderly individuals.

M. tuberculosis induces potent activation of IDO-1, but this is not essential for the immunological control of infection.

Indoleamine 2,3-dioxygenesae-1 (IDO-1) catalyses the initial, rate-limiting step in tryptophan metabolism, thereby regulating tryptophan availability and the formation of downstream metabolites, including picolinic and quinolinic acid. We found that Mycobacterium tuberculosis infection induced marked upregulation of IDO-1 expression in both human and murine macrophages in vitro and in the lungs of mice following aerosol challenge with M. tuberculosis. The absence of IDO-1 in dendritic cells enhanced the activation of mycobacteria-specific T cells in vitro. Interestingly, IDO-1-deficiency during M. tuberculosis infection in mice was not associated with altered mycobacteria-specific T cell responses in vivo. The bacterial burden of infected organs, pulmonary inflammatory responses, and survival were also comparable in M. tuberculosis-infected IDO-1 deficient and wild type animals. Tryptophan is metabolised into either picolinic acid or quinolinic acid, but only picolinic acid inhibited the growth of M. tuberculosis in vitro. By contrast macrophages infected with pathogenic mycobacteria, produced quinolinic, rather than picolinic acid, which did not reduce M. tuberculosis growth in vitro. Therefore, although M. tuberculosis induces robust expression of IDO-1 and activation of tryptophan metabolism, IDO-1-deficiency fails to impact on the immune control and the outcome of the infection in the mouse model of tuberculosis.

Meta-analysis of plasma amyloid-ß levels in Alzheimer's disease.

Plasma amyloid-ß (Aß) levels have been proposed as biomarkers of Alzheimer's disease (AD), but studies have produced inconsistent results. We present a meta-analytic review of cross-sectional studies that examined plasma Aß levels in AD and cognitively normal subjects, and longitudinal studies that used baseline plasma Aß levels to predict conversion from normal cognition to AD. Medline and EMBASE databases were searched to generate an initial list of relevant studies, and selected authors approached for additional data. Twelve cross- sectional studies (n = 1483) and seven longitudinal (n = 3920) met the inclusion criteria for meta-analysis. Random effects model was used to calculate the weighted mean difference (WMD) by Review Manager Version 4.2. In longitudinal studies, cognitively normal individuals who converted to AD had higher baseline Aß1-40 and Aß1-42 levels (WMD: 10.29, z = 3.80, p = 0.0001 and WMD: 8.01, z = 2.76, p = 0.006, respectively), and non-significantly increased Aß1-42/Aß1-40 ratio (WMD: 0.03, z = 1.65, p = 0.10). In cross sectional studies, compared to cognitively normal individuals, AD patients had marginally but non-significantly lower Aß1-42 levels (WMD:-2.84, z = 1.73, p = 0.08), but Aß1-40 levels were not significantly different (WMD: 3.43, z = 0.40, p = 0.69). Our systematic review suggests a model of differential longitudinal changes in plasma Aß levels in cognitively stable individuals versus those who go on to develop AD dementia. Baseline Aß1-40 and Aß1-42 levels in cognitively normal elderly individuals might be predictors of higher rates of progression to AD, and should be further explored as potential biomarkers.

Plasma biomarkers for mild cognitive impairment and Alzheimer's disease.

PURPOSE OF REVIEW: With the move toward development of disease modifying treatments, there is a need for more specific diagnosis of early Alzheimer's disease (AD) and mild cognitive impairment (MCI), plasma biomarkers are likely to play an important role in this. We review the current state of knowledge on plasma biomarkers for MCI and AD, including unbiased proteomics and very recent longitudinal studies. RECENT FINDINGS: With the use of proteomics methodologies, some proteins have been identified as potential biomarkers in plasma and serum of AD patients, including alpha-1-antitrypsin, complement factor H, alpha-2-macroglobulin, apolipoprotein J, apolipoprotein A-I. The findings of cross-sectional studies of plasma amyloid beta (A beta) levels are conflicting, but some recent longitudinal studies have shown that low plasma A beta 1-42 or A beta 1-40 levels, or A beta 1-42/A beta 1-40 ratio may be markers of cognitive decline. Other potential biomarkers for MCI and AD reflecting a variety of pathophysiological processes have been assessed, including isoprostanes and homocysteine (oxidative stress), total cholesterol and ApoE4 allele (lipoprotein metabolism), and cytokines and acute phase proteins (inflammation). A panel of 18 signal proteins was reported as markers of MCI and AD. SUMMARY: A variety of potential plasma biomarkers for AD and MCI have been identified, however the findings need replication in longitudinal studies. This area of research promises to yield interesting results in the near future.

Age and circadian influences on picolinic acid concentrations in human cerebrospinal fluid.

It has been suggested that picolinic acid (PIC), an endogenous metabolite of l-tryptophan, possesses neuro-protective and anti-proliferative effects within the CNS. However, the literature surrounding PIC is limited, and its exact endogenous function is not known. Picolinic acid is produced via the kynurenine pathway which has been implicated in the pathogenesis of a range of neuro-inflammatory diseases. Although not extensively studied, there have been reports of altered PIC production alongside other kynurenine metabolites in inflammatory disorders. In order to investigate whether PIC concentrations are altered with disease in the CNS, we analysed PIC levels in the CSF of 241 patients who underwent lumbar puncture as part of their standard clinical evaluation. In patients with no apparent CNS disease, CSF PIC levels were 10-fold higher in samples taken between 20:00 and 16:00 h compared with those collected between 04:00 and 12:00 h. This result suggests a diurnal variation in PIC synthesis within the CNS. In addition, we observed a direct correlation between a patient's age and their PIC concentration. No significant correlations were observed between CSF PIC levels and any specific disease state.

Microglial activation, macrophage infiltration, and evidence of cell death in the fetal brain after uteroplacental administration of lipopolysaccharide in sheep in late gestation.

OBJECTIVE: The purpose of this study was to determine whether uteroplacental delivery of endotoxin produces fetal systemic and central nervous system reactions that are suggestive of inflammation. STUDY DESIGN: Lipopolysaccharide (30 or 60 microg) was administered into the uterine artery of late gestation (135 +/- 0.3 days) pregnant sheep. Fetal blood was assayed to determine changes in levels of quinolinic acid, which is a metabolite of tryptophan that is produced by monocytes (macrophages, microglia). Fetal brains were collected after 72 hours and examined for the presence of activated microglia and parenchymal macrophages. RESULTS: The brains of treated fetuses showed microglial activation and macrophage infiltration, which varied between brain region and lipopolysaccharide dose. Cell death that had been determined by cresyl violet/acid fuchsin staining was observed in the external capsule. There was significant increase of quinolinic acid in the fetal circulation, but no lipopolysaccharide was detected. CONCLUSION: Uteroplacental inflammation results in significant microglial activation and macrophage infiltration without direct fetal exposure to endotoxin, which suggests that placental responses contribute to perinatal brain damage that is associated with infection during pregnancy.

Characterization of the kynurenine pathway in human neurons.

The kynurenine pathway is a major route of L-tryptophan catabolism producing neuroactive metabolites implicated in neurodegeneration and immune tolerance. We characterized the kynurenine pathway in human neurons and the human SK-N-SH neuroblastoma cell line and found that the kynurenine pathway enzymes were variably expressed. Picolinic carboxylase was expressed only in primary and some adult neurons but not in SK-N-SH cells. Because of this difference, SK-N-SH cells were able to produce the excitotoxin quinolinic acid, whereas human neurons produced the neuroprotectant picolinic acid. The net result of kynurenine pathway induction in human neurons is therefore predicted to result in neuroprotection, immune regulation, and tumor inhibition, whereas in SK-N-SH cells, it may result in neurotoxicity, immune tolerance, and tumor promotion. This study represents the first comprehensive characterization of the kynurenine pathway in neurons and the first description of the involvement of the kynurenine pathway as a mechanism for controlling both tumor cell neurotoxicity and persistence.

Quantitative determination of ortho- and meta-tyrosine as biomarkers of protein oxidative damage in beta-thalassemia.

Oxidative stress in thalassemia is caused by secondary iron overload and stems from blood transfusion and increased iron uptake. In this study, we hypothesized that levels of o- and m-tyrosine, products of hydroxyl radical attack on phenylalanine, would be elevated in beta-thalassemia (intermediate). This study represents the first report in which specific markers of protein oxidative damage have been quantified in thalassemia. We used GC/MS to assay o- and m-tyrosine at the femtomole level using only a few microliters of plasma. Levels of both markers were significantly higher in patients with beta-thalassemia than in controls and were positively correlated with serum ferritin, malondialdehyde, superoxide dismutase, glutathione peroxidase and glutathione. We conclude that o- and m-tyrosine are useful biomarkers of oxidative damage to proteins in thalassemia (intermediate) and may also be useful markers in other iron overload diseases. Positive correlations between o- and m-tyrosine levels and malondialdehyde as well as antioxidants such as superoxide dismutase, glutathione peroxidase and glutathione, are indicative of the broad impact of oxidative stress on blood plasma in thalassemia, with up-regulation of antioxidant proteins probably reflecting a homeostatic response to these increased stress levels.

Dynorphin knockout reduces fat mass and increases weight loss during fasting in mice.

Endogenous opioids, particularly dynorphins, have been implicated in regulation of energy balance, but it is not known how they mediate this in vivo. We investigated energy homeostasis in dynorphin knockout mice (Dyn(-/-) mice) and probed the interactions between dynorphins and the neuropeptide Y (NPY) system. Dyn(-/-) mice were no different from wild types with regards to body weight and basal and fasting-induced food intake, but fecal output was increased, suggesting decreased nutrient absorption, and they had significantly less white fat and lost more weight during a 24-h fast. The neuroendocrine and thermal responses to fasting were at least as pronounced in Dyn(-/-) as in wild types, and there was no stimulatory effect of dynorphin knockout on 24-h energy expenditure (kilocalories of heat produced) or physical activity. However, Dyn(-/-) mice showed increased circulating concentrations of 3,4-dihydroxyphenlacetic acid and 3,4-dihydroxyphenylglycol, suggesting increased activity of the sympathetic nervous system. The respiratory exchange ratio of male but not female Dyn(-/-) mice was reduced, demonstrating increased fat oxidation. Interestingly, expression of the orexigenic acting NPY in the hypothalamic arcuate nucleus was reduced in Dyn(-/-) mice. However, fasting-induced increases in pre-prodynorphin expression in the arcuate nucleus, the paraventricular nucleus, and the ventromedial hypothalamus but not the lateral hypothalamus were abolished by deletion of Y(1) but not Y(2) receptors. Therefore, ablation of dynorphins results in increases in fatty acid oxidation in male mice, reductions in adiposity, and increased weight loss during fasting, possibly via increases in sympathetic activity, decreases in intestinal nutrient absorption, and interactions with the NPYergic system.

Inhibition of indoleamine 2,3 dioxygenase activity by H2O2.

Indoleamine 2,3-dioxygenase is the first and rate limiting enzyme of the kynurenine pathway of tryptophan metabolism, has potent effects on cell proliferation and mediates antimicrobial, antitumorogenic, and immunosuppressive effects. As a potent cytotoxic effector, the mechanisms of indoleamine 2,3-dioxygenase inhibition deserve greater attention. The work presented here represents the first systematic study exploring the mechanisms by which low levels of hydrogen peroxide (10-100 microM) inhibit indoleamine 2,3-dioxygenase in vitro. Following brief peroxide exposure both enzyme inhibition and structural changes were observed. Loss of catalysis was accompanied by oxidation of several cysteine residues to sulfinic and sulfonic acids, observed by electrospray and MALDI mass spectrometry. Enzyme activity could in part be preserved in the presence of sulfhydryl containing compounds, particularly DTT and methionine. However, these structural alterations did not prevent substrate (l-tryptophan) binding. Some enzyme activity could be recovered in the presence of thioredoxin, indicating that the inhibitory effect of H(2)O(2) is at least partially reversible in vitro. We present evidence that cysteine oxidation represents one mechanism of indoleamine 2,3-dioxygenase inhibition.

Prolonged survival of a murine model of cerebral malaria by kynurenine pathway inhibition.

C57BL/6J mice infected with Plasmodium berghei ANKA develop neurological dysfunction and die within 7 days of infection. We show that treatment of infected mice with a kynurenine-3-hydroxylase inhibitor prevents them from developing neurological symptoms and extends their life span threefold until severe anemia develops.

Fate of steroid estrogens in Australian inland and coastal wastewater treatment plants.

A comparison of estrone (E1), 17beta-estradiol (E2), and 17alpha-ethinylestradiol (EE2) removal at a coastal enhanced primary and inland advanced sewage treatment plant (STP) is reported. The average concentration of estrogens in the raw sewage is similar to that reported in other studies. The sequential batch reactor at the advanced STP removed on average 85% of the incoming E1 and 96% of the E2. Further removal was observed during later microfiltration with the estrogen concentration below detection (<0.1 ng x L(-1)) after reverse osmosis. Some 6% of the influent E1+E2 was removed in the waste activated sludge. The detection of EE2 in the waste activated sludge (0.42 ng x g(-1) solids dry weight), undetectable in the raw sewage, suggests that EE2 is resistant to biological treatment in the sequential batch reactor and is primarily removed due to sorption. Little estrogen removal was observed at the enhanced primary with only 7% of E1 and 0% of E2 removed. Low removal is expected based on the degree of estrogens partitioning in the organic fraction given the relatively low solids concentration, but surprisingly, some 43% of E2, 24% of E1, and 100% of EE2 remains associated with the solids fraction in the treated effluent. Further research is necessary to determine whether the low level of estrogen removal for the coastal treatment plant will adversely affect the receiving marine environment.

Quantification of hemorphins in Alzheimer's disease brains.

The hemorphins are a family of opioid receptor-binding peptides originating from the beta-chain of hemoglobin and have been found at high levels within the central and peripheral nervous systems. In addition to opioid receptor binding, hemorphins have been shown to have a number of effects on the renin-angiotensin system, including inhibition of angiotensin-converting enzyme and angiotensin IV receptor binding. However, relatively few studies have examined the role of hemorphins in neurological diseases. Here we report the first study of hemorphins in Alzheimer's disease (AD) brains. Quantitative MALDI-TOF mass spectrometry was employed to assess levels of LVV and VV hemorphin-6 and -7 in 10 control and 10 AD brain tissue samples. LVV hemorphin-6 and total hemorphin levels were elevated in AD temporal neocortex but not in hippocampus, occipital lobe, or frontal lobe. The elevation of hemorphins is probably indicative of a vascular abnormality resulting from cerebral amyloid angiopathy associated with both neurodegenerative disease and aging.

A beta 1-42 induces production of quinolinic acid by human macrophages and microglia.

We hypothesized that the tryptophan catabolites produced through the kynurenine pathway (KP), and more particularly the excitotoxin quinolinic acid (QUIN), may play an important role in the pathogenesis of Alzheimer's disease (AD). In this study, we demonstrated that aggregated amyloid peptide A beta 1-42 induced indoleamine 2,3-dioxygenase (IDO) expression and resulted in a significant increase in production of QUIN by human primary macrophages and microglia. In contrast, A beta 1-40 and prion peptide (PrP) 106-126 did not induce any significant increase in QUIN production. These data imply that local QUIN production may be one of the factors involved in the pathogenesis of neuronal damage in AD.

Assessment of hydroxyl radical generation and radical scavenging activity of Chinese medicinal herbs using GC-MS.

Aqueous extracts of ten Chinese herbs were evaluated for their radical scavenging activity by a GC-MS method based on the Fenton reaction system. Hydroxylation of salicylate and phenylalanine is widely used as an index of hydroxyl radical formation in vivo and in vitro. A problem associated with quantifying product from such reactions is the generation of complex reaction products that increase background 'noise' and reduce sensitivity for the target product. The aim of this investigation was to develop a GC-MS methodology to assess in vitro hydroxyl radical production. In this method, hydroxyl radical was trapped by p-hydroxyphenylacetic acid to form 3,4-dihydroxyphenylacetic acid (DOPAC) which was then selectively extracted from the reaction mixture using aluminium oxide and assayed by GC-MS. Selective adsorption and desorption of the catechol nucleus from aluminium oxide was shown to eliminate interference from non-catechol reaction products effectively. This system was applied to examine the hydroxyl radical scavenging activity of different herbal extracts. The results showed that the herb Dimocaepus Longan Lour exhibited the highest radical scavenging activity of all the herbs examined. With the use of a stable isotope-labelled internal standard, this system could be readily applied to in vitro methods which use 4-hydroxybenzoic acid as a substrate for the hydroxyl radical.

Oxidative damage to proteins in yeast cells exposed to adaptive levels of H(2)O(2).

When yeast cells are exposed to sublethal concentrations of oxidants, they adapt to tolerate subsequent lethal treatments. Here, we show that this adaptation involves tolerance of oxidative damage, rather than protection of cellular constituents. o- and m-tyrosine levels are used as a sensitive measure of protein oxidative damage and we show that such damage accumulates in yeast cells exposed to H(2)O(2) at low adaptive levels. Glutathione represents one of the main cellular protections against free radical attack and has a role in adaptation to oxidative stress. Yeast mutants defective in glutathione metabolism are shown to accumulate significant levels of o- and m-tyrosine during normal aerobic growth conditions.

Extraction and chromatography-mass spectrometric analysis of the active principles from selected Chinese herbs and other medicinal plants.

Medicinal herbs have a long history of use in the practice of traditional Chinese medicine and a substantial body of evidence has, over recent decades, demonstrated a range of important pharmacological properties. Western biomedical researchers are examining not only the efficacy of the traditional herbal products but, through the use of a range of bioassays and analytical techniques, are developing improved methods to isolate and characterize active components. This review briefly describes the different extraction methodologies used in the preparation of herbal extracts and reviews the utility of chromatography-mass spectrometry for the analysis of their active components. In particular, applications of gas or liquid chromatography with mass spectrometry for the isolation and characterization of active components of ginseng are critically assessed. The analysis of toxic substances from herb extracts with mass spectrometric techniques is also discussed along with the potential for mass spectrometric methods to investigate the proteomics of herbal extracts.

Expression of the kynurenine pathway enzymes in human microglia and macrophages.

There is good evidence that the kynurenine pathway (KP) and one of its products, quinolinic acid (QUIN) play a role in the pathogenesis of neurological diseases. Monocytic cells are known to be the major producers of QUIN. However, macrophages have the ability to produce approximately 20 to 30-fold more QUIN than microglia. The molecular origin of this difference has not been clarified yet. Using unstimulated and IFN-gamma-stimulated cultures of human fcetal microglia and adult macrophages, we assayed mRNA expression of 8 key enzymes of the KP using RT-PCR and QUIN production using GC-MS. We found that after stimulation with IFN-gamma microglia produced de novo 20-fold less QUIN than macrophages. This quantitative difference in the ability to produce QUIN appears to be associated with a lower expression of 3 important enzymes of the KP in microglia: indoleamine 2,3-dioxygenase (IDO), kynureninase (KYNase) and kynurenine hydroxylase (KYN(OH)ase). These results suggest that activated infiltrating macrophages are the most potent QUIN producers during brain inflammatory diseases with playing a lesser role.