Evaluation of an optimized [18 F]fluoro-deoxy-glucose positron emission tomography voxel-wise method to early support differential diagnosis in atypical Parkinsonian disorders.

BACKGROUND AND PURPOSE: Atypical Parkinsonian disorders (APD) frequently overlap in clinical presentations, making the differential diagnosis challenging in the early stages. The present study aimed to evaluate the accuracy of the [18 F]fluoro-deoxy-glucose positron emission tomography Statistical Parametric Mapping (SPM) optimized procedure in supporting the early and differential diagnosis of APD. METHODS: Seventy patients with possible APD were retrospectively included from a large clinical cohort. The included patients underwent [18 F]fluoro-deoxy-glucose positron emission tomography within 3 months of the first clinical assessment and a diagnostic follow-up. An optimized SPM voxel-wise procedure was used to produce t-maps of brain hypometabolism in single subjects, which were classified by experts blinded to any clinical information. We compared the accuracy of both the first clinical diagnosis and the SPM t-map classifications with the diagnosis at follow-up as the reference standard. RESULTS: At first diagnosis, 60% of patients were classified as possible APD (progressive supranuclear palsy, corticobasal degeneration, dementia with Lewy bodies, multiple system atrophy) and about 40% as APD with uncertain diagnosis, providing 52% sensitivity, 97% specificity and 86% accuracy with respect to the reference standard. SPM t-map classification showed 98% sensitivity, 99% specificity and 99% accuracy, and a significant agreement with the diagnosis at follow-up (P < 0.001). CONCLUSIONS: The SPM t-map classification at entry predicted the second diagnosis at follow-up. This indicates its significantly superior role for an early identification of APD subtypes, particularly in cases of uncertain diagnosis. The use of a metabolic biomarker at entry in the instrumental work-up of APD may shorten the diagnostic time, producing benefits for treatment options and support to the patients.

Anemia Offers Stronger Protection Than Sickle Cell Trait Against the Erythrocytic Stage of Falciparum Malaria and This Protection Is Reversed by Iron Supplementation.

BACKGROUND: Iron deficiency causes long-term adverse consequences for children and is the most common nutritional deficiency worldwide. Observational studies suggest that iron deficiency anemia protects against Plasmodium falciparum malaria and several intervention trials have indicated that iron supplementation increases malaria risk through unknown mechanism(s). This poses a major challenge for health policy. We investigated how anemia inhibits blood stage malaria infection and how iron supplementation abrogates this protection. METHODS: This observational cohort study occurred in a malaria-endemic region where sickle-cell trait is also common. We studied fresh RBCs from anemic children (135 children; age 6-24months; hemoglobin <11g/dl) participating in an iron supplementation trial (ISRCTN registry, number ISRCTN07210906) in which they received iron (12mg/day) as part of a micronutrient powder for 84days. Children donated RBCs at baseline, Day 49, and Day 84 for use in flow cytometry-based in vitro growth and invasion assays with P. falciparum laboratory and field strains. In vitro parasite growth in subject RBCs was the primary endpoint. FINDINGS: Anemia substantially reduced the invasion and growth of both laboratory and field strains of P. falciparum in vitro (~10% growth reduction per standard deviation shift in hemoglobin). The population level impact against erythrocytic stage malaria was 15.9% from anemia compared to 3.5% for sickle-cell trait. Parasite growth was 2.4 fold higher after 49days of iron supplementation relative to baseline (p<0.001), paralleling increases in erythropoiesis. INTERPRETATION: These results confirm and quantify a plausible mechanism by which anemia protects African children against falciparum malaria, an effect that is substantially greater than the protection offered by sickle-cell trait. Iron supplementation completely reversed the observed protection and hence should be accompanied by malaria prophylaxis. Lower hemoglobin levels typically seen in populations of African descent may reflect past genetic selection by malaria. FUNDING: National Institute of Child Health and Development, Bill and Melinda Gates Foundation, UK Medical Research Council (MRC) and Department for International Development (DFID) under the MRC/DFID Concordat.

Motor neuron dysfunctions in the frontotemporal lobar degeneration spectrum: a clinical and neurophysiological study.

BACKGROUND: Although only a few frontotemporal lobar degeneration (FTLD) patients develop frank amyotrophic lateral sclerosis (ALS), motor neuron dysfunctions (MNDys) occur in a larger proportion of patients. The aim of this study is to evaluate MNDys and ALS in a sample of consecutively enrolled sporadic FTLD patients. METHODS: Clinical and neurophysiological evaluations (i.e. needle electromyography) assessed lower (LMN) and upper (UMN) motor neuron function at the baseline in 70 probable FTLD patients (i.e., 26 behavioural variant-bvFTD, 20 primary progressive aphasias-PPAs and 24 corticobasal syndrome-CBS). To obtain a more accurate estimation, quantitative scales were also applied (i.e. ALSFRS-r and UMN scale). Patients were screened for MAPT, GRN and C9orf72 mutations. A mean clinical follow-up of 27.8±22.4 months assessed MNDys progression and the clinical presentation of ALS. RESULTS: Five genetic cases were identified. Within the sample of sporadic patients, a relative low rate of FTLD patients was diagnosed as probable ALS (5%), while a higher proportion of patients (17%) showed clinical and neurophysiological MNDys. Thirteen patients (20%) presented with isolated clinical signs of LMN and/or UMN dysfunction, and 8 patients (12%) showed neurogenic changes at the electromyography. No differences in FTLD phenotype and disease duration were found between MNDys positive and negative patients. Clinical MNDys were highly associated with positive electromyographic findings. At follow-up, no MNDys positive patient developed ALS. CONCLUSION: Neurophysiological and clinical examinations revealed mild MNDys in FTLD patients not fulfilling criteria for ALS. This condition did not evolve at a mean follow-up of two years. These results, indicating a subclinical degeneration of corticospinal tracts and lower motor neurons, suggest that FTLD patients may be more at risk of MNDys than the general population.

In vivo microglia activation in very early dementia with Lewy bodies, comparison with Parkinson's disease.

BACKGROUND: Reactive microgliosis, hallmark of neuroinflammation, may contribute to neuronal degeneration, as shown in several neurodegenerative diseases. We in vivo evaluated microglia activation in early dementia with Lewy bodies, still not reported, and compared with early Parkinson's disease, to assess possible differential pathological patterns. METHODS: We measured the [(11)C]-PK11195 binding potentials with Positron Emission Tomography, using a simplified reference tissue model, as marker of microglia activation, and cerebral spinal fluid protein carbonylation levels, as marker of oxidative stress. Six dementia with Lewy bodies and 6 Parkinson's disease patients within a year from the onset, and eleven healthy controls were included. Clinical diagnosis was confirmed at a 4-year follow-up. RESULTS: In dementia with Lewy bodies as well as in Parkinson's disease, we found significant (p < 0.001) [(11)C]-PK11195 binding potential increases in the substantia nigra and putamen. Patients with Lewy bodies dementia had extensive additional microglia activation in several associative cortices. This was evident also at a single subject level. Significant increase of Cerebral Spinal Fluid protein carbonylation was shown in both patients' groups. CONCLUSIONS: [(11)C]-PK11195 Positron Emission Tomography imaging revealed neuroinflammation in dementia with Lewy bodies and Parkinson's disease, mirroring, even at a single subject level, the common and the different topographical distribution of neuropathological changes, yet in the earliest stages of the disease process. Focusing on those events that characterize parkinsonisms and Parkinson's disease may be the key to further advancing the understanding of pathogenesis and to taking these mechanisms forward as a means of defining targets for neuroprotection.

White matter damage in frontotemporal lobar degeneration spectrum.

White matter (WM) tract damage was assessed in patients with the behavioral variant frontotemporal dementia (bvFTD) and the 3 primary progressive aphasia (PPA) variants and compared with the corresponding brain atrophy patterns. Thirteen bvFTD and 20 PPA patients were studied. Tract-based spatial statistics and voxel-based morphometry were used. Patients with bvFTD showed widespread diffusion tensor magnetic resonance imaging (DT MRI) abnormalities affecting most of the WM bilaterally. In PPA patients, WM damage was more focal and varied across the 3 syndromes: left frontotemporoparietal in nonfluent, left frontotemporal in semantic, and left frontoparietal in logopenic patients. In each syndrome, DT MRI changes extended beyond the topography of gray matter loss. Left uncinate damage was the best predictor of frontotemporal lobar degeneration diagnosis versus controls. DT MRI measures of the anterior corpus callosum and left superior longitudinal fasciculus differentiated bvFTD from nonfluent cases. The best predictors of semantic PPA compared with both bvFTD and nonfluent cases were diffusivity abnormalities of the left uncinate and inferior longitudinal fasciculus. This study provides insights into the similarities and differences of WM damage in bvFTD and PPA variants. DT MRI metrics hold promise to serve as early markers of WM integrity loss that only at a later stage may be detectable by volumetric measures.

Effects of epoetin alfa on the central nervous system.

Erythropoietin (EPO) is a glycoprotein that has been shown to mediate response to hypoxia, and is most notably recognized for its central role in erythropoiesis. In a series of experiments using rodent models, the ability of systemically administered recombinant human erythropoietin (r-HuEPO, epoetin alfa) to cross the blood-brain barrier and affect the outcome of neuronal injury or cognitive function was evaluated. It was shown that EPO and EPO receptors are expressed at capillaries of the brain-periphery interface, and that systemically administered epoetin alfa crossed the blood-brain barrier. Compared with control animals, epoetin alfa significantly reduced tissue damage in an ischemic stroke model when administered 24 hours before inducing stroke, with significant protection still evident when epoetin alfa was administered 6 hours poststroke. Epoetin alfa reduced injury by blunt trauma when administered 24 hours before trauma, with a significantly smaller volume of tissue necrosis noted when compared with controls. The observation that epoetin alfa may reduce nervous system inflammation was confirmed when an experimental autoimmune encephalomyelitis model in which rats were shown to have significantly delayed onset and reduced severity of experimental autoimmune encephalomyelitis symptoms after treatment with epoetin alfa. Epoetin alfa also was shown to ameliorate the latency and severity of seizures, and significantly increase survival versus controls when exposed to kainate. These findings suggest future potential therapeutic uses for epoetin alfa beyond its current use to increase erythropoiesis.

Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury.

Erythropoietin (EPO), recognized for its central role in erythropoiesis, also mediates neuroprotection when the recombinant form (r-Hu-EPO) is directly injected into ischemic rodent brain. We observed abundant expression of the EPO receptor at brain capillaries, which could provide a route for circulating EPO to enter the brain. In confirmation of this hypothesis, systemic administration of r-Hu-EPO before or up to 6 h after focal brain ischemia reduced injury by approximately 50-75%. R-Hu-EPO also ameliorates the extent of concussive brain injury, the immune damage in experimental autoimmune encephalomyelitis, and the toxicity of kainate. Given r-Hu-EPO's excellent safety profile, clinical trials evaluating systemically administered r-Hu-EPO as a general neuroprotective treatment are warranted.

Tobacco smoke is a source of toxic reactive glycation products.

Smokers have a significantly higher risk for developing coronary and cerebrovascular disease than nonsmokers. Advanced glycation end products (AGEs) are reactive, cross-linking moieties that form from the reaction of reducing sugars and the amino groups of proteins, lipids, and nucleic acids. AGEs circulate in high concentrations in the plasma of patients with diabetes or renal insufficiency and have been linked to the accelerated vasculopathy seen in patients with these diseases. Because the curing of tobacco takes place under conditions that could lead to the formation of glycation products, we examined whether tobacco and tobacco smoke could generate these reactive species that would increase AGE formation in vivo. Our findings show that reactive glycation products are present in aqueous extracts of tobacco and in tobacco smoke in a form that can rapidly react with proteins to form AGEs. This reaction can be inhibited by aminoguanidine, a known inhibitor of AGE formation. We have named these glycation products "glycotoxins." Like other known reducing sugars and reactive glycation products, glycotoxins form smoke, react with protein, exhibit a specific fluorescence when cross-linked to proteins, and are mutagenic. Glycotoxins are transferred to the serum proteins of human smokers. AGE-apolipoprotein B and serum AGE levels in cigarette smokers were significantly higher than those in nonsmokers. These results suggest that increased glycotoxin exposure may contribute to the increased incidence of atherosclerosis and high prevalence of cancer in smokers.

High-performance liquid chromatographic method for guanylhydrazone compounds.

A high-performance liquid chromatographic method has been developed for a series of aromatic guanylhydrazones that have demonstrated therapeutic potential as anti-inflammatory agents. The compounds were separated using octadecyl or diisopropyloctyl reversed-phase columns, with an acetonitrile gradient in water containing heptane sulfonate, tetramethylammonium chloride, and phosphoric acid. The method was used to reliably quantify levels of analyte as low as 785 ng/ml, and the detector response was linear to at least 50 micrograms/ml using a 100 microliters injection volume. The assay system was used to determine the basic pharmacokinetics of a lead compound, CNI-1493, from serum concentrations following a single intravenous injection in rats.

Structural and functional properties of region II-plus of the malaria circumsporozoite protein.

During feeding, infected mosquitos inject malaria sporozoites into the host circulation. Within minutes, the parasites are found in the liver where they initiate the first stage of malaria infection. All species of malaria sporozoites are uniformly covered by the circumsporozoite protein (CS), which contains a conserved COOH-terminal sequence called region II-plus. We have previously shown that region II-plus is the parasite's hepatocyte-binding ligand and that this ligand binds to heparan sulfate proteoglycans (HSPGs) on the hepatocyte membrane. Using a series of substituted region II-plus peptides, we show here that the downstream basic amino acids as well as the interdispersed hydrophobic residues are required for binding of CS to hepatocyte HSPGs. We also show that this positively charged stretch of amino acids must be aggregated in order to bind to the receptor. On the basis of this information, we have synthesized a multiple antigen peptide that mimics the hepatocyte-binding ligand. This construct inhibits both CS binding to HepG2 cells in vitro as well as CS clearance in mice.

Rapid clearance of malaria circumsporozoite protein (CS) by hepatocytes.

The circumsporozoite protein (CS) covers uniformly the plasma membrane of malaria sporozoites. In vitro, CS multimers bind specifically to regions of the hepatocyte plasma membrane that are exposed to circulating blood in the Disse space. The ligand is in the region II-plus of CS, an evolutionarily conserved stretch of the protein that has amino acid sequence homology to a cell adhesive motif of thrombospondin. We have now found that intravenously injected CS constructs bind rapidly to the basolateral surface of hepatocytes, provided that the recombinant proteins contain region II-plus, and that they are aggregated. Significant amounts of CS were not retained in any other organ. The striking parallelism between these in vitro and in vivo findings with the target specificity of malaria sporozoites, reinforces the hypothesis that the attachment of the parasites to hepatocytes is via region II-plus of CS.

Malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes.

During feeding by infected mosquitoes, malaria sporozoites are injected into the host's bloodstream and enter hepatocytes within minutes. The remarkable target cell specificity of this parasite may be explained by the presence of receptors for the region II-plus of the circumsporozoite protein (CS) on the basolateral domain of the plasma membrane of hepatocytes. We have now identified these receptors as heparan sulfate proteoglycans (HSPG). The binding of CS to the receptors is abolished by heparitinase treatment, indicating that the recognition of region II-plus is via the glycosaminoglycan chains. We have purified and partially characterized the CS-binding HSPGs from HepG2 cells. They have a molecular weight of 400,000-700,000, are tightly associated with the plasma membrane, and are released from the cell surface by very mild trypsinization, a property which the CS receptors share with the syndecan family of proteoglycans.

The basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of Plasmodium falciparum sporozoites.

Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. We show here that recombinant Plasmodium falciparum circumsporozoite protein (CS) binds specifically to regions of the plasma membrane of hepatocytes exposed to circulating blood in the Disse space. No binding has been detected in other organs, or even in other regions of the hepatocyte membrane. The interaction of CS with hepatocytes, as well as sporozoite invasion of HepG2 cells, is inhibited by synthetic peptides representing the evolutionarily conserved region II of CS. We conclude that region II is a sporozoite ligand for hepatocyte receptors localized to the basolateral domain of the plasma membrane. Our findings provide a rational explanation for the target cell specificity of malaria sporozoites.

Binding of malarial circumsporozoite protein to sulfatides [Gal(3-SO4)beta 1-Cer] and cholesterol-3-sulfate and its dependence on disulfide bond formation between cysteines in region II.

Region II of the malaria circumsporozoite (CS) protein is highly conserved between the CS proteins of different species of malaria. Amino acid sequences homologous to that of region II are found in thrombospondin, properdin, von Willebrand factor and a few other proteins. We show here that the native CS protein from the rodent parasite Plasmodium berghei, and recombinant Plasmodium vivax and Plasmodium falciparum CS proteins containing region II, but not recombinant proteins lacking region II, specifically bind to sulfatides and cholesterol-3-sulfate. The binding is abolished following reduction and alkylation of the proteins. Region II contains 2 cysteines separated by only 3 amino acids, S(N), V, T, and these are the only cysteines present in our recombinant proteins. Therefore, our findings strongly suggest that the region II cysteines are linked by a disulfide bond forming a small peptide loop. We also present evidence that the recognition of sulfatides, cholesterol-3-sulfate, or other cross-reactive sulfated macromolecules by region II may be required during sporozoite invasion of liver cells. Antibodies to a peptide representing region II react with live sporozoites and with sporozoites fixed with glutaraldehyde, indicating that this region is exposed on the surface of the parasites. Furthermore, we have found that the sulfatide and cholesterol-3-sulfate recognition by the CS proteins, and the invasion of hepatocytes by P. berghei sporozoites, are specifically inhibited by dextran sulfate.

Advanced glycosylation endproducts on erythrocyte cell surface induce receptor-mediated phagocytosis by macrophages. A model for turnover of aging cells.

Glucose can react nonenzymatically with amino groups of proteins to form covalent Amadori products. With time these adducts undergo further rearrangements to form irreversible advanced glycosylation endproducts (AGE), which accumulate with protein age. A specific AGE, 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI), has been identified on proteins in vivo. We have recently shown that a macrophage receptor specifically recognizes and internalizes proteins modified by AGE such as FFI, thus preferentially degrading senescent macromolecules. Reasoning that cellular turnover may be mediated by macrophage recognition of AGE-membrane proteins, we prepared human RBCs with FFI attached chemically. Human monocytes were incubated with either FFI-RBCs, IgG-opsonized RBCs, or PBS-treated RBCs. Erythrophagocytosis of FFI-RBCs was significantly higher than that of PBS-RBCs (55 vs. 4%; p less than 0.0025) and almost as high as that of IgG-RBCs (70%), and was competitively inhibited by AGE-BSA. AGE-RBCs were also prepared by incubating RBCs with various sugars. Human monocytes showed a 15% ingestion of glucose-RBCs, and a 26% ingestion of glucose-6-phosphate-RBCs, compared to 6% for PBS-RBCs. Similarly, diabetic mouse RBCs were phagocytosed by nearly three times more cells (21%) than normal mouse RBCs when exposed to syngeneic mouse macrophages. This phagocytosis was competitively inhibited (70%) by addition of excess AGE-BSA. The in vivo half-life of 51Cr-labeled mouse FFI-RBCs injected into syngeneic mice was reduced to 7 d, as compared to a half-life of 20 d for the controls. These data suggest that the macrophage receptor for the removal of glucose-modified proteins may also mediate the endocytosis of RBCs with AGE formed on their surface, and thus be responsible in part for the removal of some populations of aging cells.