Proteopathic tau seeding predicts tauopathy in vivo

Transcellular propagation of protein aggregates, or proteopathic seeds, may drive the progression of neurodegenerative diseases in a prion-like manner. In tauopathies such as Alzheimer’s disease, this model predicts that tau seeds propagate pathology through the brain via cell–cell transfer in neural networks. The critical role of tau seeding activity is untested, however. It is unknown whether seeding anticipates and correlates with subsequent development of pathology as predicted for a causal agent. One major limitation has been the lack of a robust assay to measure proteopathic seeding activity in biological specimens. We engineered an ultrasensitive, specific, and facile FRET-based flow cytometry biosensor assay based on expression of tau or synuclein fusions to CFP and YFP, and confirmed its sensitivity and specificity to tau (∼300 fM) and synuclein (∼300 pM) fibrils. This assay readily discriminates Alzheimer’s disease vs. Huntington''s disease and aged control brains. We then carried out a detailed time-course study in P301S tauopathy mice, comparing seeding activity versus histological markers of tau pathology, including MC1, AT8, PG5, and Thioflavin S. We detected robust seeding activity at 1.5 mo, >1 mo before the earliest histopathological stain. Proteopathic tau seeding is thus an early and robust marker of tauopathy, suggesting a proximal role for tau seeds in neurodegeneration.Protein aggregation characterizes many neurodegenerative disorders, including Alzheimer’s disease (AD) and the related tauopathies. These disorders feature the accumulation of fibrillar deposits of the microtubule-associated protein tau with progressive deterioration of the central nervous system. Tau pathology and its associated brain atrophy do not appear randomly throughout the brain, but rather progress along distinct neural networks (1–5). This aspect suggests a role for transcellular spread of a pathogenic agent via neural connections. Our laboratory and others have previously hypothesized that tau aggregates—or seeds—serve as this agent of spread, transmitting the aggregated state from cell to cell via prion-like mechanisms (6–15).Mounting fundamental insights support this hypothesis. Tau seeds applied to the outside of cells bind the cell surface by attaching to heparan sulfate proteoglycans, triggering uptake by macropinocytosis (13). Upon internalization, tau seeds nucleate the fibrillization of endogenous tau monomer via templated conformational change, or seeding (8, 10). Tau seeding requires a critical unit of size for activity, as only particular species propagate the aggregated state (16). In vivo studies have described tau protein spreading from local sites to distant regions, presumably via transsynaptic movement (11, 12, 17–19). Finally, our laboratory and another recently demonstrated that tau propagates discrete amyloid conformations through the brains of animals that give rise to unique neuropathologies (18, 20).Despite this evidence, it remains unclear whether the development of proteopathic tau seeding represents a causal process of tauopathy, a downstream consequence of tau fibril accumulation, a coincident trait of neurodegeneration, or even an epiphenomenon. If proteopathic seeds are indeed a causal agent of disease, then their activity should exist in the brains of tauopathy mouse models and human subjects, precede other forms of pathology, and correlate with disease progression.To test these hypotheses, we created a highly sensitive and quantitative assay using a novel FRET-based biosensor cell line that specifically reports tau seeding activity. With this assay, we profiled the temporal evolution of tau seeding activity in the brains of P301S transgenic mice, a model of human tauopathy, and compared it to standard measurements of histopathology taken from the same animals. We find that tau seeding marks incipient tauopathy, occurring far before the onset of several standard histopathological markers. This finding implicates proteopathic tau seeding as a proximal cause of neurodegeneration, and establishes a highly quantitative and sensitive seed detection method.     

Average arterial input function for quantitative dynamic contrast enhanced magnetic resonance imaging of neck nodal metastases

Background

We evaluated the repeatability of the calculation of myocardial blood flow (MBF) at rest and pharmacological stress, and calculated the coronary flow reserve (CFR) utilizing ⁸²Rb PET imaging. The aim of the research was to prove high repeatability for global MBF and CFR values and good repeatability for regional MBF and CFR values. The results will have significant impact on cardiac PET imaging in terms of making it more affordable and increasing its use.

Methods

12 normal volunteers were imaged at rest and during pharmacological stress, with 2220 MBq of ⁸²Rb each. A GE Advance PET system was used to acquire dynamic 50-frame studies. MBF was calculated with a 2-compartmental model using a modified PMOD program (PMOD; University Hospital Zurich, Zurich, Switzerland). Two differential equations, describing a 2-compartmental model, were solved by numerical integration and using Levenberg-Marquardt's method for fitting data. The PMOD program defines 16 standard segments and calculates myocardial flow for each segment, as well as average septal, anterior, lateral, inferior and global flow. Repeatability was evaluated according to the method of Bland and Altman.

Results

Global rest and stress MBF, as well as global CFR, showed very good repeatability. No significant differences were found between the paired resting global MBF (0.63 ± 0.13 vs. 0.64 ± 0.13 mL/min/g; mean difference, -1.0% ± 2.6%) and the stress global MBF (1.37 ± 0.23 vs. 1.37 ± 0.24; mean difference, 0.1% ± 2.3%). Global CFR was highly reproducible (2.25 ± 0.56 vs. 2.22 ± 0.54, P = not statistically significant; mean difference, 1.3% ± 14.3%). Repeatability coefficients for global rest MBF were 0.033 (5.2%) and stress MBF 0.062 (4.5%) mL/min/g. Regional rest and stress MBF and CFR have shown good reproducibility. The average per sector repeatability coefficients for rest MBF were 0.056 (8.5%) and stress MBF 0.089 (6.3%) mL/min/g, and average repeatability coefficient for CFR was 0.25 (10.6%).

Conclusion

The results of the study show that software calculation of MBF and CFR with ⁸²Rb myocardial PET imaging is highly repeatable for global values and has good repeatability for regional values.     

Alcohol consumption and blood lipids in elderly coronary patients

Alcohol may have a beneficial effect on coronary heart disease (CHD) that could be mediated by elevation of high-density lipoprotein cholesterol (HDLC). Data on alcohol consumption and blood lipids in coronary patients are scarce. We studied whether total ethanol intake and consumption of specific types of beverages are associated with blood lipids in older subjects with CHD. Blood lipids were measured in 1052 myocardial infarction patients aged 60 to 80 years (78% male). Intake of alcoholic beverages, total ethanol, and macronutrients was assessed by food frequency questionnaire. Seventy percent of the subjects used lipid-lowering medication. Total cholesterol was on average 5.14 mmol/L, and HDLC was on average 1.28 mmol/L. Among men, total ethanol intake was positively associated with HDLC (difference of 0.094 mmol/L for ≥15 g/d vs 0 g/d, P = .024), whereas the association with HDLC among women was not significant (difference of 0.060 mmol/L for ≥5 g/d vs 0 g/d, P = .560) after adjustment for dietary, lifestyle, and CHD risk factors. Liquor consumption was weakly positively associated with HDLC in men (P = .045). Beer consumption in men and wine consumption in women were also positively associated with HDLC, but were not significant in the fully adjusted model. In conclusion, moderate alcohol consumption may elevate HDLC in treated post-myocardial infarction patients. This may be due to ethanol and not to other beneficial substances in alcoholic beverages. Based on this finding, further research needs to be done to examine the effects of the residual substances from different types of alcoholic beverages on HDLC.     

Cytodiagnosis of actinomycetoma

Mycetoma is a late clinical manifestation of a subcutaneous infection produced by bacteria (actinomycetoma) or fungi (eumycetoma). The distinction between eumycetoma and actinomycetoma in Fine Needle Aspiration Cytology (FNAC) is as accurate as histopathology. A 55 year old man presented with a slow growing swelling on the plantar aspect of the right foot which was present for the last 10 years. A clinical diagnosis of soft tissue tumor was made and FNAC was advised. Smears revealed mixed inflammatory infiltrate and foreign body type of giant cells along with clumps of fibrillar organisms. Gram stain done later demonstrated gram positive thin branching filaments. A diagnosis of actinomycetoma was rendered. Histopathology of the excised specimen confirmed the cytologic diagnosis of actinomycetoma. Mycetoma can be accurately diagnosed by FNAC, which is a simple, inexpensive technique for rapid diagnosis. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

Tridimensional ultrastructure and glycolipid pattern studies of Trypanosoma dionisii

Trypanosoma (Schizotrypanum) dionisii is a non-pathogenic bat trypanosome closely related to Trypanosoma cruzi, the etiological agent of Chaga's disease. Both kinetoplastids present similar morphological stages and are able to infect mammalian cells in culture. In the present study we examined 3D ultrastructure aspects of the two species by serial sectioning epimastigote and trypomastigote forms, and identified common carbohydrate epitopes expressed in T. dionisii, T. cruzi and Leishmania major. A major difference in 3D morphology was that T. dionisii epimastigote forms present larger multivesicular structures, restricted to the parasite posterior region. These structures could be related to T. cruzi reservosomes and are also rich in cruzipain, the major cysteine-proteinase of T. cruzi. We analyzed the reactivity of two monoclonal antibodies: MEST-1 directed to galactofuranose residues of glycolipids purified from Paracoccidioides brasiliensis, and BST-1 directed to glycolipids purified from T. cruzi epimastigotes. Both antibodies were reactive with T. dionisii epimastigotes by indirect immunofluorescense, but we noted differences in the location and intensity of the epitopes, when compared to T. cruzi. In summary, despite similar features in cellular structure and life cycle of T. dionisii and T. cruzi, we observed a unique morphological characteristic in T. dionisii that deserves to be explored.     

Gene polymorphism profiles of drug-metabolising enzymes GSTM1, GSTT1 and GSTP1 in an Argentinian population

Background: Glutathione S-transferases (GSTs) are drug-metabolising enzymes involved in biotransformation of carcinogens, drugs, xenobiotics and oxygen free radicals. Polymorphisms of GST genes contribute to inter-individual and population variability in the susceptibility to environmental risk factors, cancer predisposition and pharmacotherapy responses. However, data about GST variability in Argentina are lacking.

Aim: The purpose was to determine the prevalence of GSTM1, GSTT1 and GSTP1 polymorphisms in the general population from a central region of Argentina and to perform inter-population comparisons.

Subjects and methods: GSTM1 and GSTT1 gene deletions and GSTP1 c.313A?>?G were genotyped by PCR assays in 609 healthy and unrelated Argentinians.

Results: The frequencies of variant genotypes in Argentinians were GSTM1-null (45%), GSTT1-null (17%) and GSTP1-GG (11%). GSTM1-present genotype was significantly associated with GSTP1-AG or GSTP1-GG variants (p?=?0.037; p?=?0.034, respectively). Comparison with worldwide populations demonstrated that the GST distributions in Argentina are similar to those reported for Italy and Spain, whereas significant differences were observed regarding Asian and African populations (p?Conclusion: This study has determined, for the first time, the normative profile of three pharmacogenetically relevant polymorphisms (GSTM1, GSTT1 and GSTP1) in the largest Argentinian cohort described to date, providing the basis for further epidemiological and pharmacogenetic studies in this country.     

Assessment of physical education time and after-school outdoor time in elementary and middle school students in south Mexico City: the dilemma between physical fitness and the adverse health effects of outdoor pollutant exposure

Strategies to promote lifelong physical activity among children are needed to stem the adverse health consequences of inactivity. However, the health effects in growing children of long-term exposure to a polluted atmosphere are of deep concern. The atmosphere of south Mexico City (SMC) is characterized by a complex mixture of air pollutants, including ozone, particulate matter, and aldehydes. Radiological evidence suggests that small-airway disease could be present in clinically healthy, tobacco unexposed SMC children. The aim of this study was to assess, by means of a self-reported questionnaire, the physical education class times, daily outdoor after-school exposure time, and tobacco exposure in students attending public elementary and middle schools in SMC. Additionally, the time each student spent viewing television was assessed, and the authors measured each student's weight and height to determine body mass index (BMI, weight in kg divided by height in m2). The survey included 1,159 students in grades 7-9. The authors identified 2 critical periods of outdoor exposure in SMC children that coincided with significant concentrations of both ozone and particulate matter with diameters less than 10 micrometers (PM10): during school time after 11:00 A.M. and in the after-school outdoor activity period, usually extending from 1:00 P.M. to 6:00 P.M. Thirty-two percent of elementary and 61% of middle school students have physical education classes after 11:00 A.M. Students in SMC spend an average of 19.6 hr/wk outdoors in the after-school period, during which time they are engaged in light to moderate physical activities. Half of the students are exposed to tobacco smoke at home, and 7% of middle school students smoke. On the basis of BMI, 60% of students were classified as undernourished, overweight, or obese. No correlations were found between BMI and time spent viewing TV, time outdoors (on weekdays and weekends), or exposure to environmental tobacco smoke. Children and adolescents in SMC are participating in physical activities that enhance multiple components of health-related fitness. However, their activities occur outdoors, where they are exposed to high concentrations of air pollutants throughout the year. The authors believe that SMC children and adolescents must be educated, through both the school and health systems, regarding ways to obtain the necessary exercise while protecting themselves from the high concentrations of pollutants. Individuals should instruct and encourage young people to be involved in lifetime fitness activities and to eat balanced diets, if the goal is to control health-care costs, reduce disease incidence, and improve the overall quality of life of the Mexico City population.     

Differential secretion of chicken growth hormone variants after growth hormone-releasing hormone stimulation in vitro

Variants of growth hormone (GH) are present in most vertebrates. Chicken GH (cGH) undergoes posttranslational modifications that contribute to its structural diversity. Although the 22-kDa form of GH is the most abundant, some other variants have discrete bioactivities that may not be shared by others. The proportion of cGH variants changes during ontogeny, suggesting that they are regulated differentially. The effect of growth hormone-releasing hormone (GHRH) on the release of cGH variants was studied in both pituitary gland and primary cell cultures, employing sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and densitometry. GHRH (2 nM, 2 h) stimulated the secretion of most of the size variants of cGH although the amplitude of increase was not equal for each one. A differential effect on the secretion of GH size variants, particularly on the 22- (monomer) and 26-kDa (putatively glycosylated) cGH isoforms was found in both systems. In the whole pituitary culture, the proportion of the 26-kDa immunoreactive cGH increased 35% while the 22 kDa decreased 31% after GHRH treatment in comparison with the controls. In the primary cell culture system, the proportion of the glycosylated variant increased 43% whereas the monomer and the dimer decreased 22.26 and 29%, respectively, after GHRH stimulation. Activators of intracellular signals such as 1 mM 8-bromo-cAMP and 1 μM phorbol myristate acetate had a similar effect to that obtained with GHRH. The data support the hypothesis that GH variants may be under differential control and that GHRH promotes the release of a glycosylated cGH variant that has an extended half-life in circulation.