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There is a growing interest in the potential roles of misfolded protein interactions in neurodegeneration. To investigate this issue, we inoculated 3 prion strains intracerebrally into transgenic (TgM83) mice that overexpress human A53T α-synuclein. In comparison to nontransgenic controls, there was a striking decrease in the incubation periods of scrapie, classic and H-type bovine spongiform encephalopathies(C-BSE and H-BSE), with conservation of the histopathologic and biochemical features characterizing these 3 prion strains. TgM83 mice died of scrapie or C-BSE prion diseases before accumulating the insoluble and phosphorylated forms of α-synuclein specific to late stages of synucleinopathy. In contrast, the median incubation time for TgM83 mice inoculated with H-BSE was comparable to that observed when these mice were uninfected, thereby allowing the development of molecular alterations of α-synuclein. The last 4 mice of this cohort exhibited early accumulations of H-BSE prion protein along with α-synuclein pathology. The results indicate that a prion disease was triggered concomitantly with an overt synucleinopathy in some transgenic mice overexpressing human A53T α-synuclein after intracerebral inoculation with an H-BSE prion strain.

A new method for the rapid and accurate detection of pathogenic Naegleria fowleri amoebae in surface environmental water was developed. The method is based on an immunofluorescent assay combined with detection by solid-phase cytometry. In this study we developed and compared two protocols using different reporter systems conjugated to antibodies. The monoclonal antibody Ac5D12 was conjugated with biotin and horseradish peroxidase, and the presence of cells was revealed with streptavidin conjugated to both Rphycoerythrin and cyanine Cy5 (RPE-Cy5) and tyramide-fluorescein isothiocyanate, respectively. The RPE-Cy5 protocol was the most efficient protocol and allowed the detection of both trophozoite and cyst forms in water. The direct counts obtained by this new method were not significantly different from those obtained by the traditional culture approach, and results were provided within 3 h. The sensitivity of the quantitative method is 200 cells per liter. The limit is due only to the filtration capacity of the membrane used.The free-living amoeba Naegleria fowleri (3, 16), found in diverse freshwater environments, produces a rapidly fatal primary amoebic meningoencephalitis after exposure to contaminated water (7,11,12). N. fowleri infects mostly young and healthy people swimming in contaminated water. Symptoms occur in a few days, followed by a dramatic clinical course and death. Therefore, risk prevention is essential and necessitates environmental monitoring using a rapid and accurate assay to distinguish pathogenic N. fowleri from other free-living amoeba in water samples.Current methods for detection and enumeration of Naegleria species are based on culture techniques (8) followed by identification using monoclonal antibodies (19,21), PCR (10,20), or enzyme electrophoresis (15). Additionally, isolates are tested for pathogenicity in mice. These methods are timeconsuming, and novel methods are being developed to increase the sensitivity and rapidity of detection and thus reduce the amount of time required to obtain results. The main challenges for the development of an assay are to provide tools for the real-time monitoring of the pathogen in the aquatic environment which are highly quantitative and sensitive.Epifluorescence microscopy and flow cytometry are commonly used for the detection and enumeration of cells after fluorescent staining (1, 6). However, none of these techniques can be applied to the detection of low concentrations of pathogens in the aquatic environment because of their low quantitative sensitivity (10). The ChemScan system (Chemunex, Ivry, France) is a recently developed solid-phase cytometer that uses fluorescent labeling of microorganisms after concentration of organisms by filtration on a membrane in combination with an automated detection and counting system (13, 23). Solid-phase cytometry is the only technique that allows the accurate enumeration of rare events (down to one cell on a filtration membrane), providing the same sensitivity as traditional culture methods (10). This sy...

A monoclonal antibody (MAb) 81D1c2, which recognizes the N epsilon (gamma glutamyl) lysine isopeptide produced by the action of transglutaminase activity was prepared. Its reactivity towards the homologous isopeptide was about 3-fold greater than that with either N alpha (alpha glutamyl) lysine (a naturally occurring heterologous dipeptide) or N alpha (gamma glutamyl) lysine, another heterologous peptide not described so far in naturally occurring proteins. When used in an immunohistochemical study on cells in culture derived from human carcinoma of the larynx (HEp2) and from chicken embryo cells (CEC), both fixed in acetone, this MAb detected N epsilon (gamma glutamyl) lysine residues in the nucleus. The amount of N epsilon (gamma glutamyl) lysine isopeptides follows closely transglutaminase activity during the lag phase of growth of both CEC and HEp2 cells. However, during exponential growth, the 2 parameters decrease concomitantly in HEp2 cells, whereas in CEC, transglutaminase activity increases but isopeptide bond levels drop. Compared with other reported methods for measuring isopeptides, this immunohistological approach permits the localization and at least the semi-quantitative determination of N epsilon (gamma glutamyl) lysine in cells in situ.

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