Relationships of circulating CD4+ T cell subsets and cytokines with the risk of relapse in patients with Crohn's disease.

Background and aims: We aimed to analyze circulating CD4+ T cell subsets and cytokines during the course of Crohn's disease (CD). Methods and results: CD4+ T cell subsets, ultrasensitive C-reactive protein (usCRP), and various serum cytokines (IL-6, IL-8, IL-10, IL-13, IL-17A, IL-23, TNFα, IFNγ, and TGFß) were prospectively monitored every 3 months for 1 year, using multicolor flow cytometry and an ultrasensitive Erenna method in CD patients in remission at inclusion. Relapse occurred in 35 out of the 113 consecutive patients (31%). For patients in remission within 4 months prior to relapse and at the time of relapse, there was no significant difference in Th1, Th17, Treg, and double-positive CD4+ T cell subsets co-expressing either IFNγ and FOXP3, IL-17A and FOXP3, or IFNγ and IL-17A. On the contrary, in patients who remained in remission, the mean frequency and number of double-positive IL-17A+FOXP3+ CD4+ T cells and the level of usCRP were significantly higher (p ≤ 0.01) 1 to 4 months prior to relapse. At the time of relapse, only the IL-6 and usCRP levels were significantly higher (p ≤ 0.001) compared with those patients in remission. On multivariate analysis, a high number of double-positive IL-17A+FOXP3+ CD4+ T cells (≥1.4 cells/mm3) and elevated serum usCRP (≥3.44 mg/L) were two independent factors associated with risk of relapse. Conclusions: Detection of circulating double-positive FOXP3+IL-17A+ CD4+ T cell subsets supports that T cell plasticity may reflect the inflammatory context of Crohn's disease. Whether this subset contributes to the pathogenesis of CD relapse needs further studies.

Detection of Disease-associated α-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated α-synuclein.

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated α-synuclein (αS(D)) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T αS and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of αS(D) (disease-associated αS) in these mice, an ELISA was designed to specifically quantify αS(D) in sick mice. Analysis of the central nervous system in this mouse model showed the presence of αS(D) mainly in the caudal brain regions and the spinal cord. There were no differences in αS(D) distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of αS(D) immunoreactivity using an antibody against Ser129 phosphorylated αS by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of αS. The propagation of αS(D), suggesting the involvement of a "prion-like" mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

Prion-like acceleration of a synucleinopathy in a transgenic mouse model.

Our aim in this study was to investigate experimentally the possible in vivo transmission of a synucleinopathy, using a transgenic mouse model (TgM83) expressing the human A53T mutated α-synuclein. Brain homogenates from old TgM83 mice showing motor clinical signs due to the synucleinopathy and containing insoluble and phosphorylated (pSer129) α-synuclein were intracerebrally inoculated in young TgM83 mice. This triggered an early onset of characteristic motor clinical signs, compared with uninoculated TgM83 mice or to mice inoculated with a brain homogenate from a young, healthy TgM83 mouse. This early disease was associated with insoluble α-synuclein phosphorylated on Ser129, as already identified in old and sick uninoculated TgM83 transgenic mice. Although the molecular mechanisms remain to be determined, acceleration of the pathology following inoculation of mice expressing human mutated α-synuclein with tissues from mice affected by the synucleinopathy, could be consistent with "prion-like" propagation of the disease.

Transmission of prion strains in a transgenic mouse model overexpressing human A53T mutated α-synuclein.

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.

Production of a monoclonal antibody, against human α-synuclein, in a subpopulation of C57BL/6J mice, presenting a deletion of the α-synuclein locus.

Analyses using antibodies directed against α-synuclein play a key role in the understanding of the pathologies associated with neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). However, the generation of antibodies against immunogens with significant sequence similarity to host proteins such as α-synuclein is often hindered by host immunotolerance. In contrast to wild-type C57BL/6J and BALB/c mice immunized with recombinant human α-synuclein, C57BL/6S Δsnca mice presenting a natural deletion of the α-synuclein locus, bypassed the immunotolerance process which resulted in a much higher polyclonal antibody response. The native or fibrillized conformation of α-synuclein used as the immunogen did not have an impact on the amounts of specific antibodies in sera of the host. The immunization protocols resulted in the generation of the IgG AS11, raised against fibrillized recombinant human α-synuclein in C57BL/6S Δsnca mice. This monoclonal antibody, recognizing an N-terminal α-synuclein epitope, was selected for its specificity and significant reactivity in Western-blot, immunofluorescence and immunohistochemistry assays. The ability of AS11 to detect both soluble and aggregated forms of α-synuclein present in pathological cytoplasmic inclusions was further assessed using analysis of human brains with PD or MSA, transgenic mouse lines expressing A53T human α-synuclein, and cellular models expressing human α-synuclein. Taken together, our study indicates that novel antibodies helpful to characterize alterations of α-synuclein leading to neurodegeneration in PD and related disorders could be efficiently developed using this original immunization strategy.

An enzyme-linked immunosorbent assay (ELISA) for the identification of Naegleria fowleri in environmental water samples.

Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis, a fatal human disease of the central nervous system often contracted after swimming in fresh water. Identifying sites contaminated by N. fowleri is important in order to prevent the disease. An Enzyme-Linked ImmunoSorbent Assay (ELISA) has been developed for the specific identification of N. fawleri in primary cultures of environmental water samples. Of 939 samples isolated from artificially heated river water and screened by ELISA, 283 were positive. These results were subsequently confirmed by isoelectric focusing, the established reference method. A sensitivity of 97.4% and a specificity of 97% were obtained. These results indicate that this ELISA method is reliable and can be considered as a powerful tool for the detection of N. fowleri in environmental water samples.

Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry.

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 R-phycoerythrin 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.