Tau in the brain interstitial fluid is fragmented and seeding-competent.

In Alzheimer disease, Tau pathology is thought to propagate from cell to cell throughout interconnected brain areas. However, the forms of Tau released into the brain interstitial fluid (ISF) in vivo during the development of Tauopathy and their pathological relevance remain unclear. Combining in vivo microdialysis and biochemical analysis, we find that in Tau transgenic mice, human Tau (hTau) present in brain ISF is truncated and comprises at least 10 distinct fragments spanning the entire Tau protein. The fragmentation pattern is similar across different Tau transgenic models, pathological stages and brain areas. ISF hTau concentration decreases during Tauopathy progression, while its phosphorylation increases. ISF from mice with established Tauopathy induces Tau aggregation in HEK293-Tau biosensor cells. Notably, immunodepletion of ISF phosphorylated Tau, but not Tau fragments, significantly reduces its ability to seed Tau aggregation and only a fraction of Tau, separated by ultracentrifugation, is seeding-competent. These results indicate that ISF seeding competence is driven by a small subset of Tau, which potentially contribute to the propagation of Tau pathology.

Collecting antibodies and large molecule biomarkers in mouse interstitial brain fluid: a comparison of microdialysis and cerebral open flow microperfusion.

The determination of concentrations of large therapeutic molecules, like monoclonal antibodies (mAbs), in the interstitial brain fluid (ISF) is one of the cornerstones for the translation from preclinical species to humans of treatments for neurodegenerative diseases. Microdialysis (MD) and cerebral open flow microperfusion (cOFM) are the only currently available methods for extracting ISF, and their use and characterization for the collection of large molecules in rodents have barely started. For the first time, we compared both methods at a technical and performance level for measuring ISF concentrations of a non-target-binding mAb, trastuzumab, in awake and freely moving mice. Without correction of the data for recovery, concentrations of samples are over 10-fold higher through cOFM compared to MD. The overall similar pharmacokinetic profile and ISF exposure between MD (corrected for recovery) and cOFM indicate an underestimation of the absolute concentrations calculated with in vitro recovery. In vivo recovery (zero-flow rate method) revealed an increased extraction of trastuzumab at low flow rates and a 6-fold higher absolute concentration at steady state than initially calculated with the in vitro recovery. Technical optimizations have significantly increased the performance of both systems, resulting in the possibility of sampling up to 12 mice simultaneously. Moreover, strict aseptic conditions have played an important role in improving data quality. The standardization of these complex methods makes the unraveling of ISF concentrations attainable for various diseases and modalities, starting in this study with mAbs, but extending further in the future to RNA therapeutics, antibody-drug conjugates, and even cell therapies.

Alternative splicing of the TNFSF13B (BAFF) pre-mRNA and expression of the BAFFX1 isoform in human immune cells.

Human B cell activating factor (TNFSF13B, BAFF) is a tumor necrosis factor superfamily member. Binding its unique receptor (TNFRSF13C, BAFF-R) mediates gene expression and cell survival in B cells via activation of NFκB pathway. Furthermore, there is data indicating a role in T cell function. A functionally inhibitory isoform (ΔBAFF) resulting from the deletion of exon 3 in the TNFSF13B pre-RNA has already been reported. However, data on the complexity of post-transcriptional regulation is scarce. Here, we report molecular cloning of nine TNFSF13B transcript variants resulting from alternative splicing of the TNFSF13B pre-mRNA including BAFFX1. This variant is characterized by a partial retention of intron 3 of the TNFSF13B gene causing the appearance of a premature stop codon. We demonstrate the expression of the corresponding BAFFX1 protein in Jurkat T cells, in ex vivo human immune cells and in human tonsillar tissue. Thereby we contribute to the understanding of TNFSF13B gene regulation and reveal that BAFF is regulated through a post-transcriptional mechanism to a greater extent than reported to date.

N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain.

Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions.Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component (< 0.1%) of insoluble Tau.Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.

Levetiracetam but not valproate inhibits function of CD8+ T lymphocytes.

PURPOSE: To further elucidate possible immune-modulatory effects of valproate (VPA) or levetiracetam (LEV), we investigated their influence on apoptosis and cytotoxic function of CD8+ T lymphocytes in humans. METHODS: In 15 healthy subjects (9 female (60%), 35.7±12.1 years), apoptosis and cytotoxic function of CD8+ T lymphocytes were measured using flow cytometry following in vitro exposure to LEV (5 mg/L and 50 mg/L) and VPA (10mg/L and 100 mg/L). Apoptosis rates were determined after incubation with LEV or VPA for 1 h or 24 h. Cytotoxic function was assessed following 2h stimulation with mixed virus peptides, using perforin release, CD107a/b expression and proliferation. The presence of synaptic vesicle protein 2A (SV2A) was investigated in human CD8+ T lymphocytes by flow cytometry analysis, Western blot and real time polymerase chain reaction (rtPCR). RESULTS: High concentration of LEV decreased perforin release of CD8+ T lymphocytes (LEV 50 mg/L vs. CEF only: 21.4% (interquartile range (IQR) 16.5-35.9%) vs. 16.6% (IQR 12-24.9%), p=0.002). LEV had no influence on apoptosis and proliferation (p>0.05). VPA (100 mg/L) slowed apoptosis of CD8() T lymphocytes after 24h (VPA 100mg/L vs. control: 7.3% (IQR 5.4-9.5%) vs. 11.3% (IQR 8.2-15.1%), p<0.001), but had no effects on perforin release (p>0.05). SV2A protein was detected in CD8+ T lymphocytes. CONCLUSION: LEV decreased degranulation of CD8+ T lymphocytes which may contribute to the increased incidence of upper respiratory tract infections in LEV treated patients. Inhibition of SV2A may be responsible for this effect.

Sibling disability risk at onset and during disease progression in familial multiple sclerosis.

BACKGROUND: The objective of this study was to address the differences in onset and disease progression between familial and sporadic multiple sclerosis (MS) and the association within sibling pairs. METHODS: Ninety-eight siblings and their controls were included from a database of 763 sporadic MS-patients, randomly pair-matched for age, gender, clinical course, disease duration and treatment. Sixty-eight available siblings completed a prospective six-year follow-up. Outcome parameters included baseline Expanded Disability Status Scale (EDSS), age at onset, mono- or multifocal onset, disease progression and conversion to secondary progression of initially relapsing-remitting MS. For statistical analyses Wilcoxon's signed-rank statistics for categorical differences, t-statistics for continuous variables, McNemar's test for relative frequencies of categories, intra-class correlations for within sibling-pair associations, or Kaplan-Meier analysis for survival analyses were used; all two-sided at the 5% level. RESULTS: Disease onset was slightly earlier (29.01 vs. 29.44 years, p = 0.0492) and multifocal onset significantly more often (p = 0.0052) in familial than in sporadic MS. Notably, a substantial within sibling-pair correlation for disease progression (rho = 0.40; p = 0.0062) as well as a higher risk for siblings than for controls to convert into secondary progression (0.545 vs. 0.227; p = 0.018) could be observed. CONCLUSIONS: Familial MS differs from sporadic cases with respect to age at onset, multifocal involvement as first clinical event, and conversion into secondary progression. The progression rate of one out of two affected siblings may act as a predictor for the other sib.

Clonal expansions of CD4+ B helper T cells in autoimmune myasthenia gravis.

The weakness in myasthenia gravis (MG) is mediated by T helper cell (Th)-dependent autoantibodies against neuromuscular epitopes. So far, analyzing Th phenotypes or antigen specificities has yielded very few clues to pathogenesis. Here we adopt an alternative antigen-independent approach, analyzing T cell receptor (TCR) Vbeta usage/expansions in blood from 118 MG patients. We found major expansions (>or= five standard deviations above the mean of 118 healthy, individually age- and sex-matched controls) in diverse Vbeta in 21 patients (17.6%, p<0.001) among CD4+ T cells, and in 45 patients (38.1%, p<0.001) among CD8+ T cells. In informative probands, the expanded CD4+ cells consistently showed a Th cell phenotype (CD57+CXCR5+) and expressed Th1 cytokines. Furthermore, their expression of markers for activation, lymphocyte trafficking and B cell-activating ability persisted for >or=3 years. Surprisingly, we noted a selective decline in the expansions/their CD57 positivity while the probands' MG was improving. CDR3 spectratyping suggested mono- or oligoclonal origins, which were confirmed by the prevalent TCR Vbeta CDR3 sequences of Th cells cloned from repeat bleeds. Thus, our data provide evidence for persistent clonally expanded CD4+ B helper T cell populations in the blood of MG patients. These unexpected CD4+ expansions might hold valuable clues to MG immunopathogenesis.

Suppression of autoimmune encephalomyelitis by a neurokinin-1 receptor antagonist--a putative role for substance P in CNS inflammation.

Substance P (SP) is an excitatory neurotransmitter in the central and peripheral nervous system. Most of its physiological functions are mediated through binding to the neurokinin-1 receptor (NK-1R). Recently, proinflammatory properties of SP have been described. In this study we utilized T cell transfer experimental autoimmune encephalomyelitis (EAE) to investigate the role of SP in CNS autoimmune disease. Treatment with the NK-1R antagonist CP-96,345 dramatically reduced clinical and histological signs of EAE if administered before disease onset. The protective effect of CP96,345 treatment was related to a reduced expression of the adhesion molecules ICAM-1 and VCAM-1 on CNS endothelia. The cellular composition or activation status of splenocytes was not affected by CP-96,345 administration, while the secretion of proinflammatory Th1 cytokines was reduced in treated animals. Th2 cytokines remained largely unaffected by NK-1 receptor antagonist treatment. In summary, our findings suggest that the protective effect of CP96,345 treatment is mediated by stabilization of the blood-brain barrier and suppression of Th1 immunity.

Development and prevalidation of a list of structure-activity relationship rules to be used in expert systems for prediction of the skin-sensitising properties of chemicals.

The new European Union (EU) chemicals policy, as described in the White Paper entitled Strategy for a Future Chemicals Policy, has identified a need for computer-based tools suitable for predicting the hazardous properties of chemicals. Two sets of structural alerts (fragments of chemical structure) for the prediction of skin sensitisation hazard classification ("R43, may cause sensitisation by skin contact") have been drawn up, based on sensitising chemicals from a regulatory database (containing data for the EU notification of new chemicals). These alerts comprise 15 rules for chemical structures deemed to be sensitising by direct action of the chemicals with cells or proteins within the skin, and three rules for substructures that act indirectly, i.e. requiring chemical or biochemical transformation. The predictivity rates of the rules were found to be good (positive predictivity, 88%; false-positive rate, 1%; specificity, 99%; negative predictivity, 74%; false-negative rate, 80%; sensitivity, 20%). Because of the confidential nature of the regulatory database, the rules are supported by examples of sensitising chemicals taken from the "Allergenliste" now held by the Federal Institute for Risk Assessment (BfR) and the DEREK for Windows expert system. The rules were prevalidated against data not used for their development. As a result of the prevalidation study, it is proposed that the two sets of structural alerts should be taken forward for formal validation, with a view to incorporating them into regulatory guidelines.

Behaviour of beta 2-adrenoceptors on lymphocytes under continuous and pulsatile tocolysis with Fenoterol.

The present study investigates the population of beta 2-receptors on lymphocytes in pregnant women with premature labor between the 29th and 34th week of pregnancy. The population of receptors on lymphocytes correlates with that on the myometrium, which is not accessible for study during pregnancy. Fourteen patients received a pulsatile tocolysis, while ten women received a continuous tocolysis with Fenoterol. Assuming an equal population of receptors in both groups before commencement of therapy, the numbers of receptors in the patients with continuous tocolysis fell to about 35% of the initial value after 72 hours. Under pulsatile tocolysis, the numbers of receptors remained unchanged for a period of three days and was still only just below 70% of the initial value by the seventh day. Our data demonstrate that continuous administration of the short-acting beta 2-agonist Fenoterol resulted in a substantial loss of beta 2-adrenoceptors on lymphocytes. In contrast, intermittent administration of the same beta 2-adrenergic agonist prevented the onset of receptor down-regulation in pregnant women with preterm labor. Further studies are required to investigate the impact of the decreased loss of beta 2-adrenoceptor density on the good clinical experience with intermittent tocolysis.

Comparison of protein with DNA therapy for chronic myocardial ischemia using fibroblast growth factor-2.

OBJECTIVE: Treatment of coronary disease by growth factors has become an increasingly used strategy for otherwise untreatable patients and is subject to a number of clinical studies. The aim is to stimulate the development of a sufficient collateral circulation and hereby to rescue cardiac function. The objective of our study was to compare the effectiveness of fibroblast growth factor-2 (FGF-2) as protein and as naked plasmid DNA in a porcine model of chronic myocardial ischemia. MATERIALS AND METHODS: A severe stenosis of the left anterior descending artery (LAD) artery was created in healthy pigs. After 1 week, perfusion and regional and global contractility was assessed at baseline at rest and under stress. Afterwards, recombinant FGF-2 (n=6) or naked plasmid DNA encoding FGF-2 (n=7) was intramyocardially injected into the LAD territory. Control animals were left untreated (n=5). After 3 months, the animals were re-examined and underwent immunohistologic analysis. One animal received an Enhanced Green Fluorescent Protein plasmid. RESULTS: Plasmid-dependent protein synthesis was present in cardiomyocytes. FGF-2 protein as well as plasmid injections resulted in an increased number of capillaries and of arterioles compared with untreated ischemia. The improvement of the regional myocardial blood flow by FGF-2 plasmid therapy at rest might however indicate the effectiveness of the DNA application for the induction of a collateral circulation. A benefit from FGF-2 plasmid therapy was revealed with regard to regional contractility. Systemic hemodynamics were partially improved following plasFGF-2 treatment. CONCLUSIONS: In this porcine model of chronic myocardial ischemia, intramyocardial injection of FGF-2 plasmid was more effective than of FGF-2 protein in improving regional perfusion and contractility compared to untreated ischemia.