Altering Brain Amyloidosis by Intra-Lingual and Extra-Nasal Exposure of Aß Aggregates.

Extensive experimental and human-derived evidence suggest that misfolded Aß particles spread similarly to infectious prions. Moreover, peripheral administration of Aß seeds accelerates brain amyloidosis in both susceptible experimental animals and humans. The mechanisms and elements governing the transport of misfolded Aß from the periphery to the brain are not fully understood, although circulation and retrograde axonal transport have been proposed. Here, we demonstrate that injection of Aß seeds in the tongue, a highly innervated organ, substantially accelerates the appearance of plaques in Tg2576 mice. In addition, the extra-nasal exposure of Aß aggregates increased amyloid pathology in the olfactory bulb. Our results show that exposing highly innervated tissues to Aß seeds accelerates AD-like pathological features, and suggest that Aß seeds can be transported from peripheral compartments to the brain by retrograde axonal transport. Research in this direction may be relevant on different fronts, including disease mechanisms, diagnosis, and risk-evaluation of potential iatrogenic transmission of Aß misfolding.

Cross-Talk Between Histone Methyltransferases and Demethylases Regulate REST Transcription During Neurogenesis.

The RE1 Silencing Transcription Factor (REST) is a major regulator of neurogenesis and brain development. Medulloblastoma (MB) is a pediatric brain cancer characterized by a blockade of neuronal specification. REST gene expression is aberrantly elevated in a subset of MBs that are driven by constitutive activation of sonic hedgehog (SHH) signaling in cerebellar granular progenitor cells (CGNPs), the cells of origin of this subgroup of tumors. To understand its transcriptional deregulation in MBs, we first studied control of Rest gene expression during neuronal differentiation of normal mouse CGNPs. Higher Rest expression was observed in proliferating CGNPs compared to differentiating neurons. Interestingly, two Rest isoforms were expressed in CGNPs, of which only one showed a significant reduction in expression during neurogenesis. In proliferating CGNPs, higher MLL4 and KDM7A activities opposed by the repressive polycomb repressive complex 2 (PRC2) and the G9A/G9A-like protein (GLP) complex function allowed Rest homeostasis. During differentiation, reduction in MLL4 enrichment on chromatin, in conjunction with an increase in PRC2/G9A/GLP/KDM7A activities promoted a decline in Rest expression. These findings suggest a lineage-context specific paradoxical role for KDM7A in the regulation of Rest expression in CGNPs. In human SHH-MBs (SHH-α and SHH-ß) where elevated REST gene expression is associated with poor prognosis, up- or downregulation of KDM7A caused a significant worsening in patient survival. Our studies are the first to implicate KDM7A in REST regulation and in MB biology.

Transmission of cerebral amyloid pathology by peripheral administration of misfolded Aß aggregates.

Previous reports showed that brain Aß amyloidosis can be induced in animal models by exogenous administration of pre-formed aggregates. To date, only intra-peritoneal and intra-venous administrations are described as effective means to peripherally accelerate brain Aß amyloidosis by seeding. Here, we show that cerebral accumulation of Aß can be accelerated after exposing mouse models of Alzheimer's disease (AD) to Aß seeds by different peripheral routes of administration, including intra-peritoneal and intra-muscular. Interestingly, animals receiving drops of brain homogenate laden with Aß seeds in the eyes were efficiently induced. On the contrary, oral administration of large quantities of brain extracts from aged transgenic mice and AD patients did not have any effect in brain pathology. Importantly, pathological induction by peripheral administration of Aß seeds generated a large proportion of aggregates in blood vessels, suggesting vascular transport. This information highlights the role of peripheral tissues and body fluids in AD-related pathological changes.

Infusion of blood from mice displaying cerebral amyloidosis accelerates amyloid pathology in animal models of Alzheimer's disease.

Previous studies showed that injection of tissue extracts containing amyloid-ß (Aß) aggregates accelerate amyloid deposition in the brain of mouse models of Alzheimer's disease (AD) through prion-like mechanisms. In this study, we evaluated whether brain amyloidosis could be accelerated by blood infusions, procedures that have been shown to transmit prion diseases in animals and humans. Young transgenic mice infused with whole blood or plasma from old animals with extensive Aß deposition in their brains developed significantly higher levels brain amyloidosis and neuroinflammation compared to untreated animals or mice infused with wild type blood. Similarly, intra-venous injection of purified Aß aggregates accelerated amyloid pathology, supporting the concept that Aß seeds present in blood can reach the brain to promote neuropathological alterations in the brain of treated animals. However, an amyloid-enhancing effect of other factors present in the blood of donors cannot be discarded. Our results may help to understand the role of peripheral (amyloid-dependent or -independent) factors implicated in the development of AD and uncover new strategies for disease intervention.

Sex differences in T cell immune responses, gut permeability and outcome after ischemic stroke in aged mice.

INTRODUCTION: Stroke is a disease that presents with well-known sex differences. While women account for more stroke deaths, recent data show that after adjusting for age and pre-stroke functional status, mortality is higher in men. Immune responses are key determinants of stroke outcome and may differ by sex. This study examined sex differences in central and peripheral T cell immune responses, systemic effects on gut permeability and microbiota diversity and behavioral outcomes after stroke in aged mice. We hypothesized that there are sex differences in the immune response to stroke in aged animals. METHODS: C57BL/6CR mice (20-22 months) were subjected to 60 min middle cerebral artery occlusion, or sham surgery. T cells were quantified in brain and blood at 3, 7 and 15 days (d) post-stroke by flow cytometry. Peripheral effects on gut permeability and microbiota diversity, as well as neurological function were assessed up to 14 d, and at 21 d (cognitive function) post-stroke. Brain glial fibrillary acidic protein (GFAP) expression was evaluated at 42 d post-stroke. RESULTS AND DISCUSSION: Mortality (50% vs 14%, p < 0.05) and hemorrhagic transformation (44% vs 0%) were significantly higher in males than in females. No difference in infarct size at 3d were observed. Peripherally, stroke induced greater gut permeability of FITC-dextran in males at d3 (p < 0.05), and non-reversible alterations in microbiota diversity in males. Following the sub-acute phase, both sexes demonstrated a time-dependent increase of CD4+ and CD8+ T cells in the brain, with significantly higher levels of CD8+ T cells and Regulatory T cells in males at d15 (p < 0.01). Aged males demonstrated greater neurological deficits up to d5 and impaired sensorimotor function up to d15 when assessed by the corner asymmetry test (p < 0.001 and p < 0.01, respectively). A trend in greater cognitive decline was observed at d21 in males. Increased GFAP expression in the ischemic hemisphere, indicating astroglial activation and gliosis, was demonstrated in both males and females 42d post-stroke. Our findings indicate that despite a similar initial ischemic brain injury, aged male mice experience greater peripheral effects on the gut and ongoing central neuroinflammation past the sub-acute phase after stroke.

Correction to: Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

There was a typo in author Andrew Wahba's name in the initial online publication. The original article has been corrected.

Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

PURPOSE: Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown. METHODS: To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging. NK cells were labeled with a fluorine-19 (19F) MRI probe and subsequently injected either intratumorally or contralaterally to the tumor in the cerebellum and effect on tumor growth was monitored. RESULTS: The 19F probe efficiently labeled the NK cells and exhibited little cytotoxicity. Fluorine-19 MRI confirmed the successful and accurate delivery of the labeled NK cells to the cerebellum of the mice. Administration of 19F-labeled NK cells suppressed MB growth, with the same efficacy as unlabeled cells. Immunohistochemistry confirmed the presence of NK cells within the tumor, which was associated with induction of apoptosis in tumor cells. NK cell migration to the tumor from a distal location as well as activation of apoptosis was also demonstrated by immunohstochemistry. CONCLUSIONS: Our results show that NK cells present a novel opportunity for new strategies in MB treatment. Further, 19F-labeled NK cells can suppress MB growth while enabling 19F MRI to provide imaging feedback that can facilitate study and optimization of therapeutic paradigms.

Transcriptional repressor REST drives lineage stage-specific chromatin compaction at Ptch1 and increases AKT activation in a mouse model of medulloblastoma.

In medulloblastomas (MBs), the expression and activity of RE1-silencing transcription factor (REST) is increased in tumors driven by the sonic hedgehog (SHH) pathway, specifically the SHH-α (children 3 to 16 years) and SHH-ß (infants) subgroups. Neuronal maturation is greater in SHH-ß than SHH-α tumors, but both correlate with poor overall patient survival. We studied the contribution of REST to MB using a transgenic mouse model (RESTTG ) wherein conditional NeuroD2-controlled REST transgene expression in lineage-committed Ptch1 +/- cerebellar granule neuron progenitors (CGNPs) accelerated tumorigenesis and increased penetrance and infiltrative disease. This model revealed a neuronal maturation context-specific antagonistic interplay between the transcriptional repressor REST and the activator GLI1 at Ptch1 Expression of Arrb1, which encodes ß-arrestin1 (a GLI1 inhibitor), was substantially reduced in proliferating and, to a lesser extent, lineage-committed RESTTG cells compared with wild-type proliferating CGNPs. Lineage-committed RESTTG cells also had decreased GLI1 activity and increased histone H3K9 methylation at the Ptch1 locus, which correlated with premature silencing of Ptch1 These cells also had decreased expression of Pten, which encodes a negative regulator of the kinase AKT. Expression of PTCH1 and GLI1 were less, and ARRB1 was somewhat greater, in patient SHH-ß than SHH-α MBs, whereas that of PTEN was similarly lower in both subtypes than in others. Inhibition of histone modifiers or AKT reduced proliferation and induced apoptosis, respectively, in cultured REST-high MB cells. Our findings linking REST to differentiation-specific chromatin remodeling, PTCH1 silencing, and AKT activation in MB tissues reveal potential subgroup-specific therapeutic targets for MB patients.

Exploring the relationship between Endothelin-1 and peripheral inflammation in multiple sclerosis.

BACKGROUND: Identifying pathways linking neuroinflammation and neurodegeneration is essential to help prevent disability progression in people with multiple sclerosis (MS). Endothelin-1 (ET-1) is a potent vasoconstrictor thought to contribute to cerebral hypoperfusion and tissue damage in MS. Its link with the neuroinflammatory process remains poorly investigated. OBJECTIVES: To determine plasma ET-1 levels in treatment-naïve people with MS and controls, and the relationship between ET-1 and other peripheral immune mediator levels as potential markers of the disease process. METHODS: This is a retrospective study that included specimens previously collected from 35 treatment-naïve patients with clinically isolated syndrome highly suggestive of MS or definite MS and 35 sex- and age-matched controls. ET-1 plasma levels were measured by enzyme-linked immunosorbent assay (ELISA), and plasma cytokine levels [interleukin (IL)-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, interferon (IFN)-γ and tumor necrosis factor (TNF)-α] were simultaneously measured by Multiplex assay. RESULTS: ET-1 levels were significantly increased in MS patients compared to controls. No significant difference in cytokine levels between the groups were found. However, a significant increase in IFN-γ/IL-4 ratio was observed in patients with MS in comparison with controls, suggestive of Th1 skewed response. Binary logistic regression was performed to ascertain the effects of age, sex, ET-1 and cytokine levels on the likelihood of MS diagnosis. In the final model, ET-1, IL-4 and IFN-γ levels remained as predictors of MS. There was no significant correlation between ET-1 and cytokine levels. CONCLUSIONS: Patients with MS presented increased levels of ET-1 and an immune response biased towards a Th1 profile. Although both ET-1 and Th1 cytokine profile were predictors of MS diagnosis, ET-1 levels were not associated with peripheral immune markers, suggesting that these changes may occur independently.

Pharmacological inhibition of LSD1 activity blocks REST-dependent medulloblastoma cell migration.

BACKGROUND: Medulloblastoma (MB) is the most common malignant brain tumor in children. Current problems in the clinic include metastasis, recurrence, and treatment-related sequelae that highlight the need for targeted therapies. Epigenetic perturbations are an established hallmark of human MB and expression of Lysine Specific Demethylase 1 (LSD1) is elevated in MBs compared to normal tissue, suggesting that LSD1 inhibitors may have efficacy against human MB tumors. METHODS: Expression of LSD1 was examined across a publicly-available database and correlated with patient outcomes. Sonic Hedgehog (SHH) MB samples were clustered based on expression of LSD1 and LSD1-associated RE-1 silencing transcription factor (REST) target genes as well as genes involved in metastasis. Resulting clusters were examined for patient outcomes associated with LSD1 and REST expression. Human SHH MB cell lines were transduced with a REST-transgene to create isogenic cell pairs. In vitro viability and cell migration assays were used to examine the effect of LSD1 knockdown or inhibition on these parameters. RESULTS: We demonstrate that subsets of SHH MB tumors have elevated LSD1 expression coincident with increased expression of its deubiquitylase, USP7, and REST. Patients with co-elevation of USP7, REST, and LSD1 have poorer outcomes compared to those with lower expression of these genes. In SHH MB cell lines, REST elevation increased cell growth and LSD1 protein levels. Surprisingly, while genetic loss of LSD1 reduced cell viability, pharmacological targeting of its activity using LSD1 inhibitors did not affect cell viability. However, a reduction in REST-dependent cell migration was seen in wound healing, suggesting that REST-LSD1 interaction regulates cell migration. Ingenuity pathway analyses validated these findings and identified Hypoxia Inducible Factor 1 alpha (HIF1A) as a potential target. In line with this, ectopic expression of HIF1A rescued the loss of migration seen following LSD1 inhibition. CONCLUSIONS: A subset of SHH patients display increased levels of LSD1 and REST, which is associated with poor outcomes. REST elevation in MB in conjunction with elevated LSD1 promotes MB cell migration. LSD1 inhibition blocks REST-dependent cell migration of MB cells in a HIF1A-dependent manner.

Modeling amyloid beta and tau pathology in human cerebral organoids.

The typical abnormalities observed in the brain of Alzheimer's disease (AD) patients include synaptic alterations, neuronal death, brain inflammation, and the accumulation of protein aggregates in the form of amyloid plaques and neurofibrillary tangles. Despite the development of many animal and in vitro models for AD, there is a lack of an experimental approach that fully recapitulates essential aspects of the disease in human cells. Here, we report the generation of a new model to study AD, consisting of cerebral organoids (COs) produced from human-induced pluripotent stem cells (iPSCs). Under our experimental conditions, COs grow to form three-dimensional (3D) structures containing neural areas with cortical-like organization. Analysis of COs by histological and biochemical methods revealed that organoids produced from iPSCs derived from patients affected by familial AD or Down syndrome (DS) spontaneously develop over time pathological features of AD, including accumulation of structures highly reminiscent to amyloid plaques and neurofibrillary tangles. These pathological abnormalities were not observed in COs generated from various controls, including human iPSCs from healthy individuals, human iPSCs from patients affected by Creutzfeldt-Jakob disease, mouse embryonic stem cells (ESCs), or mouse iPSCs. These findings enable modeling genetic AD in a human cellular context in a 3D cortical-like tissue developed in vitro from patient-specific stem cells. This system provides a more relevant disease model compared to pre-existing methods and offers a new platform for discovery of novel targets and screening of drugs for therapeutic intervention.

Sex Differences in Adipose Tissue CD8+ T Cells and Regulatory T Cells in Middle-Aged Mice.

The prevalence of cardiovascular disease has increased among middle-aged women in the United States, yet has declined in middle-aged men. In experimental stroke, middle-aged females have larger strokes and greater inflammation than age-matched males or younger females. The mechanism underlying this shift from an "ischemia-protected" to an "ischemia-sensitive" phenotype in aging females is unknown. One potential factor is an age-related increase in systemic factors that induce inflammation. Increased abdominal fat deposition is seen in women during middle age. Adipose tissue plays a key role in obesity-induced systemic inflammation, including increased pro-inflammatory cytokines. We hypothesized that age and sex differences in adipose immune cells promote an augmented pro-inflammatory milieu in middle-aged females driven by a balance shift between pro-inflammatory and anti-inflammatory T cells. Abdominal adipose tissue immune cells from young (3-4 months) and middle-aged (15-16 months) male and female C57BL/6J mice were analyzed by flow cytometry. Plasma triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were determined with colorimetric assays. Middle-aged mice had higher adipose tissue mass compared to young mice. Lipid profiling showed no sex differences in TG and LDL, but middle-aged females had lower HDL (0.84 ± 0.07 µg/µl) than middle-aged males (1.35 ± 0.06 µg/µl). Flow cytometry data demonstrated an age-associated increase in adipose tissue CD8+ T cells that was augmented by female sex, with middle-aged females having a higher percentage of CD8+ cells (34.4 ± 3.2% of CD3+ T cells) than middle-aged males (24.4 ± 2.2%). This increase in CD8+ T-cell proportion was adipose tissue-specific, as this change was not observed in blood. Middle-aged females had higher numbers of activated (CD69+) CD8+ T cells than males. In addition, female CD8+ T cells produced higher levels of IFN-γ, TNF-α, and granzyme B ex vivo, and females had higher adipose levels of IFN-γ, RANTES and MIP-1ß than middle-aged males. In parallel, females had lower levels of regulatory T cells (Tregs), an anti-inflammatory T-cell subtype, compared to age-matched males. In conclusion, middle-aged females have a detrimental combination of elevated pro-inflammatory T cells and decreased anti-inflammatory Tregs in adipose tissue, which may promote a pro-inflammatory milieu and contribute to increased cardiovascular disease burden in aging females.

Endothelial Nitric Oxide Synthase Is Present in Dendritic Spines of Neurons in Primary Cultures.

Nitric oxide exerts important regulatory functions in various brain processes. Its synthesis in neurons has been most commonly ascribed to the neuronal nitric oxide synthase (nNOS) isoform. However, the endothelial isoform (eNOS), which is significantly associated with caveolae in different cell types, has been implicated in synaptic plasticity and is enriched in the dendrites of CA1 hippocampal neurons. Using high resolution microscopy and co-distribution analysis of eNOS with synaptic and raft proteins, we now show for the first time in primary cortical and hippocampal neuronal cultures, virtually devoid of endothelial cells, that eNOS is present in neurons and is localized in dendritic spines. Moreover, eNOS is present in a postsynaptic density-enriched biochemical fraction isolated from these neuronal cultures. In addition, qPCR analysis reveals that both the nNOS as well as the eNOS transcripts are present in neuronal cultures. Moreover, eNOS inhibition in cortical cells has a negative impact on cell survival after excitotoxic stimulation with N-methyl-D-aspartate (NMDA). Consistent with previous results that indicated nitric oxide production in response to the neurotrophin BDNF, we could detect eNOS in immunoprecipitates of the BDNF receptor TrkB while nNOS could not be detected. Taken together, our results show that eNOS is located at excitatory synapses where it could represent a source for NO production and thus, the contribution of eNOS-derived nitric oxide to the regulation of neuronal survival and function deserves further investigations.

Sex differences in stroke across the lifespan: The role of T lymphocytes.

Stroke is a sexually dimorphic disease. Ischemic sensitivity changes throughout the lifespan and outcomes depend largely on variables like age, sex, hormonal status, inflammation, and other existing risk factors. Immune responses after stroke play a central role in how these factors interact. Although the post-stroke immune response has been extensively studied, the contribution of lymphocytes to stroke is still not well understood. T cells participate in both innate and adaptive immune responses at both acute and chronic stages of stroke. T cell responses also change at different ages and are modulated by hormones and sex chromosome complement. T cells have also been implicated in the development of hypertension, one of the most important risk factors for vascular disease. In this review, we highlight recent literature on the lymphocytic responses to stroke in the context of age and sex, with a focus on T cell response and the interaction with important stroke risk factors.

Prions efficiently cross the intestinal barrier after oral administration: Study of the bioavailability, and cellular and tissue distribution in vivo.

Natural forms of prion diseases frequently originate by oral (p.o.) infection. However, quantitative information on the gastro-intestinal (GI) absorption of prions (i.e. the bioavailability and subsequent biodistribution) is mostly unknown. The main goal of this study was to evaluate the fate of prions after oral administration, using highly purified radiolabeled PrP(Sc). The results showed a bi-phasic reduction of PrP(Sc) with time in the GI, except for the ileum and colon which showed sustained increases peaking at 3-6 hr, respectively. Plasma and whole blood (125)I-PrP(Sc) reached maximal levels by 30 min and 3 hr, respectively, and blood levels were constantly higher than plasma. Upon crossing the GI-tract (125)I-PrP(Sc) became associated to blood cells, suggesting that binding to cells decreased the biological clearance of the agent. Size-exclusion chromatography revealed that oligomeric (125)I-PrP(Sc) were transported from the intestinal tract, and protein misfolding cyclic amplification showed that PrP(Sc) in organs and blood retained the typical prion self-replicating ability. Pharmacokinetic analysis found the oral bioavailability of (125)I-PrP(Sc) to be 33.6%. Interestingly, (125)I-PrP(Sc) reached the brain in a quantity equivalent to the minimum amount needed to initiate prion disease. Our findings provide a comprehensive and quantitative study of the fate of prions upon oral infection.

Strain-dependent profile of misfolded prion protein aggregates.

Prions are composed of the misfolded prion protein (PrP(Sc)) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrP(Sc) aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrP(Sc) aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrP(Sc) aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrP(Sc) aggregates and the incubation periods for the strains studied. The relative presence of PrP(Sc) in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrP(Sc) aggregates in prion-induced neurodegeneration.

Intravenous administration of bone marrow-derived mesenchymal stem cells induces a switch from classical to atypical symptoms in experimental autoimmune encephalomyelitis.

Potent immunosuppressive and regenerative properties of mesenchymal stem cells (MSCs) position them as a novel therapy for autoimmune diseases. This research examines the therapeutic effect of MSCs administration at different disease stages in experimental autoimmune encephalomyelitis (EAE). Classical and atypical scores of EAE, associated with Th1 and Th17 response, respectively, and also Treg lymphocytes, were evaluated. MSCs administration at the onset (EAE+MSConset) induced an important amelioration of the clinical signs and less lasting effect at the peak of EAE (EAE+MSCpeak). No effect was observed when MSCs were applied after EAE stabilization (EAE+MSClate). Surprisingly, EAE atypical signs were detected in EAE+MSCpeak and EAE+MSClate mice. However, no correlation was found in Th17/Th1 ratio. Interestingly, regardless of time administration, MSCs significantly reduced IL-6 and also T-bet, RORγT, and Foxp3 mRNA levels in brain samples of EAE mice. The downregulation of IL-6 could restore the well-functioning of the blood-brain barrier of EAE mice, correlated with a decreased number of brain infiltrating leukocytes. These results suggest that the inflammatory status is important to be considered for administering MSCs in autoimmune pathologies, leading to a further research to clarify the effect of MSCs for multiple sclerosis.

Titration of biologically active amyloid-ß seeds in a transgenic mouse model of Alzheimer's disease.

Experimental evidence in animal models suggests that misfolded Amyloid-ß (Aß) spreads in disease following a prion-like mechanism. Several properties characteristics of infectious prions have been shown for the induction of Aß aggregates. However, a detailed titration of Aß misfolding transmissibility and estimation of the minimum concentration of biologically active Aß seeds able to accelerate pathological changes has not yet been performed. In this study, brain extracts from old tg2576 animals were serially diluted and intra-cerebrally injected into young subjects from the same transgenic line. Animals were sacrificed several months after treatment and brain slices were analyzed for amyloid pathology. We observed that administration of misfolded Aß was able to significantly accelerate amyloid deposition in young mice, even when the original sample was diluted a million times. The titration curve obtained in this experiment was compared to the natural Aß load spontaneously accumulated by these mice overtime. Our findings suggest that administration of the largest dose of Aß seeds led to an acceleration of pathology equivalent to over a year. These results show that active Aß seeds present in the brain can seed amyloidosis in a titratable manner, similarly as observed for infectious prions.

Mesenchymal stem cells generate a CD4+CD25+Foxp3+ regulatory T cell population during the differentiation process of Th1 and Th17 cells.

INTRODUCTION: Mesenchymal stem cells (MSCs) are adult, multipotent, stem cells with immunomodulatory properties. The mechanisms involved in the capacity of MSCs to inhibit the proliferation of proinflammatory T lymphocytes, which appear responsible for causing autoimmune disease, have yet to be fully elucidated. One of the underlying mechanisms studied recently is the ability of MSCs to generate T regulatory (Treg) cells in vitro and in vivo from activated peripheral blood mononuclear cells (PBMC), T-CD4+ and also T-CD8(+) cells. In the present work we investigated the capacity of MSCs to generate Treg cells using T-CD4(+) cells induced to differentiate toward the proinflammatory Th1 and Th17 lineages. METHODS: MSCs were obtained from mouse bone marrow and characterized according to their surface antigen expression and their multilineage differentiation potential. CD4(+) T cells isolated from mouse spleens were induced to differentiate into Th1 or Th17 cells and co-cultured with MSCs added at day 0, 2 or 4 of the differentiation processes. After six days, CD25, Foxp3, IL-17 and IFN-γ expression was assessed by flow cytometry and helios and neuropilin 1 mRNA levels were assessed by RT-qPCR. For the functional assays, the 'conditioned' subpopulation generated in the presence of MSCs was cultured with concanavalin A-activated CD4(+) T cells labeled with carboxyfluorescein succinimidyl ester. Finally, we used the encephalomyelitis autoimmune diseases (EAE) mouse model, in which mice were injected with MSCs at day 18 and 30 after immunization. At day 50, the mice were euthanized and draining lymph nodes were extracted for Th1, Th17 and Treg detection by flow cytometry. RESULTS: MSCs were able to suppress the proliferation, activation and differentiation of CD4(+) T cells induced to differentiate into Th1 and Th17 cells. This substantial suppressive effect was associated with an increase of the percentage of functional induced CD4(+)CD25(+)Foxp3(+) regulatory T cells and IL-10 secretion. However, using mature Th1 or Th17 cells our results demonstrated that while MSCs suppress the proliferation and phenotype of mature Th1 and Th17 cells they did not generate Treg cells. Finally, we showed that the beneficial effect observed following MSC injection in an EAE mouse model was associated with the suppression of Th17 cells and an increase in the percentage of CD4(+)CD25(+)Foxp3(+) T lymphocytes when administrated at early stages of the disease. CONCLUSIONS: This study demonstrated that MSCs contribute to the generation of an immunosuppressive environment via the inhibition of proinflammatory T cells and the induction of T cells with a regulatory phenotype. Together, these results might have important clinical implications for inflammatory and autoimmune diseases.