Objective: Low vitamin D status has been associated with multiple sclerosis (MS) prevalence and risk, but the therapeutic potential of vitamin D in established MS has not been explored. Our aim was to assess the tolerability of high-dose oral vitamin D and its impact on biochemical, immunologic, and clinical outcomes in patients with MS prospectively.Methods: An open-label randomized prospective controlled 52-week trial matched patients with MS for demographic and disease characteristics, with randomization to treatment or control groups. Treatment patients received escalating vitamin D doses up to 40,000 IU/day over 28 weeks to raise serum 25-hydroxyvitamin D [25(OH)D] rapidly and assess tolerability, followed by 10,000 IU/day (12 weeks), and further downtitrated to 0 IU/day. Calcium (1,200 mg/day) was given throughout the trial. Primary endpoints were mean change in serum calcium at each vitamin D dose and a comparison of serum calcium between groups. Secondary endpoints included 25(OH)D and other biochemical measures, immunologic biomarkers, relapse events, and Expanded Disability Status Scale (EDSS) score. Results: Classification of evidence:This trial provides Class II evidence that high-dose vitamin D use for 52 weeks in patients with multiple sclerosis does not significantly increase serum calcium levels when compared to patients not on high-dose supplementation. The trial, however, lacked statistical precision and the design requirements to adequately assess changes in clinical disease measures (relapses and Expanded Disability Status Scale scores), providing only Class level IV evidence for these outcomes. Neurology ® 2010;74:1852-1859 GLOSSARY ALP ϭ alkaline phosphatase; ALT ϭ alanine aminotransferase; AST ϭ aspartate aminotransferase; EAE ϭ experimental autoimmune encephalitis; EDSS ϭ Expanded Disability Status Scale; IL ϭ interleukin; LS ϭ least squares; MMP-9 ϭ matrix metalloproteinase-9; MS ϭ multiple sclerosis; PTH ϭ parathyroid hormone; TCS ϭ T-cell score; TIMP-1 ϭ tissue inhibitory of metalloproteinase-1; TNF␣ ϭ tumor necrosis factor-␣.Multiple sclerosis (MS) has a well-documented geographic distribution, with increasing prevalence and risk with increasing distance from the equator.1-4 Limited sunlight and UVB exposure, MS risk factors based on observational studies, are intermediaries between latitude and MS.2-5 Low serum 25(OH)D also appears to be a risk factor, and is a direct product of skin exposure to UVB. [4][5][6][7] e-Pub ahead of print on April 28, 2010, at www.neurology.org.

PTEN-induced putative kinase 1 (Pink1) is a recently identified gene linked to a recessive form of familial Parkinson's disease (PD). The kinase contains a mitochondrial localization sequence and is reported to reside, at least in part, in mitochondria. However, neither the manner by which the loss of Pink1 contributes to dopamine neuron loss nor its impact on mitochondrial function and relevance to death is clear. Here, we report that depletion of Pink1 by RNAi increased neuronal toxicity induced by MPP ؉ . Moreover, wild-type Pink1, but not the G309D mutant linked to familial PD or an engineered kinase-dead mutant K219M, protects neurons against MPTP both in vitro and in vivo. Intriguingly, a mutant that contains a deletion of the putative mitochondrial-targeting motif was targeted to the cytoplasm but still provided protection against 1-methyl-4-phenylpyridine (MPP ؉ )/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity. In addition, we also show that endogenous Pink1 is localized to cytosolic as well as mitochondrial fractions. Thus, our findings indicate that Pink1 plays a functional role in the survival of neurons and that cytoplasmic targets, in addition to its other actions in the mitochondria, may be important for this protective effect.Parkinson's disease ͉ neurodegeneration ͉ neuroprotection P arkinson's disease (PD) is a movement disorder with progressive loss of dopamine neurons in the substantia nigra pars compacta (SNc). The molecular events responsible for the loss of dopaminergic neurons in PD remain poorly understood. One common feature of PD is the dysfunction of mitochondria, which results in reduced complex I activity in the SNc (1, 2). Experimentally, inhibitors of complex I of the mitochondrial respiratory chain can recapitulate this selective dopaminergic neuronal loss and consequent behavioral deficits (1, 3-5). These observations support the hypothesis that nigral dopamine neurons are highly vulnerable to stress arising from mitochondrial dysfunction.Recently, several genes have been identified that cause PD (6). These genes include ␣-synuclein, parkin, PTEN-induced putative kinase 1 (Pink1), DJ-1, and LRRK2. Although several of the genes have been partially localized to the mitochondria, Pink1 is the only gene with a putative mitochondrial targeting motif. Several studies have shown that the mitochondrial targeting motif at the Nterminal region of Pink1 is sufficient to direct proteins to the mitochondria (7). Pink1 was initially identified as a PTEN-inducible transcript and contains a serine/threonine kinase domain (8). Interestingly, the G309D mutation of the kinase domain leads to a mild decrease in mitochondrial complex I activity, elevation of superoxide radicals, and increased lipid peroxidation (9). Studies with Drosophila lacking Pink1 showed mitochondrial pathology with the similar phenotype as seen in Parkin knockout flies (10, 11). The above observations suggest that mitochondrial dysfunction may be linked to the Pink1 PD phenotype.The mechanisms by which Pi...

To explore the potential of expanded healthy donor-derived allogeneic CD4 and CD8 double-negative cells (DNT) as a novel cellular immunotherapy for leukemia patients. Clinical-grade DNTs from peripheral blood of healthy donors were expanded and their antileukemic activity and safety were examined using flow cytometry-based killing assays and xenograft models against AML patient blasts and healthy donor-derived hematopoietic cells. Mechanism of action was investigated using antibody-mediated blocking assays and recombinant protein treatment assays. Expanded DNTs from healthy donors target a majority (36/46) of primary AML cells, including 9 chemotherapy-resistant patient samples , and significantly reduce the leukemia load in patient-derived xenograft models in a DNT donor-unrestricted manner. Importantly, allogeneic DNTs do not attack normal hematopoietic cells or affect hematopoietic stem/progenitor cell engraftment and differentiation, or cause xenogeneic GVHD in recipients. Mechanistically, DNTs express high levels of NKG2D and DNAM-1 that bind to cognate ligands preferentially expressed on AML cells. Upon recognition of AML cells, DNTs rapidly release IFNγ, which further increases NKG2D and DNAM-1 ligands' expression on AML cells. IFNγ pretreatment enhances the susceptibility of AML cells to DNT-mediated cytotoxicity, including primary AML samples that are otherwise resistant to DNTs, and the effect of IFNγ treatment is abrogated by NKG2D and DNAM-1-blocking antibodies. This study supports healthy donor-derived allogeneic DNTs as a therapy to treat patients with chemotherapy-resistant AML and also reveals interrelated roles of NKG2D, DNAM-1, and IFNγ in selective targeting of AML by DNTs. .

Purpose: To expand clinical-grade healthy donor-derived double-negative T cells (DNT) to a therapeutically relevant number and characterize their potential to be used as an "offthe-shelf" adoptive cellular therapy (ACT) against cancers.Experimental Design: We developed methods to expand DNTs under GMP conditions and characterized their surface molecule expression pattern using flow cytometry-based high-throughput screening. We investigated the off-the-shelf potential of clinical-grade DNTs by assessing their cytotoxicity against various cancer types and their off-tumor toxicity in vitro and in xenograft models and determining the effect of cryopreservation under GMP conditions on cell viability and cytotoxicity. Further, we determined the susceptibility of DNTs to conventional allogeneic T cells in vitro and in vivo.Results: Clinical-grade DNTs expanded 1,558 AE 795.5-fold in 17 days with >90% purity. Expanded DNTs showed potent in vitro cytotoxic activity against various cancer types in a donor-unrestricted manner. DNTs enhanced the survival of mice infused with a lethal dose of EBV-LCL and significantly reduced leukemia engraftment in xenograft models. Expanded DNTs cryopreserved using GMP-compliant reagents maintained viability and anticancer functions for at least 600 days. Live allogeneic DNTs did not induce cytotoxicity of alloreactive CD8 þ T cells in vitro, and coinfusion of DNTs with peripheral blood mononuclear cells (PBMC) from a different donor into mice resulted in coengraftment of DNTs and PBMC-derived allogeneic conventional T cells in the absence of cytotoxicity toward DNTs, suggesting the lack of hostversus-graft reaction.Conclusions: We have established a method to generate therapeutic numbers of clinical-grade DNTs that fulfill the requirements of an off-the-shelf ACT.

Intracellular accumulation of insoluble ␣-synuclein in Lewy bodies is a key neuropathological trait of Parkinson disease (PD).Neither the normal function of ␣-synuclein nor the biochemical mechanisms that cause its deposition are understood, although both are likely influenced by the interaction of ␣-synuclein with vesicular membranes, either for a physiological role in vesicular trafficking or as a pathological seeding mechanism that exacerbates the propensity of ␣-synuclein to self-assemble into fibrils. In addition to the ␣-helical form that is peripherally-attached to vesicles, a substantial portion of ␣-synuclein is freely diffusible in the cytoplasm. The mechanisms controlling ␣-synuclein exchange between these compartments are unknown and the possibility that chronic dysregulation of membrane-bound and soluble ␣-synuclein pools may contribute to Lewy body pathology led us to search for cellular factors that can regulate ␣-synuclein membrane interactions. Here we reveal that dissociation of membrane-bound ␣-synuclein is dependent on brain-specific cytosolic proteins and insensitive to calcium or metabolic energy. Two PD-linked mutations (A30P and A53T) significantly increase the cytosoldependent ␣-synuclein off-rate but have no effect on cytosolindependent dissociation. These results reveal a novel mechanism by which cytosolic brain proteins modulate ␣-synuclein interactions with intracellular membranes. Importantly, our finding that ␣-synuclein dissociation is up-regulated by both familial PD mutations implicates cytosolic cofactors in disease pathogenesis and as molecular targets to influence ␣-synuclein aggregation.

MS-associated, abnormal T cell reactivities were suppressed in vivo by cholecalciferol at serum 25(OH)D concentrations higher than 100 nmol/liter.

Trogocytosis is a process which involves the transfer of membrane fragments and cell surface proteins between cells. Various types of T cells have been shown to be able to acquire membrane-bound proteins from antigen-presenting cells and their functions can be modulated following trogocytosis. However, it is not known whether induced regulatory T cells (iTregs) can undergo trogocytosis, and if so, what the functional consequences of this process might entail. In this study, we show that iTregs can be generated from CD80 2/2 CD86 2/2 double knockout (DKO) mice. Using flow cytometry and confocal fluorescence microscopy, we demonstrate that iTregs generated from DKO mice are able to acquire both CD80 and CD86 from mature dendritic cells (mDCs) and that the acquisition of CD86 occurs to a higher extent than that of CD80. Furthermore, we found that after co-incubation with iTregs, dendritic cells (DCs) downregulate their surface expression of CD80 and CD86. The trogocytosis of both CD80 and CD86 occurs in a cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), CD28 and programmed death ligand-1 (PDL1)-independent manner. Importantly, we showed that iTregs that acquired CD86 from mDCs expressed higher activation markers and their ability to suppress naive CD4 1 T-cell proliferation was enhanced, compared to iTregs that did not acquire CD86. These data demonstrate, for the first time, that iTregs can acquire CD80 and CD86 from mDCs, and the acquisition of CD86 may enhance their suppressive function. These findings provide novel understanding of the interaction between iTregs and DCs, suggesting that trogocytosis may play a significant role in iTreg-mediated immune suppression.

Regulatory T (Treg) cells suppress immune responses by downregulating the expression of costimulatory molecules CD80 and CD86 on dendritic cells (DCs) through cytotoxic T lymphocyte antigen 4 (CTLA4). However, it is unclear whether inducible Treg (iTreg) cellscan hamper immune responses via the same mechanism. Moreover, whether a reverse signal sent by CTLA4 alone is sufficient to prevent maturation of DCs has never been evaluated. Here, we demonstrate that stimulation of DCs with CTLA4, either expressed by inducible Treg cells or by cross-linking with CTLA4Fc fusion protein, can significantly inhibit LPS-induced CD80 and CD86 mRNA and protein expression in both mouse and human DCs. Importantly, CTLA4Fc-treated DCs had reduced ability to stimulate CD4 + and CD8 + T-cell proliferation and cytokine production in both syngeneic and allogeneic settings. We also investigated the molecular mechanism involved in the induction of tolerogenic DCs by CTLA4. We determined that the interaction of CTLA4 with its high affinity ligand CD80 on DCs induces STAT3 phosphorylation followed by reduction of NF-κB activity, leading to suppression of CD80 and CD86 gene transcription and protein production. Our work opens new windows for the generation of tolerogenic DCs that could ultimately be used for treating autoimmune diseases and transplant rejection. Keywords: Dendritic cells r Immune regulation r Regulatory T cells r ToleranceAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionDendritic cells (DCs) play a central role in the initiation and regulation of immune responses [1]. Although traditionally viewed as immunity inducers, in the absence of inflammatory danger signals, DCs participate in the maintenance of peripheral tolerance [2] and can induce T-cell deletion, anergy and generation and expansion of regulatory T (Treg) cells [3]. These tolerogenic DCs show decreased expression of costimulatory molecules such as CD80Correspondence: Dr. Li Zhang e-mail: lzhang@uhnres.utoronto.ca and CD86 and reduced production of proinflammatory cytokines [4]. Several in vitro methods have been developed in order to generate tolerogenic DCs [5]. Additionally, a growing body of experimental data highlights the role of Treg cells in maintaining tolerance through the suppression of DC immunostimulatory capacity [6][7][8][9]. However, the molecular mechanisms by which Treg cells modulate DC function remain obscure. Elucidation of the mechanisms governing DC maturation may facilitate their use in treating a variety of immune-related conditions including autoimmune diseases and transplantation.CD4 + CD25 + Foxp3 + Treg cells play a pivotal role in maintaining self-tolerance and preventing autoimmunity. At least two major subtypes of CD4 + CD25 + Foxp3 + Treg cells have been identified: Thymus-derived natural T regulatory (nTreg) cells and C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu1144 Aleksandra Kowalczyk et al. Eur. J. Immunol. 2014. 44: 1143-1155 ...