Intranasal delivery of exosomes from human adipose derived stem cells at forty-eight hours post injury reduces motor and cognitive impairments following traumatic brain injury.

The neuroprotective role of human adipose-derived stems cells (hASCs) has raised great interest in regenerative medicine due to their ability to modulate their surrounding environment. Our group has demonstrated that exosomes derived from hASC (hASCexo) are a cell-free regenerative approach to long term recovery following traumatic brain injury (TBI). Previously, we demonstrated the efficacy of exosome treatment with intravenous delivery at 3 h post TBI in rats. Here, we show efficacy of exosomes through intranasal delivery at 48 h post TBI in mice lengthening the therapeutic window of treatment and therefore increasing possible translation to clinical studies. Our findings demonstrate significant recovery of motor impairment assessed by an elevated body swing test in mice treated with exosomes containing MALAT1 compared to both TBI mice without exosomes and exosomes depleted of MALAT1. Significant cognitive improvement was seen in the reversal trial of 8 arm radial arm water maze in mice treated with exosomes containing MALAT1. Furthermore, cortical damage was significantly reduced in mice treated with exosomes containing MALAT1 as well as decreased MHCII+ staining of microglial cells. Mice without exosomes or treated with exosomes depleted of MALAT1 did not show similar recovery. Results demonstrate both inflammation related genes and NRTK3 (TrkC) are target genes modulated by hASC exosomes and further that MALAT1 in hASC exosomes regulates expression of full length TrkC thereby activating the MAPK pathway and promoting recovery. Exosomes are a promising therapeutic approach following TBI with a therapeutic window of at least 48 h and contain long noncoding RNA's, specifically MALAT1 that play a vital role in the mechanism of action.

T cell infiltration and upregulation of MHCII in microglia leads to accelerated neuronal loss in an α-synuclein rat model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is the second most prevalent movement disorder characterized by up to 80% loss of dopamine (DA) neurons and accumulation of Lewy body deposits composed of α-synuclein (α-syn). Accumulation of α-syn is associated with microglial activation, leading to a pro-inflammatory environment linked with the pathogenesis of PD. Along with microglia, CD4 and CD8 T cells are observed in SNpc. The contribution of T-cells to PD development remains unclear with studies demonstrating that they may mediate neurodegeneration or act in a neuroprotective manner. METHODS: Here, we assessed the contribution of T cells to PD neurodegeneration using an adeno-associated virus (AAV) coding human wild-type α-syn or GFP injected into the substantia nigra pars compacta (SNpc) in T cell deficient (athymic nude) and T cell competent (heterozygous) rats. The rats were behaviorally assessed with cylinder test to test paw bias. Following behavior testing, brains were collected and analyzed for markers of dopamine neuron, microglial activation, T cells, and α-syn expression. RESULTS: Injection of AAV9-α-syn unilaterally into the SN of T cell competent rats resulted in a significant paw bias in comparison to the controls at 60 days post-injection. Conversely, T cell-deficient rats injected with AAV9-α-syn showed no deficit in paw bias. As expected, injected T cell competent rats demonstrated a significant increase in microglial activation (MHCII staining) as well as significant dopaminergic neuron loss. In contrast, the T cell-deficient counterparts did not show a significant increase in microglial activation or significant neuron loss compared to the control animals. We also observed CD4 and CD8 T cells in SNpc following microglial MHCII expression and dopaminergic neuron loss. The time course of T cell entry correlates with upregulation of MHCII and the peak loss of TH+ cells in the SNpc. CONCLUSION: These data demonstrate that T cell infiltration and microglial upregulation of MHCII are involved in α-synuclein-mediated DA neuron loss in this rat model of PD.

Identification of phagocytic cells, NK-like cytotoxic cell activity and the production of cellular exudates in the coelomic cavity of adult zebrafish.

Coelomic cavity (CC) cells of mature zebrafish harvested by lavage with media or trypsin-EDTA contained 0.80-1.20 x 10(5) and 2.0-3.5 x 10(5) cells, respectively. Media lavage was composed of granulocytes (60-80%), lymphocytes (10-20%), and NCC (4-10%). Granulocytes had large electron dense cytoplasmic paracrystalline granules and a segmented nucleus; they expressed plastin-1, myeloid specific peroxidase and MCSF mRNA; and they were NCAMP-1(+). Lymphocytes had B- and T-cell specific mRNA and were NCAMP-1(-) and NCCRP-1(-). NCC were 3 microm, NCAMP-1(+) and NCCRP-1(+) and did not express B- and T-cell specific mRNA. Additionally, trypsin lavage contained monocytes (marginated chromatin, low nuclear:cytoplasm ratio, sparse cytosolic granules) and macrophages (non-segmented nuclei, no margination of chromatin, abundant electron dense granules). E. coli injected into the CC were phagocytosed in a dose and time dependent fashion by granulocytes, monocytes and macrophages. NCC lysed mammalian target cells and NCAMP-1 expressing hybridoma cells in redirected lysis assays.

Cellular expression and antimicrobial function of a phylogenetically conserved novel histone 1x-like protein on mouse cells: a potential new class of pattern recognition receptor.

A H1x-like protein (i.e., NCAMP-1) is expressed on the membrane and in GEs from fish NK-like cells. In the present study, we identify the imprinting control region mouse NCAMP-1 ortholog using NCAMP-1 polyclonal antibodies and mAb. Polychromatic flow cytometry revealed NCAMP-1 expression on PBLs (Gr-1(+) PMNs were 21.1% NCAMP-1(+); DX-5(+) NK cells were 12.2% NCAMP-1(+)), mesenteric LN cells (CD11c(+) DCs were 23.2% NCAMP-1(+); Gr-1(+) PMNs were 24.8% NCAMP-1(+); CD21(+) B cells were 17.8% NCAMP-1(+)), and splenocytes (CD11c(+) were 39.6% NCAMP-1(+); Gr-1(+) PMNs were 40.9% NCAMP-1(+); DX-5(+) NK cells were 24.3% NCAMP-1(+); CD21(+) B cells were 28.5% NCAMP-1(+)). Western blot analysis using pNCAMP-1 and GEs from RAW 264.7 cells produced a 32-kDa signal. GEs from RAW 264.7 cells produced a significant reduction in Escherichia coli CFU. This antimicrobial killing activity was inhibited by pretreatment of the extract with (polyclonal) anti-NCAMP-1. Treatment with preimmune serum did not reduce bacterial cell killing. Confocal microscopy using NCAMP-1 and LAMP-1 mAb demonstrated that NCAMP-1 was located on the membrane and in cytosolic vesicles of RAW 264.7 cells and did not appear to colocalize with LAMP-1. NCAMP-1 may participate as a bifunctional protein on cells. It is expressed on the membranes of phagocytic cells, NK cells, and APCs in mice as well as in the granules of macrophages. In phagocytic cells, NCAMP-1 may participate in a nonregulated exocytosis pathway of cellular secretion.