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Multiple Sclerosis and Neuroimmunology
Brain-Targeted Autoimmunity: Beyond Multiple Sclerosis View all 10 Articles
Roland S. LIBLAU
Institut National de la Santé et de la Recherche Médicale (INSERM), France
Rishein Gupta
University of Texas at San Antonio, United States
Catharina C. Gross
University of Münster, Germany
Front. Immunol., 29 January 2019 | https://doi.org/10.3389/fimmu.2019.00121
Evidence for Innate and Adaptive Immune Responses in a Cohort of Intractable Pediatric Epilepsy Surgery Patients
Geoffrey C. Owens1*, Alejandro J. Garcia2, Aaron Y. Mochizuki2,3, Julia W. Chang1,4, Samuel D. Reyes1, Noriko Salamon5, Robert M. Prins1,6,7,8, Gary W. Mathern1,4,7,9 and Aria Fallah1,7,9
1Department of Neurosurgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
2Division of Hematology Oncology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
3Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
4Intellectual and Developmental Disabilities Research Center, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
5Department of Radiological Sciences, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, CA, United States
6Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
7Brain Research Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
8Parker Institute for Cancer Immunotherapy, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
9Mattel Children's Hospital, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
Brain-infiltrating lymphocytes (BILs) were isolated from resected brain tissue from 10 pediatric epilepsy patients who had undergone surgery for Hemimegalencephaly (HME) (n = 1), Tuberous sclerosis complex (TSC) (n = 2), Focal cortical dysplasia (FCD) (n = 4), and Rasmussen encephalitis (RE) (n = 3). Peripheral blood mononuclear cells (PBMCs) were also isolated from blood collected at the time of the surgery. Cells were immunostained with a panel of 20 antibody markers, and analyzed by mass cytometry. To identify and quantify the immune cell types in the samples, an unbiased clustering method was applied to the entire data set. More than 85 percent of the CD45+ cells isolated from resected RE brain tissue comprised T cells; by contrast NK cells and myeloid cells constituted 80–95 percent of the CD45+ cells isolated from the TSC and the FCD brain specimens. Three populations of myeloid cells made up >50 percent of all of the myeloid cells in all of the samples of which a population of HLA-DR+ CD11b+ CD4− cells comprised the vast majority of myeloid cells in the BIL fractions from the FCD and TSC cases. CD45RA+ HLA-DR− CD11b+ CD16+ NK cells constituted the major population of NK cells in the blood from all of the cases. This subset also comprised the majority of NK cells in BILs from the resected RE and HME brain tissue, whereas NK cells defined as CD45RA− HLA-DR+ CD11b− CD16− cells comprised 86–96 percent of the NK cells isolated from the FCD and TSC brain tissue. Thirteen different subsets of CD4 and CD8 αβ T cells and γδ T cells accounted for over 80% of the CD3+ T cells in all of the BIL and PBMC samples. At least 90 percent of the T cells in the RE BILs, 80 percent of the T cells in the HME BILs and 40–66 percent in the TSC and FCD BILs comprised activated antigen-experienced (CD45RO+ HLA-DR+ CD69+) T cells. We conclude that even in cases where there is no evidence for an infection or an immune disorder, activated peripheral immune cells may be present in epileptogenic areas of the brain, possibly in response to seizure-driven brain inflammation.
It has been estimated that by 15 years of age, approximately one percent of children will have experienced at least one seizure (1). For reasons that may not always be understood ~10% of children go on to develop medically refractory epilepsy, which is defined as a failure of two or more antiepileptic drugs to control seizures, and at least one seizure per month for ≥18 months (2). Recurrent seizures may severely impair a child's cognitive development leading to lifelong learning and behavioral difficulties. For children with drug-resistant epilepsy, surgery may be the only option to obtain seizure freedom, but will result in neurological deficits if the zone of resection involves eloquent cerebral cortex.
Many of the children who are candidates for epilepsy surgery suffer from rare neurological disorders including Rasmussen encephalitis (RE), Tuberous Sclerosis Complex (TSC), Focal Cortical Dysplasia (FCD), and Hemimegalencephaly (HME). RE patients present with partial (focal) seizures; magnetic resonance images (MRI) may indicate inflammation and atrophy in the affected cerebral hemisphere (3). The inflammation may spread through the affected cerebral hemisphere, but generally does not cross over to the contralateral hemisphere (4). Histopathological examination of resected brain tissue and brain biopsies show T cells in perivascular spaces, leptomeninges, and in small clusters scattered throughout the affected gray and white matter (5, 6). Clonally focused T cells have been found in resected RE brain tissue strongly implicating an antigen driven immune response in disease etiology (7–10).
In TSC patients, germ line and somatic dominant loss of function mutations in the genes encoding hamartin (TSC1) or tuberin (TSC2) can cause the development of benign tumors and, abnormally differentiated cortical neuronal progenitors that may cause focal seizures (11). Tuberin and harmatin are components of a complex that regulates the activity of the protein kinase, mTOR (12). Similarly, FCD and HME can be caused by activating somatic mutations in the MTOR gene, and in genes that regulate mTOR activity (13–15). Histopathological examination of resected brain tissue and analysis of RNA transcripts has shown that FCD and TSC lesions may be associated with an inflammatory reaction (11, 16–19).
In the present study we report on the characterization, by mass cytometry, of brain-infiltrating lymphocytes (BILs) isolated from surgical resections of epileptogenic tissue to treat FCD, TSC, and HME, as well as RE, and of peripheral blood mononuclear cells (PBMCs) prepared from blood collected at the time of surgery from the same cases. Immune cell profiling showed that activated T cells were present in brain tissue from all of the cases examined, and that the relative abundance of adaptive and innate lymphoid cells, and myeloid cells markedly differed between the RE and non-RE cases.
Patient Cohort
Under UCLA IRB approval (IRB#18-000154) brain tissue and blood were collected at surgery as part of UCLA's Pediatric Epilepsy Surgery Program. All of the patients or their parents or legal guardians provided informed consent for the use of the surgical remnant and blood for research purposes. All specimens were collected using the same standard operating procedures. De-identified patient information including age at seizure onset, age at surgery, gender, and affected cerebral hemisphere was collected with informed consent (Table S1).
Isolation of Peripheral Blood Lymphocytes and Brain-Infiltrating Lymphocytes
PBMCs were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Piscataway, NJ). BILs were isolated from collagenase-treated brain tissue by fractionation on a step gradient (20). Briefly, brain tissue was diced manually on ice in dissociation solution (HBSS with 20 mM HEPES pH 7.0, 5 mM glucose, and 50 U/ml penicillin/streptomycin). Tissue fragments were incubated with agitation at room temperature overnight in dissociation solution containing 0.5 mg/ml Type IV collagenase (Worthington Biochemical Corp., Lakewood, NJ) and 5% filtered human serum (Mediatech Inc., Manassas, VA). The dissociated tissue was fractionated on a 30%: 70% Percoll® (SigmaAldrich, St. Louis, MO) step gradient in RPMI containing 20 mM HEPES. PBMCs and BILs were cryopreserved in 90% human serum /10% DMSO.
Multiparameter Mass Cytometry
The panel comprised the following markers: CD45 (89Y or 154Sm), CD196 (141Pr), CD19 (142Nd), CD69 (144Nd), CD4 (145Nd), CD8 (146Nd), CD25 (149Sm), CD103 (151Eu), CD45RA (155Gd), CD183 (156Gd), CD56 (163Dy), CD45RO (164Dy), CD16 (165Ho), TCRγδ (168Er), CD3 (170Er), CD195 (171Yb), HLA-DR (174Yb), CD194 (175Yb), CD127 (176Yb), and CD11b (209Bi). All metal-tagged antibodies (Abs) were obtained from Fluidigm (San Francisco, CA) except the CD8 and TCRγδ antibodies, which were conjugated in-house. PBMCs and BILs were stained according to Fluidigm's protocol. In brief, cells were thawed, and washed in phosphate-buffered saline (PBS); prior to staining BILs were filtered through a 40 μm sieve to remove any aggregates. Cells were resuspended in 1 ml of PBS and stained for 5 min with 1 μM Cisplatin. After quenching the staining with Maxpar® cell staining buffer (Fluidigm), cells were incubated with the cocktail of Abs in 100 μl of Maxpar® cell staining buffer for 30 min at room temperature. The Ab cocktail for staining PBMCs contained the CD45 Ab conjugated to Samarium 154 (154Sm) while the BIL Ab cocktail contained the CD45 Ab tagged with Yttrium 89 (89Y). Following two wash steps, cells were fixed overnight at 4°C in Maxpar® fixation and permeabilization buffer containing 0.125 μM Intercalator-Ir (Fluidigm). BILs and PBMCs from the same surgery were combined at this point and washed twice with Maxpar® cell staining buffer and a further two times with water. Having barcoded the BILs and PBMCs with a different metal-conjugated CD45 Ab it was possible to analyze them as single sample, thus the larger number of PBMCs served as a carrier for the smaller number of BILs that were isolated from brain tissue. Cells were resuspended in 10 percent EQ™ Four Element Calibration Beads (Fluidigm) containing Cerium (140/142Ce), Europium (151/153Eu), Holmium (165Ho), and Lutetium (175/176Lu). Samples were acquired on a Helios® cytometry time of flight (CyTOF) system (Fluidigm) at an event rate of 300–500 events/s. Post-acquisition data normalization was done using bead-based normalization in the CyTOF software. Prior to analysis, data were gated to eliminate normalization beads, debris, dead cells, and doublets.
The analysis of FCS files was initially carried out using Cytobank (21). For each surgery case, the marker expression on the BILs was resolved by first gating live singlets on CD45 (89Y). Conversely marker expression on PBMCs was resolved by first gating on CD45 (154Sm). The data were then split into two separate FCS files using software tools in Cytobank (21). To define subsets of immune cells in each BIL and PBMC population, the entire high dimensional dataset (comprising 20 FCS files) was converted into a matrix of pair-wise similarities by implementing the t-based stochastic neighbor embedding (t-SNE) algorithm, followed by a density-based clustering method (ClusterX) (22). The resulting 2-D plots from this procedure were exported to CorelDraw2017 as portable document format files (Corel Corporation, Ottawa, Canada). Individual FCS files were analyzed using FlowJo® software (FlowJo LLC, Ashland, OR); 2-D contour plots were exported as scalable vector graphic (svg) files to CorelDraw2017. The median level of expression of each marker was used to assign a phenotypic identity to each cluster. Heat maps were generated using Morpheus software (www.broadinstitute.org) and exported to CorelDraw2017 as svg files. Principal component analysis (PCA) was performed using PAST software (https://palaeo-electronica.org/2001_1/past/issue1_01.htm).
PBMCs and BILs isolated from 10 pediatric epilepsy cases were analyzed by CyTOF using a panel of antibodies designed to identify populations of adaptive lymphoid cells, innate lymphoid cells, and myeloid cells. Implementing the mass cytometry data analysis pipeline developed by Chen et al. (21) generated 46 clusters, corresponding to putatively distinct populations of CD45+ cells (Figure 1). Clusters were classified as T cells, NK cells, and myeloid cells based on the relative expression of 19 immune cell markers. Immune cell clusters were divided into a CD3+ (n = 30, median CD3 expression values of 4.648–6.283) and a CD3− group (n = 16, median expression values of 0.001–0.81). The CD3+ group was subdivided into subsets of CD4, CD8, and γδ T cells based on the level of expression of these three phenotypic markers (Figure 2). The CD3− group was further divided into five NK cell subsets, ten myeloid and one B cell population based on the expression of CD56 and CD19 (Figure 2; Table S2).
Figure 1. tSNE plots showing the relative number of different immune cells in BILs and PBMCs from the pediatric epilepsy surgeries. The expression of 20 immune cell markers was analyzed by CyTOF. To define subsets of CD45+ cells in each BIL and PBMC population, the entire high dimensional dataset (comprising 20 FCS files) was converted into a matrix of pair-wise similarities by implementing the t-based stochastic neighbor embedding (t-SNE) algorithm, followed by a density-based clustering method (ClusterX). The clusters were assigned as either T cells, NK cells, myeloid cells, or B cells based on the median expression values of specific immune cell markers (CD3, CD4, CD8, TCR γδ, CD11b, CD56, and CD19).
Figure 2. Assignment of immune cell phenotypes. The median expression values of 19 immune cell markers, calculated by the Cytofit software, were used to assign a phenotype to each cluster of CD45+ cells (Table S2). The data were first separated into CD3+ and CD3− clusters, and the CD3+ populations were further subdivided into CD4+, CD8+, γδ subsets. The CD3− populations were categorized as myeloid, natural killer cell, or B cell based on the expression of CD56 and CD19. Heat maps generated from the median expression values included all the markers that were expressed on cells in the CD3+ CD4+, CD3+ CD8+, CD3+ γδ+, CD3− CD56+, CD3− CD19+/− clusters, respectively. The median expression value of the two different CD45 antibody metal conjugates used to stain the PBMC and BIL fractions reflects the relative number of PBMCs and BILs in each cluster.
Visual inspection of the t-SNE plots (Figure 1) showed that there were clear differences between the BILs from each surgical case compared with the corresponding PBMCs. On the other hand, the profiles of BILs from the two TSC (Case IDs 460 and 462) and the four FCD cases (Case IDs 475, 490, 494, and 495) appeared to be very similar and distinct from the three RE cases (Case IDs 472, 484, and 497), and dissimilar from the HME (Case ID 485), which appeared more similar to the RE cases. Principal components analysis based on the relative abundance of all of the clusters in each sample (percentages of CD45+ cells) confirmed this observation, and also showed that the immune cell profiles of PBMCs from all of the cases were very similar (Figure 3). From the magnitude of the PCA loading values (Table S3), Clusters 1 and 8 accounted for the largest amount of variance in the first component, thus, the relative numbers of NK and myeloid cells in the BIL fractions appears to explain in large measure the observed difference between the TSC and FCD BILS compared with the RE and HME BILs. As summarized in Figure 4, CD45+ cells from the FCD and TSC brain tissue specimens comprised far more NK cells and myeloid cells than CD45+ RE BILs, which in agreement with previous studies (5, 6, 20, 23), were predominantly CD8+ αβ T and γδ cells, The HME BILs comprised approximately equal numbers of T cells, NK cells and myeloid cells.
Figure 3. Clustering of BILs and PBMCs from pediatric epilepsy surgeries. The relative number of cells in each cluster in each sample (as a percentage of the total number of CD45+ cells analyzed in each sample) was used to implement a principal component analysis (PCA). The first two components could account for 65.72% of the total variance in the data set. The 2D PCA plot shows that the BIL fractions are clearly different from the corresponding PBMCs, and that BILs from the RE and HME cases are distinct from those from the FCD and TSC cases.
Figure 4. Immune cell profiles of BILs. Donut plots show the relative number of lymphoid cells, myeloid cells and B cells in the BIL fractions isolated from resected brain tissue. Lymphoid cells are divided into CD4, CD8, and γδ T cells and NK cells. Each chart corresponds to one of the surgery cases, and are arranged according to the clinical diagnosis (TSC: 460, 462; FCD: 475, 490, 494, 495; HME: 485; RE: 472, 484, 497). The data are presented as percentages of the number of CD45+ cells in each BIL fraction.
Three populations of myeloid cells (Clusters 1, 7, and 45) (Figure 2) made up >50 percent of all of the myeloid cells in all of the samples (Figure 5; Table S4). The Cluster 1 myeloid population comprised the vast majority of CD11b+ cells in the BIL fractions from the FCD and TSC cases (Figure 5), whereas Cluster 7 CD11b+ myeloid cells were more abundant in two of the three RE BIL fractions (Case IDs 472 and 484), and in all of the PBMCs (Figure 5). The only other marker that defined the Cluster 1 population was HLA-DR, thus it was not possible to assign a definitive phenotype to cells in this population, but they are likely to be macrophages. Likewise, Cluster 7 cells, which are CD3− CD4lo could constitute a dendritic cell or monocyte population, but additional markers are also required to adequately define this population.
Figure 5. Heat maps showing the subsets of T cells, NK and myeloid cells that account for the majority of lymphoid and myeloid cells in each BILs and PBMC fraction. The heat maps show the relative abundance of each cluster as a percentage of the total number of CD4 αβ T cells, CD8 αβ T cells, γδ T cells, NK cells or myeloid cells, respectively in each BIL and PBMC fraction (Table S4).
Cluster 3 NK cells are CD56+ CD45RA+ HLA-DR− CD11b+ CD16+ (Figure 2), and constituted the major population of NK cells in the blood from all of the cases (Figure 5). This subset also comprised the majority of NK cells that were found in BILs from the resected RE and HME brain tissue (Figure 5). By contrast, Cluster 8 NK cells, defined as CD56+ CD45RA− HLA-DR+ CD11b− CD16− (Figure 2) comprised 86–96 percent of the NK cells isolated from the FCD and TSC brain tissue (Figure 5; Table S4). We confirmed the existence of Cluster 3 and 8 NK cell populations by manually curating the original FCS files from FCD case 490 using the FlowJO software package (Figure S1A).
Six subpopulations of CD4+ αβ T cells (Clusters 2, 5, 9, 12, 15, and 23), four of CD8+ αβ T cells (Clusters 4, 10, 13, and 14), and three subsets of γδ T cells (Clusters 11, 26, and 42) accounted for over 80% of the CD3+ T cells in all of the BIL and PBMC samples, although there were marked differences in the proportion of the different T cell subsets between the BIL and PBMC samples (Figure 5; Table S4). Clusters 2, 9 and 23 CD4+ T cells constitute naïve T cell populations (CD45RA+ CD45RO− CD127int) (Figure 2); different median levels of expression of CD4 and CD127 appear to account for the generation of two clusters of naïve conventional T cells (Clusters 2 and 23), whereas expression of CD56 by Cluster 9 CD45RA+CD45RO−CD4+ T cells indicates that they may be unconventional NKT cells.
Clusters 5 and 15 define two different subpopulations of antigen-experienced CCR4+ CD4+ T cells (Figure 2). Based on the expression of CD25, the interleukin-2 receptor α chain (IL-2R) and lack of CD127, the interleukin-7 receptor α chain (IL-7R), Cluster 15 cells appear to be conventional regulatory T cells (Tregs) (24), and were found almost exclusively in the BIL fractions (Figure 5). Additional minor populations of CD45RO+ CD25+ CCR4+ CD4 T cells (Clusters 21, 22, 30, and 38) found predominantly in the blood may also be Tregs (Figure 2). Cluster 5 CD4 cells were found in both the brain and blood, and may be both IL-7 and IL-2 dependent (Figures 2, 5). Cluster 12 defined an activated effector memory population of CD4 T cells (CD45RO+ HLA-DR+ CD69+) (Figure 2) that was found in all of the BIL fractions (Figure 5). These cells also expressed the chemokine receptors CXCR3 and CCR5 thus may have trafficked to the epileptogenic brain area that was resected, possibly due to local inflammation (25).
Cluster 4 CD8 T cells were primarily found in the PBMC fractions and appear to correspond to an activated unprimed subtype (CD45RA+ CD127+) since they also expressed the chemokine receptor CXCR3 (26). A CD45RA+ CD45ROlo subpopulation (Cluster 14) made up most of the remaining CD8 T cells in the blood (Figures 2, 5; Table S4). Cells in Clusters 10 and 13 appear to be activated (CD69+ HLA-DR+) effector memory subtypes, and were found almost exclusively in the brain (Figures 2, 5). Based on the marker panel used in this study, the only difference between these two clusters was the expression of the resident memory marker CD103. Fewer Cluster 13 CD8+ resident memory T cells (TRM) were found in the brain compared with Cluster 10 T cells with one exception, Case ID 484, a RE patient. In agreement with previous work (27), the majority of the CD8+ T cells isolated from resected brain tissue from this patient were TRM cells. Likewise, the majority of γδ T cells isolated from a second RE case (Case ID 472) were TRM cells (Cluster 26, Figure 5) (27). Cluster 26 and Cluster 42 γδ T cells expressed the activation markers CD69 and HLA-DR and were almost exclusively found the brain (Figures 2, 5). These two activated subsets comprised 88–98 percent of the γδ T cells in the RE BIL fractions, whereas a third subset, Cluster 11, a CCR5+ CXCR3+ effector memory population (CD45RA+ CD45ROint CD127+) contributed up 70 percent of the γδ T cells in the other cases (Table S4). The Cluster 11 subset also made up the vast majority of γδ T cells in the blood from all of the cases (Figure 5). We verified the existence of all of the major T cell populations defined by the ClusterX clustering algorithm by manually curating several of the original FCS files (Figures S1B–D).
The donut plots in Figure 6 summarize the proportion of CD69+ HLA-DR+ T cells found in each of the BIL fractions from the 10 surgery cases. At least 90 percent of the T cells isolated from the RE cases and 80 percent of the T cells from the HME case were activated. Even though T cells made up only a small fraction of the CD45+ cells isolated from the TSC and FCD cases, 40–66 percent of the T cells were also activated at the time of surgery.
Figure 6. The proportion of activated T cells in the BIL fractions. Donut plots show the relative number of CD69+ HLA-DR+ CD4, CD8, and γδ T cells as percentage of the CD3+ cells in each BIL fraction. Each chart corresponds to one of the surgery cases, and are arranged according to the clinical diagnosis (TSC: 460, 462; FCD: 475, 490, 494, 495; HME: 485; RE: 472, 484, 497). The different activated subsets are color coded; the gray area denotes the percentage of T cells that did not express activation markers.
The absence of CD8+ TRM cells in the BIL fraction from RE case ID 497 suggested that the disease may not have progressed as far in this patient compared with the other two RE cases in the study cohort (Cases IDs 472 and 484). A [18F]-deoxyglucose positron emission tomography (FDG-PET) brain scan of the patient made before the surgery showed hypometabolism extending over the entire affected hemisphere, with no definitive areas of atrophy. In the other two RE cases, areas of atrophy were clearly visible (Figure 7). Case ID 497 was the youngest of the three RE patients at the time of seizure onset and underwent surgery after the shortest time following onset of seizures (Table S1).
Figure 7. [18F]-deoxyglucose positron emission tomography (FDG-PET) identified areas of atrophy in RE cases 472 and 484 (red arrows). In RE case 497 there were no obvious signs of atrophy in the affected hemisphere, rather decreased glucose metabolism over the entire hemisphere was apparent.
We have used CyTOF to gain a better understanding of the involvement of peripheral immune cells in intractable epilepsy in children. We implemented an unsupervised clustering method to resolve the resulting high dimensional data into the main subtypes of adaptive and innate lymphoid cells present in resected epileptogenic brain tissue. Our small cohort study, and other recent work (28) implicate cellular immunity not only in RE, but in intractable epilepsy in children where an infection or immune disorder is not suspected. With the caveat that we only analyzed a limited number of surgery cases, we found a clear difference in the relative number of innate vs. adaptive peripheral immune cells in fractions of CD45+ cells isolated from resected TSC and FCD brain tissue compared with involved RE brain tissue. The FCD and TSC BIL fractions were dominated by a single subset of NK cells (Cluster 8) and a population of myeloid cells (Cluster 1) that are likely to be macrophages, possibly classically activated pro-inflammatory M1 macrophages. Adding CD38 and CD163 antibodies to the staining panel will allow us to distinguish between M1 and M2 macrophages, respectively (29, 30). In an animal seizure model, infiltrating monocytes were shown to exacerbate neuronal damage (31), indicating that the abundant myeloid population that we found in the FCD and TSC BIL fractions may be pathologically relevant.
Most NK cells in the blood express a low level of CD56 (CD56dim) and are CD16+, thus can engage in antibody-dependent cell-mediated cytotoxicity by binding antibody-coated target cells via the low affinity Fcγ receptor III (CD16) (32). Fewer circulating NK cells express a high level of CD56 (CD56bright); these cells generally lack CD16, but can produce pro-or anti-inflammatory cytokines (32). CD56bright NK cells are considered to be more immature than CD56dim NK cells in that they mostly do not express killer cell immunoglobulin-like receptors (KIRs) (33). Unlike CD56dim NK cells they express CD62L (L-selectin) and CD197 (CCR7), cell surface molecules that facilitate homing to secondary lymph nodes (32). CD56dim NK cells express KIRs and CD57 and are considered to be intrinsically more cytotoxic (32, 33). The most abundant NK cells that we found in the blood of all of the patients in our study cohort presumably correspond to CD56dimCD16+ NK cells (Cluster 3). This subset also comprised the majority of NK cells present in the BIL fractions from the RE and HME patients (Figure 5). Adding CD62L, CD197, and CD57 to the marker panel will clarify the phenotype of Cluster 3 NK cells.
By comparison to the NK cells in the PBMC fractions, the majority of NK cells found in the FCD and TSC BIL fractions expressed a lower level of CD56 suggesting that they correspond to a CD56dim subset (Cluster 8) (Figure 2, Figure S1A). Unlike canonical CD56dim NK cells, Cluster 8 cells did not express CD16, however populations of CD56dim NK cells expressing little or no CD16 have been previously described (34). Lack of CD16 may be due to the fact that the Cluster 8 NK cells were highly activated; it has been reported that the Fcγ receptor III is proteolytically cleaved in degranulating CD56dim NK cells (35). In contrast to Cluster 3 NK cells, Cluster 8 NK cells also expressed HLA-DR (Figure 2, Figure S1A), which suggests that they may be adaptive NK cells (32, 33). A third population of NK cells (Cluster 33), only found in appreciable numbers in BILs from the HME case (Case ID 485) expressed CD69 along with CCR5, CXCR3, and CD103 suggesting that this cluster may correspond to an activated resident subset (32).
BIL fractions from the RE cases were predominantly CD8 αβ and γδ effector memory and resident memory T cells with fewer effector memory CD4 T cells, confirming previous work (20, 27). We and others have shown that T cells found in RE brain are clonally focused (7–10), which strongly implicates an antigen-driven adaptive immune response in the etiology of RE, and is consonant with the idea that RE is an autoimmune disease (36). Finding TRM CD8 αβ and γδ T cells in affected RE brain tissue indicates that an earlier immune response had occurred that left a resident memory population in place. Compared with RE cases 472 and 484 however, no TRM cells were present in the BILs isolated from RE case 497, suggesting that this case may represent an early stage of the disease. In support of this idea, brain atrophy, a feature of late phases of the disease (4), was not evident in an FDG-PET brain scan of RE case 497, but was in RE cases 472 and 484 (Figure 7).
In pathological specimens from FCD and TSC surgeries scattered T cells have been previously observed (16, 17), and in our study we found activated CD4, CD8, and γδ T cells in the BIL fractions from the FCD and TSC cases (Figure 6). Determining the clonal diversity of these T cells by sequencing Vβ chain third complementarity determining regions may indicate whether they are unspecific bystanders or presage an adaptive response against self-antigens. Binding of self-peptides would however depend on the MHC alleles carried by the individual (37–39). The proportion of activated T cells in the single HME case (Case ID 485), which exceeded the number of NK cells (Figure 4), may indicate that an autoimmune response had already occurred in this very young patient who suffered from severe seizures. Future recall assays in which T cells isolated from resected brain tissue are cocultured with autologous neurons and glia differentiated from patient-derived induced pluripotent stems cells will directly answer whether the T cells are autoreactive.
The immune cell profiles of peripheral blood from all of the cases comprised both naïve and antigen experienced T cells. γδ T cells that were CD127+, CD45RO+, CXCR3+, and CCR5+ comprised the majority of γδ T cells in all of the PBMC samples, as well as in the FCD and TSC BILs (Cluster 11; Figures 2, 5, Figure S1D). They likely express Vδ2/Vγ9 TCR receptors (40) since Vδ2/Vγ9 γδ T cells make up a large fraction of circulating γδ T cells (41). Expression of CXCR3 and CCR5 would allow these γδ T cells to access sites of inflammation (26). CXCR3 expression by naïve CD45RA+ CD8 T cells (Cluster 4; Figure 2, Figure S1C) indicates that they are activated (42), and that development into effector T cells may be enhanced (43, 44). Whether the presence of these T cell subsets in the blood is directly related to inflammation in the brain remains to be determined. In future work we plan to compare the immune cell profiles of blood collected at the time of surgery and after recovery from surgery. Removal of epileptogenic brain tissue, the potential source of inflammation may change the relative frequency of specific circulating T cell subsets. Such subsets could potentially be used as biomarkers to assess the extent brain inflammation in children presenting with intractable epilepsy.
The distribution of the two most abundant CCR4+ CD4 T cell subsets (Clusters 5 and 15) differed between the blood and brain. Cluster 15 cells were almost exclusively found in BIL fractions, and appear to be activated effector memory-like Tregs (CD25+ CD45RO+CD69+HLA-DR+) (Figures 2, 5, Figure S1B). Tregs are also defined by expression of the transcription factor FOXP3 (24); adding this intracellular marker to the CyTOF panel will confirm the identity of these CD4 T cells. In a cohort of pediatric epilepsy surgery patients, a negative correlation between seizure frequency and the relative number of Tregs in resected epileptogenic brain was recently reported (28). In our study cohort, the relative number of Cluster 15 Tregs cells ranged from 2.61 to 31.53 percent of CD4 T cells in the BIL fractions (Table S4). However, the limited number of self-reports of seizure activity available do not allow us to draw any conclusions about the number of seizures and the percent Tregs in our patient cohort. Cluster 5 CD45RO+CCR4+ CD4 T cells (Figure 2, Figure S1B), which express both IL-2Rs and IL-7Rs, were found in both BIL and PBMC fractions, and may correspond to a subset of CD4 T cells that express CD25 and CD127 and low levels of FOXP3, and are not suppressive in vitro (24). Adding the FOXP3 Ab to the marker panel may help clarify the identity of this subset of CD4 T cells.
Extensive animal studies have shown that inflammation in the brain is both a cause and a consequence of seizure activity (45). Attention has focused on the innate response of brain resident microglia and astrocytes to seizures and on the pro-inflammatory molecules produced by these cells as drivers of epileptogenesis (46–48). In seizure models, chemokines and cytokines produced by activated microglia and astrocytes can cause blood brain barrier leakage, and can attract peripheral lymphoid and myeloid cells to the brain (31, 49–51). Our data suggest that this could be happening in children with chronic pharmacoresistant seizures. Intractable epilepsy might be viewed as a "sterile" infection in which T cells and other immune cells traffic to epileptogenic areas of the cerebral cortex in response to local seizure-driven inflammation. Activated T cells and NK cells for example could permanently alter brain circuitry by directly or indirectly damaging neurons (52–55). Adjunctive treatments that block the recruitment of pro-inflammatory peripheral lymphoid and myeloid cells to the brain (56–61) may therefore be therapeutically beneficial.
GO designed the study, analyzed the data, and wrote the paper. AG assisted with study design and performed the CyTOF. AM assisted with analysis of the data. JC and SR coordinated collection of surgical specimens and prepared BILs and PBMCs. RP assisted with study design and helped draft the paper. NS provided FDG-PET results. GM supported the project. AF provided support for the work, and provided surgical specimens and patient information.
This work was supported by The RE Children's Project and NIH/NCATS/UCLA CTSI UL1TR001881, and in part by NIH R01 NS083823 and U01 MH108898. GM was partly supported by the Davies/Crandall Endowed Chair for Epilepsy Research at UCLA.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2019.00121/full#supplementary-material
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Keywords: brain, epilepsy, inflammation, peripheral immune cells, mass cytometry (CyTOF)
Citation: Owens GC, Garcia AJ, Mochizuki AY, Chang JW, Reyes SD, Salamon N, Prins RM, Mathern GW and Fallah A (2019) Evidence for Innate and Adaptive Immune Responses in a Cohort of Intractable Pediatric Epilepsy Surgery Patients. Front. Immunol. 10:121. doi: 10.3389/fimmu.2019.00121
Received: 15 September 2018; Accepted: 15 January 2019;
Published: 29 January 2019.
Roland S. Liblau, Institut National de la Santé et de la Recherche Médicale (INSERM), France
Catharina C. Gross, University of Münster, Germany
Rishein Gupta, University of Texas at San Antonio, United States
Copyright © 2019 Owens, Garcia, Mochizuki, Chang, Reyes, Salamon, Prins, Mathern and Fallah. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Geoffrey C. Owens, geoffreyowens@mednet.ucla.edu


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Named Entity Results, James Guthrie
Horace Greeley, The American Conflict: A History of the Great Rebellion in the United States of America, 1860-65: its Causes, Incidents, and Results: Intended to exhibit especially its moral and political phases with the drift and progress of American opinion respecting human slavery from 1776 to the close of the War for the Union. Volume I.	 37 1 Browse Search
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Rebellion Record: a Diary of American Events: Documents and Narratives, Volume 1. (ed. Frank Moore), chapter 65 (search)
Doc. 63.--meeting at Louisville, Ky. Mr. Guthrie's speech. the Hon. James Guthrie rose amid tremendous cheering. He said: Fellow-citizens, my voice is not very strong, and I fear it cannot be heard all over this great assemblage, but I will try to make it heard. Events press upon us with haste, and we scarcely know what is to come next. When Mr. Lincoln was elected President we all felt that the remedy for a sectional President was in the Union and under the Constitution. We knew wthe Hon. James Guthrie rose amid tremendous cheering. He said: Fellow-citizens, my voice is not very strong, and I fear it cannot be heard all over this great assemblage, but I will try to make it heard. Events press upon us with haste, and we scarcely know what is to come next. When Mr. Lincoln was elected President we all felt that the remedy for a sectional President was in the Union and under the Constitution. We knew we had a Senate against him, and hoped that we had the House against him; and there would have been if all men had stood at their posts as Kentucky has stood. But certain States chose to take the remedy into their own hands, and dissolve their connexion with the Union; South Carolina first, and then seven other States followed. They have organized a separate Government, and one exercising governmental authority. Louisville spoke early, decidedly, and firmly against a sectional party in the Uni
Rebellion Record: a Diary of American Events: Documents and Narratives, Volume 1. (ed. Frank Moore), chapter 265 (search)
w-citizens, on both sides, from slaughter, and we would commit the interests of our distracted country to His hands who can bring forth peace and order out of strife and confusion when man's wisdom utterly fails. J. J. Crittenden, President. Jas. Guthrie, H. R. Gamble, Of Missouri. Wm. A. Hall, Of Missouri. J. B. Henderson, Of Missouri. Wm. G. Pomeroy, Of Missouri. R. K. Williams, Archibald Dixon, F. M. Bristow, Joshua F. Bell, C. A. Wickliffe, G. W. Dunlap, J. F. Robinson, Jno. B.ecessary for your own best interest, and in humble submission trust and look to that Almighty Being, who has heretofore so signally blessed us as a nation for His guidance through the gloom and darkness of this hour. J. J. Crittenden, Pres't. James Guthrie, R. K. Williams, Archibald Dixon, F. M. Bristow, Joshua F. Bell, C. A. Wickliffe, G. W. Dunlap, C. S. Morehead, I have signed the foregoing address, because I approve of the policy therein indicated, of refusing to furnish troops to


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The topic of financial technology will have been discussed at length within this current edition of Cayman Financial Review. No doubt experts in the field will have informed readers on these exciting new trends within the financial services sector, and the many benefits such developments will bring to the industry. As my field of expertise is real estate, I thought it appropriate, as perhaps a parallel discussion, to talk about a new market trend that has opened up in the Cayman Islands recently; one which, I believe, has the potential to be something of a game changer when it comes to home ownership.
There are plenty of great houses on the market that would suit buyers looking for their own spacious accommodation to call home, however, the new development of Stone Island, located at the prestigious Yacht Club development, breaks new ground when it comes to home ownership, cutting an innovative swathe through the current market.
Up until this point, buyers could choose from a home that would most likely be looked after by the individual owner, in terms of landscaping, pool, deck, dock, etc. If a buyer is looking for a "lock and leave" type of home that is under the control of a strata management company, these have generally taken the form of a condominium. The beauty of Stone Island is that it combines owning a separate home tailored to the buyer's individual needs with the ease of owning a strata-operated property, where all the hard work of home ownership (i.e. the external upkeep) is undertaken by a third party.
By way of some background, Stone Island is a gated and fully maintained waterfront community that will have 44 luxury residences within its location, each with approximately 4,500-square-feet of open-concept living space. Buyers can choose to upgrade their residence with a number of additional features available, including building in an elevator, a built-in outdoor barbecue, as well as the inclusion of superior finish packages, among other options.
Located in a well-established and prestigious residential community, Stone Island offers easy access to the ocean and is just a short walk from Seven Mile Beach. The Residences of Stone Island are at the heart of a thriving yet quiet community, and enjoy the added benefits of being close proximity to The Cayman Islands Yacht Club, which has a full-service marina, restaurants and amenities.
In addition to employing the highest building standards, fixtures and finishes, which include poured concrete, foam insulation, LED lighting, impact-rated windows and smart-home technology, the developers have also gone the extra mile to produce lush landscaping, an additional separate swimming pool to each home's individual pool, tennis courts, a full-sized gym and on-site security. Exclusivity has been written into every detail – for example, the homes have been carefully designed so that you don't glimpse the entrances to other homes as you drive up to your property and cars are kept off the driveways and out of sight in a specially built garage. You can buy a home for a similar price to these residences at Stone Island, but house buyers will not have access to the extensive amenities that this development is offering, principally due to the scale of the development and the fact that it will be operated by a strata management company. This is why I am excited by this new concept.
It is especially relevant that we are able to offer new niche markets for potential buyers because of the recent upswing in demand for Cayman Islands property by overseas buyers. It is vital that Cayman is able to keep up with the appetite for second (or third homes) for wealthy retirees, in particular. This new type of home ownership will appeal to the overseas buyer who might be looking to buy a sizeable home in the Cayman Islands, but who also wants to be able to lock it up and leave it whenever they travel back home. This was traditionally only a benefit derived from owning a condo within a strata.
The security that will be on site gives additional peace of mind for people only living in their home for part of the year. Local residents who may be retiring will also find such a set-up an attractive lifestyle choice, with people reaching the end of their working life looking to spend just part of their time residing in Cayman.
There are currently three model homes already completed, to give would-be buyers a glimpse of how they could customize a home at Stone Island to suit their individual needs.
I believe that The Residences at Stone Island will be ground breaking for Cayman's real estate market and it's a precedent that stands the island in good stead for continued positive growth in the years to come.


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Hundreds of Season Ticket Holders headed to King Power Stadium for a special edition of Your 90 Minutes on Wednesday - featuring Brendan Rodgers.
Over previous seasons, Your 90 Minutes has functioned as an open forum for supporters to have their say on a particular topic concerning anything from ticketing to Fox Memberships.
Wednesday's session, however, handed members of the Blue Army with the opportunity to get to know the Football Club's new manager and to hear his plans for the future of Leicester City.
As City look ahead to a sixth consecutive season in the Premier League with a young and vibrant squad, work has also commenced on a brand-new state-of-the-art training facility in north Leicestershire.
Brendan spoke to the audience in two sittings of 45 minutes at King Power Stadium.
Since his appointment in February, meanwhile, Rodgers has won four out of five Premier League matches in charge of the Foxes, propelling them up to seventh in the table with five matches to play.
The free event, made available to Season Ticket Holders, was staged in the Walkers Hall at King Power Stadium, two days before City welcome Newcastle United to Leicester (8pm BST kick-off).
Hosted by Club Ambassador Alan 'The Birch' Birchenall, Rodgers took advantage of the opportunity to emphasise that supporters remain at the heart of the Football Club's thinking at King Power Stadium.
For next season, my ambition is to challenge the top six. It has been clear over the last few seasons who they are. That's the ambition, to really push to get into there.
In his opening speech, The Birch promised all those in attendance at the free event that they would 'get what they paid for' but it naturally became a night to remember for everyone in attendance.
Asked by Foxes fan Robert Gwyther-Canton to elaborate on the Club's ambitions next season and over the campaigns which follow, Brendan said: "For next season, my ambition is to challenge the top six.
"It has been clear over the last few seasons who they are. That's the ambition, to really push to get into there. And of course, the ambition is to win a cup competition and do it in a style that excites."
Hundreds of Leicester supporters were in attendance on Wednesday.
Anthony Boyle, meanwhile, asked Brendan about his life away from the rigours of managing a club of Leicester City's size and the Northern Irishman discussed his love of travelling and studying football tactics.
Describing himself as a 'student of the game', Brendan also provided an insight into his tactical approach to managing the Foxes, expressing his desire to play a 'fast, dynamic' brand of football.
Brendan's words on Khun Vichai Srivaddhanaprabha, our beloved Chairman who tragically passed away in 2018, particularly struck a chord with those in attendance, who felt such affection for Khun Vichai.
We're an emotional club, we've been through so much, over the past couple of years. We've got to come together and use Khun Vichai's memory to connect and build a legacy he can be proud of.
"As a manager, I feel as though I've missed something," he said. "To get a feel of the man, Khun Vichai, and the memories he has created here, I feel like I've missed something, both as a man and as a manager.
"We're an emotional club, we've been through so much, over the past couple of years. We've got to come together and use Khun Vichai's memory to connect and build a legacy he can be proud of."
Rodgers is also a manager who is renowned for trusting young players from the Academy to thrive in the first team - and he provided his thoughts on the Club's current set-up at Belvoir Drive.
The Birch oversaw proceedings by asking questions on behalf of the Foxes faithful.
"It's something Leicester have always done well, produce players from the local area," he said, replying to Steve Durham. "Age is no barrier for me. We had the younger groups watch training today.
"I want them to see and to understand the training we do and the model of our training. We've got some great youth coaches at the Club and the Academy is very well structured."
Concluding the evening, Brendan had a message for the Foxes fans to take with them as the Club approaches the final matches of the 2018/19 season and heads into an exciting future.
"The style will always be the same," he said. "We have to attack, to run, to press. My job as a manager is to entertain the supporters. I want you to enjoy coming here and watching your team."


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Home / Fighters & Attack / HAWKER SEA FURY
Title: Hawker Sea Fury
Originally designed for the RAF, who cancelled the order as WW2 drew to a close, the Sea Fury was taken up by the Royal Navy and first flew in 1945. It was the last propeller driven fighter to serve with the Royal Navy. Over 860 were built and it found a ready export market with at least seven other air arms operating the aircraft.
This title includes the Flight Manual (called Pilot's Notes) for all variants of the Sea Fury operated by the Royal Navy. The files have been scanned from the original flight manuals and retain any colour pages.
HAWKER SEA FURY quantity
Royal Navy Pilot's Notes for the Sea Fury X, AP4018A-PN dated August 1947 with approx 43 pages.
Royal Navy Pilot's Notes for the Sea Fury FB.11, AP4018B-PN, 3rd Edition dated May 1952 with approx 61 pages.
Royal Navy Pilot's Notes for the Sea Fury 20, AP4018C-PN dated June 1950 with approx 63 pages.
CESSNA A-37 DRAGONFLY
SUKHOI SU-27 FLANKER
DOUGLAS A-26 INVADER


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Do the Ethiopian Nationalities have cause to celebrate their so called "Day" in the Afar Region? : Afar Party statement
Editor's note: The following is ARDUF (Afar Revolutionary Democratic Unity Front) statement on "forthcoming Nations, Nationalities and People's Day celebration in the Afar Region."
Contact e-mail arduf1978@gmail.com
Do the Ethiopian Nationalities have cause to celebrate their so called "Day" in the Afar Region?
This year's Nations, Nationalities and Peoples "Day" is to be celebrated in December 2017, in Samara, the capital city of the Afar Region. The forthcoming is the 12th round of those Jamborees prepared in the name of Ethiopian Nations, Nationalities and Peoples "Day". A brochure issued by the federal authorities' states that Nations, Nationalities and Peoples day is celebrated in "commemoration of the people of Ethiopia's ultimate triumph against the repressive and oppressive regime that had deprived them of their rights in their own nation". They are of course, referring to pre-1991 oppressive regimes and not the present regime. The Ethiopian Nations, Nationalities and Peoples are being deprived of their legitimate rights and fundamental freedom and liberty by the current regime in their own nation. This is the tip of the ridiculous mockery that provoke outrage and wrath in those regions in particular and the whole country in general. It is not only" the peace between its people" that is conspicuously and unmistakable absent in those days between Ethiopian peoples.
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This huge Carnival Festivities of this round of Nationalities "DAY" will be celebrated massively, while 99% of the Afar population do not have basic necessities of life, such as water, food, clothing, shelter, housing and healthcare. Meanwhile, thousands of Oromo citizens who were displaced from Ethiopian Somali Region are being tracked in Lorries and freight to safer places in their home region, namely; Oromia. It is reported that 50,000 Oromos were so far displaced from Ethiopian Somali Region. The scene looks like any African civil war dramas we are all familiar with. Somalis are displacing Oromos from Jijiga only but from neighbouring Somali Republics like Somali-land & Punt-land. Here in Ethiopia Konsos fight Burjis, Afars fight IssaSomalis, Amharas fight Tigrais in Wokayet-Tsegede and Neuers fight Anwaks and so forth. It looks as if it is every body's war against everybody. Settled farmers against cattle herders, natives versus new-comers and so forth. Everybody seems to be at each other's throat. Allowing the so called Liyu Hayle (Liyu Polis) armed militias of every regions to engage in illegal acts of armed aggression to occupy each other's territories by use of force is a serious disaster especially when the local militias are under ambitious and out of control local prospective war lords are in collusion with greedy TPLF army Commanders. The so called Liyu Hayle forces, in all regions, have categorically failed to maintain law and order, peace and security within their local jurisdictions. As a result of TPLF failed policy and bad governance the recurrent clashes are crossing the threshold into fullblown bloody civil war.
In spite of all those upheavals, the preparation in Afar land is being finalized for the forthcoming "Day" of Nationalities under the slogan "peace among its Peoples." The Festival flyer states that the stadium, collection hall, internal roads and other infrastructure constructions continue to be build. The Festivities Preparatory Committee making the poor Afar people to donate money and livestock. The Committee announced that 701 camels, 10,513 goats and sheep and 175 cattle were received from Afar herders. This is from a Region where people live on handouts from the central government because of the recurrent drought, man-made hunger, oppression, discrimination, gross human rights abuses and deprivation of economic and social development packages. The ruling officials of the Afar region are proud to build "1 Stadium" for 1 billion and 600 million Ethiopian Birr, while their people are facing unprecedented severe economic and social hardship and health problem. Afar region is the most neglected, marginalised and underdeveloped regions in the country. Investors are called upon to invest in the Afar Region. The Afar Diasporas are also invited to come to celebrate the big "Day". We are told delegates were even sent to European capital cities, such as Brussels and London to convince the Diasporas Communities to attend the Festival. Everything is ready for the big Jamboree but the only conspicuously absentee is the "peace and development" which they are supposed to "celebrate" on "their Big Day".
The Ethiopian Nations, Nationalities and Peoples have little to "celebrate". Above all, the Afars of all groups have definitely little cause to celebrate. Afars will join their fellow Ethiopians in Samara in displaying their music and dancing in their traditional dress and decoration but having little to pay tribute to. On the contrary, today they are all busy in bundling together to get rid of this called TPLF, while TPLF which in turn is busy in hoodwinking everybody in its irresponsible behaviour of instigating one group against another group to maintain its divide and rule policy. They are deliberating in Mekelle City, the capital of Tigray State on how to refine their "divide and rule" machinations. But, the Ethiopian Nations, Nationalities and Peoples have too little "to celebrate on the so called "Nationalities Day" but every cause to stand and rebel in unison to uproot TPLF/EPRDF, the cause of all their misery and calamity.
Victory to ARDUF (UGUUGUMO)
Long live the Struggle of Ethiopian People!
Military Command Centre (MCC)
Information Desk of ARDUF
Afar Revolutionary Democratic Unity Front (ARDUF)
To publish article, statements and position papers on this page, please e-mail content to editor@borkena.com
Afar Region, Afar Revolutionary Democratic Unity Front, ARDUF, Ethiopia, Ethiopian Nations


It's cold outside. And in the case of my box, it's very cold inside, as well.
I've chronicled my issue with the temperature shutting down most outdoor activities in the Midwest. The rower becomes the cardio norm, and although we have roughed out a 100-yard lap at our affiliate, running workouts are almost nonexistent.
I don't know what the percentage is of CrossFit affiliates that have heated facilities, but I do know that the one I train at does not. In the summer, we simply open the doors wide and use very large "barn" fans to push the heat and humidity through the box as we grind out our WODs. But when temperatures bottom out in January and February (we are looking at air temps about -12 degrees this Wednesday), the option of pumping heat into our space isn't an easy fix.
Part of the issue is that our affiliate is in a massive warehouse that offers thousands of feet of space but whose original purpose was to store and refrigerate frozen seafood and other perishable items. When we first moved to this spot, the owners had to have a professional company come in to remove the multi-ton refrigeration units from the rafters of the building.
I don't know the science behind training in cold temps or whether being cold (externally) is somehow detrimental to the body's ability to train. I'm sure you could find studies that support both sides of the issue if you search hard enough. What I do know is the feel of an icy barbell in the early morning is something that has taken me some time to get used to. I know that jumping up to grab a Rogue pull-up bar and being able to hold on with frozen fingers might not be for everyone.
We all have different interpretations of what is "cold." Last week, I interviewed a CrossFit Games athlete and asked her about the weather where she was in California because I heard that it had snowed a little. She remarked that it was "freezing" there because the temps had dipped below 50 degrees and it was tougher to train when it got "that cold." I then informed her that it had hit 9 degrees here and that the wind chill was about -1 when I had gone into our box to do a version of "Annie," which included air squats as a third component along with the traditional double-unders and sit-ups.
She agreed that mine sounded worse.
I did hear recently that the work put in during these chilly months are the most important in terms of performance during the Open and Regionals and, if you are one of the very chosen few, the CrossFit Games. While that won't apply to an old grinder like me, I know that I always put in at least 20 WODs in January, regardless of the lack of warmth at our box.
Like my coaches always told me growing up, "The more you move, the warmer you'll feel." Can't argue with that logic.
What is your experience training in cold weather (either outside or at a place that doesn't have heat like ours). Share your stories and experiences in the comments.


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The further you get into "The Fate of the Furious" the more it reminds you of one of Sylvester Stallone's "Expendables" films. It has a huge cast of a-list talent - that is even larger than the previous installment. You can just imagine executive producer Amanda Lewis calling folk with the line "Hey, wanna make a bunch of money?"
James Wan, the director of "Furious 7" didn't return so the pitch went to F. Gary Gray ("Straight Outta Compton", "Law Abiding Citizen", "The Italian Job") who has a good track record for making money. Jason Statham and Dwayne Johnson were gimmes. The price tag went up a bit more with the addition of Charlize Theron and Helen Mirren. They also talked Kurt Russell to return with Scott Eastwood as Mr Nobody and Little Nobody respectively. Luke Evans' Owen Shaw character was revived as was Elsa Patasky's Elena character.
The rest of the gang - Michelle Rodriguez, Tyrese Gibson, Ludacris, and Nathalie Emmanuel were a piece of cake as the Fast & Furious franchise are the biggest projects in their respective filmographies. "Furious 7" earned over $1.5 billion globally; no wonder this production budget topped out at $250 million.
The story was inked by Chris Morgan, the writer of the previous two F&F films, so there is some continuity...sorta.
The film opens with a really cool drag race between two vintage cars in Havana driven by Dom (Vin Diesel) and Cuban local played by Celestino Cornielle - Dom was driving an old beat-up Chevy that is literally falling apart.
Soon thereafter Hobbs (Johnson) is approached to swipe a EMP bomb ala "Mission: Impossible". But what Hobbs doesn't know is that Dom has been recruited by a dark agent named Cipher (Theron) who wants him to swipe the EMP for her own dubious purposes.
With Dom now turned to the dark side, Hobbs aligns with Mr Nobody and Little Nobody who aligns him with none other than Deckard Shaw (Statham), who is currently in prison. No problem getting out of prison is just part three of a series of over the top action sequences.
Like all the previous films, we quickly learn the superhero capabilities of a car. Provided it is a superfast car! But here is exactly where so much of the film becomes a boated farce. The cars are faster than heat seeking missiles (Mach 1.7) but not faster than a nuclear sub (top speed of 40 mph). That is a serious continuity and logic flaw, flaws that are hard to overlook even in a over-the-top action farce.
Hobbs deflects rubber bullets off his chest and wipes out entire armies with his massive arms. At nearly 50 years old, Statham can perform acrobatic maneuvers while kicking ass and taking names. And yet Tyrese Gibson and Ludacris can't find the words to say to Nathalie Emmanuel.
The motive behind the attacks by Cipher are to simply take over the world, much like Blofeld from the James Bond series. There is no explanation of revenge for a long ago wrong or stated amounts of monetary benefits... she just wants to take over the world by getting Russia and the United States into a nuclear war. How she convinces Dom to join her is stale and also contrived based on the recurring "it's all about family" theme.
For big splashes, the film is more than adequate, but it is seriously lacking a cohesive story. No worries though, two others are already scheduled as soon as this one earns a billion.
"The Fate of the Furious" is an overload of stunts and special effects that barely come together in a nonsensical plot. For fans of non-stop over-the-top action, it is a winner, but don't expect an ounce of substance. -- GEOFF BURTON


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Mind Matters Blog
Exercise is the new primary prescription for those with mental health problems - Neuroscience News
Exercise is an amazing benefit to both physical and mental health and something anybody is capable of in some way. This article largely discusses the use of exercise in inpatient treatment facilities, but it is equally as beneficial in outpatient treatment. A little movement can go a long way!
Summary: Adding exercise as a treatment for patients in psychiatric care facilities has positive benefits and is proving to be more effective than medications or psychotherapy alone. Exercise helps reduce mental health disorder symptoms such as anxiety, depression, anger and psychomotor agitation. Researchers say exercise also helps to develop a more balanced and integrated sense of self for those with mental health disorders.
Source: University of Vermont
When it comes to inpatient treatment of a range of mental health and mood disorders — from anxiety and depression to schizophrenia, suicidality and acute psychotic episodes — a new study advocates for exercise, rather than psychotropic medications, as the primary prescription and method of intervention. Findings from the study reveal that physical exercise is so effective at alleviating patient symptoms that it could reduce patients' time admitted to acute facilities and reliance on psychotropic medications.
"The general attitude of medicine is that you treat the primary problem first, and exercise was never considered to be a life or death treatment option. Now that we know it's so effective, it can become as fundamental as pharmacological intervention," explains David Tomasi, a lecturer at the University of Vermont, psychotherapist and inpatient psychiatry group therapist at the University of Vermont Medical Center and lead researcher of the study.
Practitioners at inpatient psychiatric facilities — often crowded, acute settings in which patients experience severe distress and discomfort — typically prescribe psychotropic medications first, rather than natural remedies like physical exercise, to alleviate patients' symptoms such as anger, anxiety and depression. In fact, Tomasi estimates that only a handful of inpatient psychiatric hospitals in the U.S. provide psychotherapist-supported gym facilities exclusively for these patients. Instead, practitioners rely on classical psychotherapeutic and pharmacological frameworks to treat psychiatric symptoms, which they monitor to determine when a patient is ready to be discharged from the facility.
Tomasi, in collaboration with UVMMC's Sheri Gates and Emily Reyns, built a gym exclusively for roughly 100 patients in the medical center's inpatient psychiatry unit, and led and introduced 60-minute structured exercise and nutrition education programs into their treatment plans. The psychotherapists surveyed patients on their mood, self-esteem and self-image both before and after the exercise sessions to gauge the effects of exercise on psychiatric symptoms.
Patients reported lower levels of anger, anxiety and depression, higher self-esteem, and overall improved moods. Tomasi, Gates and Reyns found an average of 95 percent of patients reported that their moods improved after doing the structured exercises, while 63 percent of the patients reported being happy or very happy, as opposed to neutral, sad or very sad, after the exercises. An average of 91.8 of patients also reported that they were pleased with the way their bodies felt after doing the structured exercises.
"The fantastic thing about these results is that, if you're in a psychotic state, you're sort of limited with what you can do in terms of talk therapy or psychotherapy. It's hard to receive a message through talk therapy in that state, whereas with exercise, you can use your body and not rely on emotional intelligence alone" explains Tomasi.
"The priority is to provide more natural strategies for the treatment of mood disorders, depression and anxiety," he adds. "In practice, we hope that every psychiatric facility will include integrative therapies — in our case, exercise in particular — as the primary resource for their patients' psycho-physical wellbeing."
https://neurosciencenews.com/exercise-mental-health-14069/?fbclid=IwAR1gvz3bSZKF07qkjwB-nk19K0wbnJRLP0haO_Vc3AEWcPVqD1Oj7Q57G1E
Krysta Oehm, the founder of Mind Matters.
Mind Matters is a private practice dedicated to providing the best mental health, counseling, and wellness services to those in Lincoln and its surrounding communities.
hello@mindmatterslincoln.com
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Jelenlegi: EUROPE FROM A REGIONAL PERSPECTIVE? – WHAT CAN FLEXIBLE COOPERATION BRING TO EUROPE?
EUROPE FROM A REGIONAL PERSPECTIVE? – WHAT CAN FLEXIBLE COOPERATION BRING TO EUROPE?
The Institute for Foreign Affairs and Trade in cooperation with the State Secretariat for European Union Affairs of the Prime Minister's Office have the pleasure to invite you the event titled STRONGER TOGETHER MAPPING THE NEW PATH FOR THE EUROPEAN UNION (Fifth session): EUROPE FROM A REGIONAL PERSPECTIVE? – WHAT CAN FLEXIBLE COOPERATION BRING TO EUROPE?
to be held on 24 May (Wednesday) at 2.30 p.m.
Yet during the Brexit process, the dynamics among the Member States would possible greatly change, besides French and German axis will be shaped depending on their domestic election as well.
In a large-Member States-dominated European Union, Visegrad 4 countries could lose more from their advocacy's role, not to mention that United Kingdom was a significant ally for the Central-Eastern European countries in market-oriented economy and completing the Single Market.
On the other hand, another important ally for the V4 countries is traditionally Germany, whose political and economic interests partially, but decisively are bound to the CEE region. Besides Germany's interests, the four countries of the Visegrad cooperation's trade are directed mainly to Germany, making important partners for each other.
In a changing structure of the European Union, regional policies could be even more highlighted, including the future of the Cohesion and Structural Policies, especially from V4's point of view.
This roundtable discussion aims to find answers to the future of the European Union especially focusing on the regional cooperation with speakers from Germany and the V4 countries.


Really liked the last one but the other two were effective too, and very thoughtful.
Thanks for starting the thread - these are all good reads. I'm not a poet myself, but can always appreciate the imagery that poems conjure up.
I'll be on the lookout for some to add to this . . .


text: sudo apt-get -y install webshells

redpajama_set_name: RedPajamaGithub


Estonian startup Shipitwise makes it possible to send any type of personal goods from one door to another, without the hassle in between. The company recently raised 176k EUR with a 2.67m valuation.
Sending a huge wooden horse from London to Tallinn is a double trouble. You don't have to try it in order to realise how complicated the process can be. It's difficult to transport a human-size statue to a post office, and neither do you know how to package the item.
That's the exact problem that Shipitwise CEO, Aleksander Gansen faced in winter 2014 together with Olari Miiter and Sander Gansen. Back home, they decided to do something about it. That's how Shipitwise – a full-service shipping platform for small businesses and individuals – was born. The core founding team included Aleksander Gansen, Olari Miiter, Ragmar Saksing, Anna Medjanskaja, Helen Kokk, and Sander Gansen.
Shipitwise's business model is to provide door-to-door shipping for any types of goods – mobiles, guitars, beer samples, even a 200kg egg. They work with logistics partners to build up new chains of logistics consisting of different couriers, and cure the inefficiencies in worldwide logistics.
Shipping an item with Shipitwise is a child's play. You answer 3 simple questions: What? Where from? Where to? Soon, an offer will land in your inbox. As you agree to the price, your goods will be packaged and taken care of, without any extra input (e.g. pre-packaging) from your part. A few days later, the package will reach its destination.
Shipitwise has big plans to build a better logistics chain for everyone. One that's fully optimized and able to ship non-standardized items such as pets, motorbikes, and furniture. The company's already partnered up with many logistics chains and is looking to find business partners such as ski resorts and other international companies looking to ship items on a regular basis.
Having recently closed a 176 200 EUR financing round with Funderbeam with a 2.67M valuation, Shipitwise now has wind on their sails. According to Funderbeam, the estimated total serviceable market is €200m to provide shipping services to small businesses and individuals. The startup estimates their annual revenue to exceed €5m by the end of 2018.
Between building a new logistics platform and servicing an ever growing number of clients, there's also room for celebration – the 14-people team will open their new office on July 28.


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Tony Hawk's Pro Skater 5 Gets a Major Bug-Fixing Update
Travis Boswell
Published on November 28, 2015 | Updated on November 29th, 2015 at 06:12 am Travis Boswell
Home » News » Tony Hawk's Pro Skater 5 Gets a Major Bug-Fixing Update
Tony Hawk's Pro Skater 5 didn't stick the landing when it launched on consoles earlier this year. The game was plagued with bugs, awkward animations, long loading times and poor frame rates. Today, developers Robomodo have released a large patch to fix some of the game's issues.
The patch for Tony Hawk's Pro Skater 5 is 7.8 GB on PlayStation 4 and Xbox One. It contains some tweaks to commonly reported glitches as well as a major content addition, including some new guest skaters. Here are the full patch notes.
Additional levels have been added (Underground, Wild West)
Additional character campaigns have been added (Teenage Mutant Ninja Turtles, Tyler the Creator)
Lighting and visuals have been improved for several levels
Ability to regain Special Meter during a single combo has been added
Per-Mission Leaderboards have been added
Added option to "Return to Freeskate" while in a Mission or Mode
Added option to "Return to Freeskate" while search for the other player for Co-op Missions or Modes
Tutorial has been updated
Many Pro Skaters' Textures have been updated
Several Pro Skaters have recived Sponsor update
Create-a-Park Levels have been updated
Increased the total number of Stat points a Campaign can earn from 128 to 150
Several gameplay mechanics within the levels have been updated
Grinds were updated in several of the levels
The User Generated Content Rating System has been updated
Added Likes/Dislikes to the User Generated Levels
New Challenge UI Flow for matching during FreeSkate has been added
Leaderboard functionality was updated
Several multiplayer modes have been added and updated
Modes added include "Daily Grind," "Skate Hoarding" and "Manual Mayhem"
More options for user preference have been added including:
Double tapping grind button to activate slam
Opt in/out of Global Voice Chat
Several UI audio effects have been added
Misc – minor UI fixes and improvements have been made
The patch notes don't mention improvements to the game's frame rate, which was an issue that affected most of the game's players at launch. Still, this update does add bonus content for free and fix some of the game's common issues.
Will you try Tony Hawk's Pro Skater 5 with these updates or will you wait until more fixes are made? Let us know in the comments.
Cards Against Humanity Is Letting You Give Them $5 for Black Friday
Get into the Spirit Early with Azure Striker Gunvolt's New Christmas Mode
Related Topics:Bugs, fix, glitches, thps, tony hawk's pro skater 5, update


Can my vehicle tow the caravan?
Our Caravans have full details of the ATM (Weight of the van loaded with water, gas and belongings) in our caravan specifications, please contact your vehicle manufacturer or tow ball installer to determine if your vehicle is able to tow the caravan you would like to hire.
do i need electric brakes installed in my vehicle?
Our Caravans have electric brakes so you need to have an electric brake controller installed in your vehicle. If your vehicle does not have one then we have a portable electric brake controller which you can hire as an extra in some circumstances. Please contact us with your vehicle details.
what type of trailer plug is suitable to tow the vans?
Most of our vans have 12 pin plugs but we have adapters to suit other types of plugs like 7 pin round or 7 pin flat trailer plugs.
will the fridge in the caravan work while I'm driving?
You cannot run the fridge if you have either a 7 pin round or 7 pin flat plug in your vehicle. If you have a 12 pin trailer plug or Anderson plug installed in your vehicle then most of our vans have the facility for the fridge to work while driving. Please contact us for more details. You cannot run the fridge on gas while driving!
is the caravan supplied with gas?
We do not have an extra fee for gas but there will be at least one bottle full of gas. When you run out of gas you are required to refill the bottle at your own expense.
Yes, you can camp somewhere where there is no power or water and still have lights in the van (runs from battery charged by the solar panel on the roof) and hot water by gas (if applicable) and also the fridge will run on gas. Most of our vans have two water tanks ranging from 60 litres to 240 litres depending on which van you are hiring.
can i still hire if I have never towed a van before?
Some of our vans are only suitable for experienced towers with vehicles that are set up for towing. If you have had experience towing a trailer and reversing it then we have lighter and smaller vans which would be suitable for you. Please send us an enquiry.
what age do i have to be to hire a van?
The minimum age to hire a van is 25 years old with an open drivers licence and the maximum age is 75 years old.
can i take the caravan offroad?
No, our terms and conditions specifies no unsealed roads apart from a short distance up to 10 km from the main road to a camping area.
You will need your own bedding including pillows, sheets and blankets. Also your own towels, clothing, personal items and food. Our vans are fitted out with basic cooking and kitchen equipment only.
Yes, all of our caravans are fully insured. The bond payment that you give will be used as the excess on the insurance policy if it is necessary to make a claim.


The City of Del Mar offers several beautiful locations (indoor and outdoor venues) to host your special event. Our knowledgeable staff will serve as your guide to acquire all of the necessary documents needed to plan your event.
The Powerhouse includes cool, trellis-covered verandas with perfectly framed views of palms and surf that invites the visitor to sit, relax and absorb the beauty of Del Mar.
Powerhouse Park is the larger grassy area north of the railroad tracks. Powerhouse Park has a children's tot lot, two separate water fountains, a shower, and a public restroom. Bicycle racks are located at both park locations as well as handicapped access pathways for people with disabilities. There are two concrete walkways which lead down to the beach.
This beautiful and intimate park is on a cliff overlooking and adjacent to the Pacific Ocean. Seagrove Park is located at the very west end of 15th Street at Coast Boulevard.
Our facilities are extremely popular and booked well in advance. Please contact the Community Services team at cs@delmar.ca.us for more information.