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Vaccines and Antiviral Agents
Effect of Transplantation of Bone Marrow-Derived Mesenchymal Stem Cells on Mice Infected with Prions
Chang-Hyun Song, Osamu Honmou, Natsuo Ohsawa, Kiminori Nakamura, Hirofumi Hamada, Hidefumi Furuoka, Rie Hasebe, Motohiro Horiuchi
Chang-Hyun Song
1Laboratory of Prion Diseases, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo 060-0818
Osamu Honmou
2Departments of Neural Repair and Therapeutics
Natsuo Ohsawa
Kiminori Nakamura
3Molecular Medicine, Sapporo Medical University, South-1st, West-16th, Chuo-ku, Sapporo 060-8543
Hirofumi Hamada
Hidefumi Furuoka
4Department of Pathobiological Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro 080-8555, Japan
Rie Hasebe
Motohiro Horiuchi
For correspondence: horiuchi@vetmed.hokudai.ac.jp
Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to migrate to brain lesions in experimental models of ischemia, tumors, and neurodegenerative diseases and to ameliorate functional deficits. In this study, we attempted to evaluate the therapeutic potential of MSCs for treating prion diseases. Immortalized human MSCs (hMSCs) that express the LacZ gene were transplanted into the unilateral hippocampi or thalami of mice, and their distributions were monitored by the expression of β-galactosidase. In mice infected with prions, hMSCs transplanted at 120 days postinoculation (dpi) were detected on the contralateral side at 2 days after transplantation and existed there even at 3 weeks after transplantation. In contrast, few hMSCs were detected on the contralateral side for mock-infected mice. Interestingly, the migration of hMSCs appeared to correlate with the severity of neuropathological lesions, including disease-specific prion protein deposition. The hMSCs also migrated to a prion-specific lesion in the brain, even when intravenously injected. Although the effects were modest, intrahippocampal and intravenous transplantation of hMSCs prolonged the survival of mice infected with prions. A subpopulation of hMSCs in the brains of prion-infected mice produced various trophic factors and differentiated into cells of neuronal and glial lineages. These results suggest that MSCs have promise as a cellular vehicle for the delivery of therapeutic genes to brain lesions associated with prion diseases and, furthermore, that they may help to regenerate neuronal tissues damaged by prion propagation.
Prion diseases are fatal neurodegenerative disorders of humans and animals that are strongly associated with the conversion of normal prion protein (PrPC) to a disease-specific isoform of prion protein (PrPSc). Many inhibitors of PrPSc formation, investigated by using cells persistently infected with prions or an in vitro conversion reaction, have been reported as candidates for therapeutics (55). Several compounds or active/passive immunization with PrP showed a prophylactic effect when administered before, simultaneously with, or just after inoculation with prions (14, 19, 43, 50, 52). However, only a few compounds, such as amphotericin B and its derivative pentosan polysulfate, porphyrin derivatives, and certain amyloidophilic compounds, have been shown to be effective at prolonging survival when administered in the middle or late stage of prion infection (10, 13, 25, 27). In clinical trials, pentosan polysulfate seems to extend the survival of several patients beyond the mean but appears unable to arrest the progression of the disease (4, 45).
Recently, we demonstrated that intraventricular infusion of an anti-PrP monoclonal antibody (MAb) could antagonize disease progression even when initiated after clinical onset, although the distribution of the MAb was largely restricted to the hippocampus and thalamus (53). Thus, improved delivery of the MAb may enhance its beneficial effects. Additionally, because antagonizing PrPSc formation is not sufficient to restore degenerated lesions, it is necessary to pursue ways to regenerate degenerated neuronal tissues.
Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells of mesodermal origin. They can differentiate into mesenchymal lineages, such as osteoblasts, adipocytes, and myocytes (15, 41, 44). Remarkably, they also trans-differentiate into nonmesodermal cell types, including neuronal and glial lineages (48, 61). A number of studies have shown that MSCs migrate to damaged neuronal tissues following cerebral or systemic transplantation in animal models of ischemia (2, 7), spinal cord injury (23), brain tumors (37), Parkinson's diseases (21, 30), and Niemann-Pick disease (24). The introduction of MSCs in these model contexts resulted in functional recovery; however, the precise mechanisms for restoration remain to be elucidated (36, 38).
In this study, we investigated the therapeutic potential of MSCs for prion diseases. Although the use of mouse MSCs is suitable for studying the effect of MSCs on mice infected with prions, we used immortalized human MSCs (hMSCs) here because of the lack of appropriate methods for the isolation of mouse MSCs at the beginning of the study. In addition, hMSCs can be readily expanded in cell culture; their phenotypes remain similar to those of the primary human MSCs (26); and hMSCs are reported to avoid allogeneic rejection when they are transplanted into rat brains in a model of ischemia (38). Here we show that the hMSCs can migrate to neuropathological lesions in prion-infected mice and that their transplantation prolongs the survival of such mice. In addition, we also show that hMSCs that have migrated to prion-specific lesions secrete trophic factors and differentiate into cells of neuronal and glial lineages.
MSCs.A retroviral vector, Rx-LacZ-bsr, containing the expression units of the lacZ gene and a gene conferring blasticidin resistance, were generated as described elsewhere (63). The recombinant retrovirus was used to transfect human bone marrow-derived MSCs that had been immortalized with the human telomerase catalytic subunit gene (26), and hMSCs were selected in the presence of 10 μg/ml of blasticidin. hMSCs stably expressing β-galactosidase (β-Gal) were cultured with Dulbecco's modified Eagle medium (DMEM) (Sigma Chemical Co., St. Louis, MO) containing 10% fetal bovine serum under a humidified atmosphere of 5% CO2 at 37°C.
Mice and prion inoculation.Animal experiments were performed according to protocols approved by the Institutional Committee for Animal Experiments. Four-week-old female Jcl:ICR mice were purchased from CLEA Japan, and all mice were acclimatized for a week prior to use. Mice were intracerebrally inoculated with 20 μl of a 10% (wt/vol) brain homogenate from Jcl:ICR mice infected with the scrapie strain Obihiro or Chandler. Mice assigned to the mock-infected group were intracerebrally inoculated with 20 μl of a 10% (wt/vol) brain homogenate from age-matched uninfected Jcl:ICR mice. All mice were maintained on ad libitum feed and water with a 12-h light/dark cycle.
Transplantation of hMSCs.For transplantation of cells into the hippocampus or thalamus, mice were anesthetized by intramuscular injection of xylazine (10 mg/kg) and ketamine (50 mg/kg) and were placed on a stereotaxic apparatus (Narishige, Japan). After a linear scalp incision, burr holes were drilled to accommodate stereotaxic placement into the left hippocampus (2.0 mm caudal and 2.1 mm lateral to the bregma; depth, 2 mm) or thalamus (2.0 mm caudal and 1.5 mm lateral to the bregma; depth, 3.2 mm). hMSCs (1 × 105 cells in 2 μl phosphate-buffered saline [PBS]) were transplanted over a period of 15 min using a Hamilton syringe with a 31-gauge needle set in a micromanipulator. For transplantation of hMSCs via a peripheral route, 1 × 106 hMSCs were injected intravenously through the tail vein.
Immunohistochemistry.Mouse brains were frozen in Tissue-Tek OCT compound (Sakura, Japan), and cryosections (10 μm thick) were prepared as described elsewhere (53). Coronal sections were dried and fixed with ice-cold methanol for 15 min. A mouse anti-β-Gal MAb (catalog no. Z3783; Promega, Madison, WI) was conjugated with Alexa Fluor 488 by using a protein labeling kit (Molecular Probes, Eugene, OR) for the detection of hMSCs by direct staining. The following antibodies were used for the detection of various tropic factors: rabbit polyclonal antibodies against nerve growth factor (NGF) (Santa Cruz Biotechnology, Santa Cruz, CA), brain-derived neurotropic factor (BDNF) (Chemicon, Temecula, CA), neurotrophin 3 (NT3) (Chemicon), and neurotrophin 4/5 (NT4/5) (Santa Cruz Biotechnology); a rabbit MAb against vascular endothelial growth factor (VEGF) (clone EP1176Y; Abcam, Cambridge, MA); and a mouse MAb against ciliary neurotropic factor (CNTF) (clone A-11; Santa Cruz Biotechnology). As neuronal markers, we used a mouse MAb against microtubule-associated protein 2 (MAP2) (clone HM-2; Sigma Chemical Co.) for neurons, rabbit polyclonal antibodies (Dako, Denmark) against glial fibrillary acidic protein (GFAP) for astrocytes, and a mouse MAb against cyclic nucleotide phosphodiesterase (CNPase) (clone 11-5B; Chemicon) for oligodendrocytes. All sections were incubated with primary antibodies for 1 h at 37°C. To detect trophic factors and neural markers, the sections were subsequently incubated with an Alexa Fluor 546-conjugated anti-mouse antibody or an Alexa Fluor 555-conjugated anti-rabbit antibody (Molecular Probes) for 1 h at room temperature. After a wash with PBS, sections were mounted with Vectashield containing propidium iodide or 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) and were examined with a Nikon C1 laser confocal microscope. To exclude the possibility of nonspecific reactions between the Alexa Fluor 546-conjugated anti-mouse antibody and mouse tissues, we carried out the immunostaining without primary antibodies and confirmed that the level of nonspecific binding of the Alexa Fluor 546-conjugated anti-mouse antibody was negligible.
For the detection of PrPSc accumulation and astrocytosis, mouse brains were fixed in 10% formalin and embedded in paraffin. Coronal sections (thickness, 4 μm) were subjected to hematoxylin-eosin (HE) staining or immunohistochemistry as described elsewhere (17, 53).
Proliferation assay.To detect proliferating cells in the brain, 50 mg of bromodeoxyuridine (BrdUrd; Sigma Chemical Co.) per kg of body weight in PBS with 0.007 M NaOH was administered to mice intraperitoneally twice a day for a week. BrdUrd administration was initiated soon or 2 weeks after the transplantation of hMSCs into the hippocampus. Mice were sacrificed 24 h after the last BrdUrd administration, and the brains of these mice were then prepared for cryosectioning. The sections were pretreated with 2 M HCl for 30 min at 37°C, followed by a neutralization step with 0.1 M borate buffer for 15 min at room temperature. BrdUrd in nuclei was detected using a fluorescein isothiocyanate-conjugated anti-BrdUrd MAb (clone B33.1; Abcam).
Cell migration assay.Prion- or mock-infected mice were sacrificed at 120 days postinoculation (dpi), and the brains of these mice were homogenized to 20% in DMEM (Sigma Chemical Co.). The homogenates were centrifuged at 10,000 × g for 10 min at 4°C, and the resulting supernatants were filtered (pore size, 0.22 μm). The brain extracts were aliquoted and stored at −80°C until use. Migration of hMSCs to brain extracts was analyzed using a QCM 24-well colorimetric cell migration assay kit (Chemicon). The hMSCs (approximately 80% confluent) were starved by incubation with serum-free medium 1 day before use. Then hMSCs were harvested, and a cell suspension (5 × 104 cells) was added to the insert well. The lower chambers were supplied with serum-free DMEM containing 1.0 or 0.1% brain extract. Twenty-four hours after incubation, hMSCs that had migrated through the polycarbonate membrane were extracted, and the absorbance at 560 nm was measured according to the manufacturer's instructions.
Distribution of hMSCs to the neuropathological lesions of prion disease.To test if hMSCs migrate to brain lesions caused by prion infection, we transplanted hMSCs into the left hippocampi of prion- or mock-infected mice at 120 dpi and monitored the distribution of β-Gal-positive hMSCs at 2 days and 1, 2, and 3 weeks after transplantation. In mock-infected mice, hMSCs were detected in the left hippocampus (transplanted side), but few hMSCs were detected in the contralateral hippocampus at 2 days to 3 weeks after transplantation (Fig. 1a). In contrast, hMSCs were detected both on the transplanted sides and on the contralateral sides of the hippocampi of mice infected with strain Chandler even 2 days after transplantation. Thereafter, hMSCs were constantly observed on both sides of the hippocampus during the observation period (every week after transplantation up to 3 weeks [Fig. 1a]). For each experimental group, we examined three mice at each time point and confirmed the similar results. One to 3 weeks after transplantation, hMSCs were also detected in the cortices, cerebella, medullae oblongatae (see Fig. 3b), and thalami (data not shown) of mice infected with prions, where intense PrPSc accumulations and astrocytosis were observed (see Fig. S1 in the supplemental material). Ramified hMSCs were observed in the corpus callosum; their morphologies differed from those observed in the contralateral hippocampus (Fig. 1b).
Distribution of hMSCs to the neuropathological lesions of prion disease. At 120 dpi, hMSCs (1 × 105 cells) were transplanted into the left hippocampi or thalami of mice infected with strain Chandler and into those of age-matched mock-infected mice. Mice were sacrificed at 2 days or at 1, 2, or 3 weeks posttransplantation. Cryosections were stained with an Alexa Fluor 488-conjugated anti-β-Gal MAb (green) and counterstained with propidium iodide (red). (a) hMSCs in the hippocampus. Ipsilateral (left) and contralateral (right) hippocampi 2 days (2d) and 3 weeks (3w) posttransplantation are shown. The rightmost panels show magnified images of the regions boxed in the panels immediately to the left. Bar, 200 μm. (b) Morphology of hMSCs in the corpora callosa (CC) and contralateral hippocampi (Hc) of mice infected with strain Chandler at 2 days and 3 weeks posttransplantation. Bar, 20 μm. (c) hMSCs in the thalamus. Ipsilateral (left) and contralateral (right) thalami 3 weeks posttransplantation are shown. Bar, 200 μm. (d) Migration of hMSCs to the hypothalamus. (Top and center) Results of immunostaining for PrPSc and HE staining of the hypothalami of mice infected with strain Obihiro or Chandler are shown at 150 dpi. Bar, 100 μm. (Bottom) The hMSCs were detected in the hypothalamus (β-Gal) 3 weeks after transplantation into the left hippocampus. Bar, 200 μm.
Transplantation of hMSCs into the left thalamus led to similar results. In mock-infected mice, hMSCs remained in the transplanted area, and few hMSCs migrated to the contralateral thalamus or to other regions. In contrast, many hMSCs were detected in the contralateral thalami (Fig. 1c) and hippocampi (data not shown) of mice infected with prions by 3 weeks posttransplantation.
We noticed a striking difference in the neuropathology of the hypothalami of mice infected with strain Obihiro versus strain Chandler. Specifically, PrPSc accumulations, astrocytosis, and spongiosis in the hypothalami of mice infected with strain Obihiro are more severe than those for mice infected with strain Chandler (Fig. 1d; see also Fig. S1 in the supplemental material). Consistent with the severity of neuropathological lesions, more hMSCs migrated to the hypothalami of mice infected with strain Obihiro than to those of mice infected with strain Chandler (Fig. 1d; β-Gal). These results suggest that hMSCs are capable of migrating to brain lesions caused by prion infection.
Migration of hMSCs in response to prion-specific lesions.To confirm the migration of hMSCs to lesions where PrPSc accumulates, we transplanted hMSCs into the left hippocampi of mice infected with strain Chandler at 73, 100, and 120 dpi, and we analyzed their migration to the contralateral (right) side a week after transplantation. When hMSCs were transplanted at 73 dpi, many hMSCs were detected on the transplanted side but fewer hMSCs were detected in the contralateral hippocampus. In contrast, migration of hMSCs to the thalamus, where moderate PrPSc deposition had already occurred, was clearly observed (data not shown). In addition, more hMSCs were detected in the contralateral hippocampus when the transplantation was carried out at later time points (Fig. 2a). To compare the migration of hMSCs quantitatively, the total area of the hMSCs in the contralateral hippocampus (areas positive for β-Gal) was measured using the NIH Image J program. Compared to the migration of hMSCs to the contralateral hippocampus a week after the transplantation at 73 dpi, 2.8 and 4.1 times more hMSCs were detected when the transplantation was done at 100 and 120 dpi, respectively (Fig. 2b). We examined at least two mice from each experimental group and confirmed the consistency of the findings. Since PrPSc accumulation and astrocytosis in the hippocampi of mice infected with strain Chandler were first detected around 90 dpi and the levels of PrPSc accumulation and astrocytosis increased gradually thereafter (see Fig. S1 in the supplemental material), the migration of hMSCs to the contralateral hippocampus appeared to correlate with the severity of pathological changes.
Migration of hMSCs in response to prion-specific lesions. hMSCs (1 × 105 cells) were transplanted into the left hippocampi of mice infected with strain Chandler at 73, 100, or 120 dpi and into those of mock-infected mice at 120 dpi. One week after transplantation, the migration of hMSCs (green) to the contralateral hippocampus (right) was analyzed. The rightmost panels show magnified images of the regions boxed in the panels immediately to the left. Bar, 200 μm. (b) Quantification of hMSC migration. Areas positive for β-Gal in the contralateral hippocampus (regions of interest [ROI]) were measured using NIH Image J. The graph shows the levels of ROIs for mice transplanted at 100 and 120 dpi relative to that for a mouse transplanted at 73 dpi. (c) Migration of hMSCs to extracts from the brains of prion-infected mice. Insert wells containing an hMSC suspension were placed in the lower chambers, which contained 1.0 or 0.1% brain extract in serum-free DMEM, and were incubated for 24 h. The mean migration of hMSCs to brain extracts from mock-infected mice was arbitrarily set at 1, and the relative migration to brain extracts from mice infected with strain Chandler is indicated. Means and standard deviations from three independent assays (triplicate in each assay) are shown. *, P < 0.05.
Next, we analyzed the migration of hMSCs to brain extracts from prion-infected mice in vitro. Insert wells of a QCM 24-well colorimetric cell migration assay kit containing hMSCs were placed in the lower chambers, which contained 1.0 or 0.1% brain extract from prion- or mock-infected mice, and were incubated for 24 h. Cells that had migrated to the back sides (facing the lower chamber) of the membranes of the insert wells were quantified according to the supplier's instructions. Compared to the migration to brain extracts from mock-infected mice, approximately 2 and 1.5 times more hMSCs had migrated into the lower chambers containing 1.0% and 0.1% brain extracts from prion-infected mice, respectively (Fig. 2c). This suggests that chemoattractive factors that promote the migration of hMSCs are produced by the lesion caused by prion infection.
Migration of hMSCs into the brain after intravenous transplantation.MSCs have been reported to migrate to a site of brain injury even when they are introduced via intravenous injection (37, 38). To test if a similar phenomenon could be observed in prion-infected mice, hMSCs were intravenously inoculated into mice infected with strain Chandler or into mock-infected mice at 120 dpi. In mice infected with strain Chandler, hMSCs were observed in the hippocampus and thalamus even at 2 days after transplantation (data not shown). The cells showed a symmetrical distribution and appeared to increase in number in these tissues by 3 weeks posttransplantation (Fig. 3a; see also Fig. S2 in the supplemental material). In contrast, few MSCs were detected in the brains of mock-infected mice, demonstrating that the hMSCs migrated to the brain lesions caused by prion propagation. At each time point, we examined two mice for each experimental group and confirmed the consistency of the findings. The hMSCs were also well distributed in other brain regions, including the cerebral cortex, cerebellum, and medulla oblongata (Fig. 3b); however, consistent with the results shown in Fig. 3b, they did not migrate well to the hypothalamus (see Fig. S2 in the supplemental material). There was no difference in the area of hMSC distribution following intravenous versus intrahippocampal transplantation except at the corpus callosum. More hMSCs were observed in the corpus callosum after transplantation into the hippocampus than after intravenous injection, suggesting that cells migrate to the contralateral side through the corpus callosum after intrahippocampal transplantation (2).
Migration of hMSCs into the brain after intravenous transplantation. hMSCs (1 × 106 cells) were intravenously injected into mice infected with strain Chandler and into mock-infected mice at 120 dpi. Three weeks after injection, cryosections were prepared and stained with an anti-β-Gal MAb. (a) Presence of hMSCs in the left and right hippocampus (Hc) and thalamus (Tl) 3 weeks postinjection. The rightmost panels show magnified images of the regions boxed in the panels immediately to the left. Bar, 200 μm. (b) Distribution of hMSCs transplanted into the left hippocampus (Intra-Hc) or introduced via intravenous injection (IV). At 120 dpi, the hMSCs were transplanted into the left hippocampi (1 × 105 cells) or injected via the tail veins (1 × 106 cells) of mice infected with strain Chandler. The presence of hMSCs in the corpus callosum (CC), cortex (Cx), cerebellum (Cb), and medulla oblongata (MO) 3 weeks posttransplantation is shown. These brain regions are boxed on the images taken from Paxinos and Franklin and reprinted with permission of the publisher (39). Bars, 200 μm.
Effects of transplantation of hMSCs on the survival of prion-infected mice.To examine whether the transplantation of hMSCs can ameliorate prion diseases, hMSCs were transplanted into the left hippocampi of mice infected with strain Chandler at 90 dpi. Figure 4 shows the survival curve for these mice. The intrahippocampal transplantation of hMSCs prolonged the survival of mice infected with strain Chandler (158 ± 6 days; n = 5) over that of the nontransplanted control group (150 ± 2 days; n = 15). Thus, hMSC transplantation prolonged mean survival by only 8 days, but this difference was statistically significant (P < 0.01 by the log rank test). We also transplanted hMSCs via the tail veins of mice infected with strain Chandler at 120 dpi. The transplantation of hMSCs via this peripheral route appeared not to be effective for nearly half of the mice; however, the remaining mice survived beyond the mean survival of the nontransplanted control group. Although the mean survival of hMSC-transplanted mice (154 ± 6 days; n = 7) was only a little longer than that of the control group, this difference was also significant (P < 0.05 by the log rank test). The fact that survival time was prolonged even when hMSCs were transplanted via a peripheral route after clinical onset (120 dpi) suggests that hMSCs have therapeutic potential for prion diseases. Since both intracerebral and intravenous transplantation of hMSCs prolonged the survival of prion-infected mice, we further analyzed the transplanted hMSCs.
Prolongation of survival of prion-infected mice by transplantation of hMSCs. For intracerebral (i.c.) transplantation, hMSCs (1 × 105 cells) were transplanted into the left hippocampi of mice infected with strain Chandler at 90 dpi (n = 5). For intravenous (i.v.) transplantation, 1 × 106 hMSCs were injected via the tail vein at 120 dpi (n = 7). The hMSC-transplanted and nontransplanted control (Cont) (n = 15) mice were observed until they reached the terminal stage of the disease. The graph shows survival curves.
Proliferation of hMSCs after transplantation.To examine the proliferation state of hMSCs that had migrated to lesions, BrdUrd was systemically administered after the transplantation of hMSCs to the left hippocampus. Three weeks posttransplantation, many BrdUrd-labeled nuclei were detected in the contralateral hippocampi and thalami of mice infected with strain Chandler (see Fig. S3 in the supplemental material), where many hMSCs had migrated (Fig. 1a). In contrast, few BrdUrd-labeled nuclei were detected in the contralateral hippocampi and thalami of mock-infected mice (see Fig. S3 in the supplemental material), although a few cells with BrdUrd-labeled nuclei were detected on the transplanted side (data not shown). BrdUrd-labeled cells were also observed in the cerebella and medullae oblongatae of mice infected with strain Chandler (data not shown). We examined two mice for each experimental group and confirmed the similar results. These results suggest that transplanted hMSCs are capable of proliferating in the microenvironment caused by prion propagation.
Expression of trophic factors in hMSCs.It is known that MSCs migrate to a site of injury in the brain and produce various trophic factors (8, 29). To ask if something similar happens in the case of prion disease, we next assayed the production of trophic factors in our model system. hMSCs were transplanted into the left thalamus at 120 dpi, and one mouse in each group was sacrificed and examined for the production of human trophic factors at 2 days and 1 and 3 weeks after transplantation. Immunoreactivities for human BDNF, NT3, and VEGF in the ipsilateral thalami of mice infected with strain Chandler became more intense from 2 days to 3 weeks posttransplantation. In contrast, no obvious increases, but rather decreases, in the signals of these trophic factors were observed for mock-infected mice (Fig. 5). Additionally, the expression of NGF, NT4/5, and CNTF was also upregulated (data not shown). These results suggest that hMSCs produce a variety of trophic factors in response to the neurodegeneration caused by prion infection.
Expression of trophic factors in hMSCs. hMSCs (1 × 105 cells) were transplanted into the left thalami of mice infected with strain Chandler and into those of mock-infected mice at 120 dpi. Two days (2d), 1 week (1w), and 3 weeks (3w) posttransplantation, cryosections were prepared and doubly stained with an anti-β-Gal MAb, for hMSCs (green), and an antibody against a human trophic factor (BDNF, NT3, or VEGF) (red). Nuclei were counterstained with DAPI (blue). Bar, 20 μm.
Interestingly, only subpopulations of the hMSCs appeared to be positive for NT3 and BDNF. In addition, parts of the regions positive for these factors did not overlap with β-Gal staining. Since these antibodies are specific to human trophic factors and will not react with the corresponding mouse trophic factors, the presence of NT3 and BDNF in areas negative for β-Gal may represent trophic factors secreted from hMSCs and bound to mouse brain cells.
Differentiation of hMSCs.MSCs are known to differentiate into cells of neuronal and glial lineages in vivo and in vitro (11, 48, 61). We next asked if hMSCs differentiate into neuronal and glial cells in response to the lesions of prion diseases. At 3 weeks after transplantation into the thalamus, hMSCs positive for the neurodifferentiation marker MAP2, GFAP, or CNPase were detected in the brains of mice infected with strain Chandler (Fig. 6), although a relatively small number of hMSCs were positive for each marker. In contrast, no hMSCs positive for MAP2, GFAP, or CNPase were observed in the brains of mock-infected mice (data not shown), suggesting that neuronal and glial differentiation of hMSCs occurs in response to the neurodegeneration caused by prion infection. The GFAP-positive hMSCs were detected in the hippocampus, thalamus, and medulla oblongata. In contrast, MAP2-positive hMSCs were detected primarily in the hippocampus, cortex, and cerebellum, and CNPase-positive hMSCs were detected mainly in the cortex (data not shown).
Differentiation of hMSCs into cells of neuronal and glial lineages. At 120 dpi, hMSCs (1 × 105 cells) were transplanted into the left thalami of mice infected with strain Chandler. Three weeks posttransplantation, cryosections were prepared and doubly stained with an anti-β-Gal MAb, for hMSCs (green), and an antibody against a marker for neurons (MAP2), astrocytes (GFAP), or oligodendrocytes (CNPase) (red). Nuclei were counterstained with DAPI (blue). Arrows indicate hMSCs positive for each marker protein. The brain region for MAP2 and GFAP is the ipsilateral hippocampus, while that for CNPase is the cerebral cortex. Bar, 20 μm.
The primary purpose of this study was to evaluate the potential of MSCs for treating prion diseases in a mouse model. For this purpose, the use of mouse MSCs would have been desirable; however, no appropriate method for the isolation of mouse MSCs from bone marrow had been established by the beginning of this study. On the other hand, it was well known that MSCs avoid allogeneic rejection (47). Thus, we adopted hMSCs to the mouse model and showed that hMSCs responded to the neuropathological lesions of prion diseases and may have therapeutic potential. Transplantation of MSCs is known to ameliorate neurological dysfunctions in experimental models (7, 23, 24, 30, 37). In clinical trials in which autologous MSCs are transplanted into patients with multiple system atrophy (28) or amyotrophic lateral sclerosis (33), or into patients who have suffered a stroke (3), there is some evidence of a beneficial effect without any adverse effects. How the introduction of MSCs leads to improved outcomes is not yet clear. However, the transplanted MSCs are known to migrate and home to a site of injury. Moreover, in this context, MSCs are expected to restore injured tissues by protecting neural tissues via secretion of various trophic factors (8), promotion of angiogenesis (20), stimulation of the proliferation and differentiation of endogenous neural stem cells (36), integration into tissues by differentiation or cell fusion (1), and modulation of the local immune response (64).
Here we showed that the level of migration of MSCs to the contralateral side of the mouse brain correlates with the degree of PrPSc accumulation and the severity of histopathological changes in the prion-infected brain (Fig. 1d and 2). Although no antigen-specific humoral or cellular immune response is provoked in prion diseases, microglial activation and astrocytosis are prominent features of the diseases. Indeed, the expression of inflammatory cytokines and chemokines, which are likely to be produced in glial cells, is upregulated in the middle to late stage of prion infection (5, 6). In addition, brain extracts from prion-infected mice promoted chemotaxis of hMSCs in vitro. These results suggest that certain factors produced in the brains of prion-infected mice act as chemoattractive factors for hMSCs, although it is not clear whether the effects of mouse factors on human MSCs are as efficient as those on homologous MSCs. Monocyte chemoattractive protein 1 (known as CCL2), interleukin-8 (IL-8), and macrophage inflammatory protein 1α (known as CCL3) have been reported to enhance the migration of MSCs to ischemic brain tissue (57, 58). It was recently reported that an interaction between stromal cell-derived factor 1 (known as CXCL12), produced in ischemic brain lesions, and CXCR4, expressed on MSCs, plays an important role in the migration of MSCs (59). Because the inflammatory response and glial activation are common events in many neurological disorders, it is conceivable that a similar mechanism may account to some extent for the migration of MSCs to brain lesions associated with prion disease. For instance, we have found that the expression of CCL3 is upregulated in the thalami and medullae of prion-infected mice (unpublished observation). Experiments are under way to identify the chemoattractive factors by use of an in vitro chemotaxis assay.
MSCs have been reported to migrate to a site of brain injury even when intravenously injected (37, 38). Consistent with previous reports, our results showed here that hMSCs transplanted via intravenous injection travel to areas of brain lesions in prion-infected mice (Fig. 3). In prion diseases, although impairment of the blood-brain barrier (BBB) was observed in the cerebellum (56), no significant impairment of the BBB was observed in the hippocampus or cerebral cortex at the time of clinical onset or even at a later stage (42, 56). Thus, passive translocation of MSCs to the brain parenchyma through a disrupted BBB seems unlikely. Instead, active transendothelial migration of MSCs, similar to the recruitment of leukocytes and monocytes from the bloodstream to an inflammation site, is expected to be involved in the engraftment of MSCs transplanted via intravenous injection. Vascular cell adhesion molecule 1 and p-selectin expressed on the endothelium are important for the adhesion of MSCs to the endothelium via the β1 integrin VLA-4 (16, 46, 51). Proinflammatory cytokines, such as tumor necrosis factor alpha and IL-1β, upregulate the expression of adhesion molecules in endothelial cells (32). Indeed, tumor necrosis factor alpha and IL-1β are upregulated during the course of prion disease (6, 49), suggesting that these cytokines induce the adherence of MSCs to the endothelium and their subsequent transendothelial migration to the brain lesions. Understanding how the migration of MSCs to brain lesions affected by prion diseases is regulated, and further elucidation of the mechanisms underlying the tropism of MSCs, may provide new insight into the engraftment of MSCs as it relates to the progression and possible treatment of neurodegenerative diseases.
The ability of MSCs to migrate to a site of injury has been given particular attention, because it suggests that these cells can act as a vehicle for gene therapy in addition to aiding in the regeneration of degenerated tissues. Indeed, MSCs expressing genes of therapeutic potential showed a greater positive effect on functional recovery than unmodified MSCs (24, 37, 38). Transgenic expression of anti-PrP antibodies (22), a fusion protein between PrPC and the Fc portion of immunoglobulin (PrP-Fc) (34), and dominant-negative PrP mutants (40) inhibited prion propagation. In addition, expression of anti-PrP Fab fragments and PrP-Fc in the brain by virus vectors has been reported to antagonize prion propagation in the brain (18, 62). Furthermore, intraventricular infusion of an anti-PrP MAb slowed the formation of neuropathological lesions and prolonged the survival of prion-infected mice even when the MAb was administered at clinical onset (53). However, large macromolecules, such as immunoglobulins, are expected to be delivered to the lesions inefficiently. Indeed, the distribution of MAbs was restricted primarily to the hippocampus and thalamus, even when the MAbs were infused directly into the lateral ventricle (53). Therefore, the observation that hMSCs target and home to brain lesions associated with prion diseases indicates the potential utility of hMSCs as a cellular vehicle for the delivery of therapeutic genes to brain lesions.
We showed here that microenvironments in the brain lesions associated with prion disease stimulate MSCs to produce various trophic factors: BDNF, NGF, VEGF, and others. These trophic factors are reported to have antiapoptotic effects, to promote nerve fiber regeneration, and to induce endogenous cell proliferation and angiogenesis in injured brains (9, 29, 31). It remains to be elucidated whether the prolonged survival of prion-infected mice by hMSC transplantation can be attributed to the secretion of trophic factors from hMSCs. Although hMSCs alone may have the ameliorative effect to some extent, they could not arrest the disease progression caused by prion propagation. Similarly, it has been shown that antagonizing prion propagation can slow disease progression but cannot ameliorate functional deficits (13, 25, 53). Thus, it seems possible that the combination of MSCs with inhibitors of prion propagation would have a synergistic effect in the treatment of prion diseases.
Replacement of damaged neurons with differentiated MSCs or their fusion with MSCs after MSC transplantation is an attractive possible route to the restoration of neurological functions (11, 54, 60). In this study, we showed that small populations of MSCs were differentiated into cells expressing neuronal, astrocyte, or oligodendrocyte markers. Because only a small portion of transplanted MSCs differentiated into a neuronal and a glial lineage in vivo, it seems unlikely that the prolongation of survival could be attributed directly to differentiation. However, induction of neuronal differentiation in vitro prior to transplantation improves functional outcomes in a rat model of Parkinson's disease and cerebral infarction (12, 35). Therefore, appropriate preconditioning may enhance the effects of trans-differentiation on the restoration of degenerated tissues.
To our knowledge, this is the first report showing the therapeutic potential of MSCs for prion diseases. We showed that hMSCs home to the lesions, produce trophic factors, and differentiate into neuronal and glial lineage cells in response to the microenvironment in the lesions. As we are already aware, not only inhibition of prion propagation but also regeneration of damaged nervous tissues is required for recovery from prion diseases. Thus, a combination of genes possessing antiprion effects with MSCs, which can deliver therapeutic genes and have potential for neuroprotection and the regeneration of damaged tissues, may provide an effective treatment for prion diseases.
This work was supported by the Regional New Consortium R&D Projects of the Ministry of Economy, Trade and Industry of Japan; by a grant from the global COE Program (F-001), a Grant-in-Aid for Science Research (A) (grant 18208026), and a Grant-in-Aid for Exploratory Research (grant 20658070) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; and by a grant from the Ministry of Health, Labor and Welfare of Japan (grant 20330701). This work was also partly supported by a grant for Strategic Cooperation to Control Emerging and Reemerging Infections and by the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Received 23 January 2009.
Accepted 9 March 2009.
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Journal of Virology May 2009, 83 (11) 5918-5927; DOI: 10.1128/JVI.00165-09
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Until the 2015 constitutional reform, the investigation and accusation commission (Comisión de Investigación y Acusación) of the House of Representatives was recommending to the plenary the indictment of the President, Constitutional Court justices, Supreme Court justices, Superior Council of the Judiciary members, Council of State justices and the Attorney General. These senior officials of the State were said to benefit from a "constitutional fuero", first enshrined in the 1886 Constitution and kept by the 1991 Constitution, although cabinet ministers lost their special constitutional protection in 1991.
The House's accusation commission had been very criticized over the years, said to grant immunity to any senior official accused of corruption or wrongdoing. Between 1886 and 2014, only one of Colombia's 40 presidents, Gustavo Rojas Pinilla, was charged and sentenced by Congress in 1959 (after the end of his term), and that ruling was overturned by the Supreme Court seven years later. Between 1992 and 2014, the accusations commission received a total of 3,496 complaints, of which 56% were closed and 44% still pending. No case resulted in impeachment, and in fact only one case ever made its way to the floor of the House, that of President Ernesto Samper for the Proceso 8000.
The government's constitutional reform in 2015 stripped the accusations commission of most of its power and restricted the existing fuero constitucional to the President. In its stead, the 2015 reform created the Comisión de Aforados, which will investigate and indict the aforementioned judges and Attorney General, even if they may have ceased to exercise their functions. The commission will be made up of five members elected by a joint session of Congress for individual eight-year terms, from lists sent by the Council of Judicial Government and elaborated through a public competition. The eligibility, ineligibility and incompatibility rules for the commission will be the same as for members of the Supreme Court of Justice.
In the case of investigations for unworthiness to serve for misconduct, the new commission shall present its charges, when necessary, to the House of Representatives, which will only be able to suspend or remove the accused from office. The House decision can be appealed to the Senate, which holds the final word. In such cases, the commission will have 60 days to present an accusation and 30 days to decide, In the case of investigations for other crimes, the commission shall present charges to the Supreme Court of Justice for further prosecution.
The new commission will be formed after a transitional period of one year, during which the accusations commission retains its original responsibilities.
^ "Constitución Política de 1991 (Artículo 176)". Secretaría General del Senado.
^ a b "Colombia 1991 (rev. 2013)". Constitute Project.
^ "Constitución Política de 1991 (Artículo 262)". Secretaría General del Senado.
^ "Decreto por el cual se deroga el decreto 2788 de 2013 y se publica el número de Representantes a la Cámara que se elegirán por circunscripciones territoriales y circunscripciones especiales el 9 de marzo de 2014". Decree No. 2897 of 16 December 2013 (PDF) (in Spanish).
^ a b "Los penosos números de la Comisión de Acusaciones". Semana. 5 March 2015.


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Effects of isolation on their development & health
> Ramkumar92 > A caregivers guide regarding the impacts of social isolation on the elderly.
The most pervasive impacts of social isolation on health
Social withdrawal: An initially adaptive behavior that becomes maladaptive when expressed excessively. Abazyan et al., 2010.
Researching the Effects of Social Isolation. Covid-19 Measures, Loneliness and Depression. Life in isolation isn't that much fun.
And while some people are coping with the current circumstances and the social distancing imposed as a result of the Covid-19 measures, others are having a real hard time. Regardless of contemporary technologies, social distancing can lead to loneliness and sadness. Combined with worries about the current state of affairs and the infection rate, this loneliness could result in a higher risk of depression. Though social distancing in Singapore isn't as severe as it has gotten to be in other parts of the world, there are still some concerns about the long-term mental health impact from the situation caused by the pandemic.
The Toll of Social Distancing and Loneliness Regardless of the circumstances, there will always be people experiencing feelings of loneliness. Coronavirus is affecting the world at a time when more people than ever before are living on their own. Chronic isolation and serotonin: how social distancing can affect the brain. Isolation and mental health: Signs, impact, and how to cope. Although physical distancing is an effective way to slow the spread of many viruses, it can also lead to feelings of loneliness and isolation, especially in older adults.
To help support the mental well-being of you and your loved ones during this difficult time, visit our dedicated hub to discover more research-backed information. People are distancing themselves from others to help prevent the spread of SARS-CoV-2, which causes COVID-19. However, isolation has a significant impact on health, contributing to conditions such as depression, anxiety, and dementia.
For this reason, the World Health Organization (WHO) encourage people to continue socializing at a distance, such as by phone or via online platforms. This article discusses how isolation and mental health affect each other. According to the WHO, social support networks can have a significant positive effect on health. The risks of social isolation.
Overview CE credits: 1 Learning objectives: After reading this article, CE candidates will be able to: Identify the effects of social isolation and loneliness on physical, mental and cognitive health.Explore how loneliness differs from social isolation.Discuss evidence-based interventions for combating loneliness.
For more information on earning CE credit for this article, go to www.apa.org/ed/ce/resources/ce-corner.aspx. According to a 2018 national survey by Cigna, loneliness levels have reached an all-time high, with nearly half of 20,000 U.S. adults reporting they sometimes or always feel alone. Such numbers are alarming because of the health and mental health risks associated with loneliness. "There is robust evidence that social isolation and loneliness significantly increase risk for premature mortality, and the magnitude of the risk exceeds that of many leading health indicators," HoltLunstad says. Who is most likely? Loneliness and Social Isolation Linked to Serious Health Conditions. Social isolation, loneliness in older people pose health risks.
Human beings are social creatures.
Our connection to others enables us to survive and thrive. The Costs of Social Isolation: Loneliness and COVID-19 - Psychiatry Advisor. The dizzying spread of coronavirus disease 2019 (COVID-19), as well as the ensuing social distancing restrictions and public health interventions, have contributed to an epidemic of another sort: loneliness.
The most at-risk population, adults aged over 80 years, face estimated fatality risks from the virus of 9.3% compared with just 0.2% in the general population.1 Under ordinary circumstances, where individuals have control over the number and quality of their interactions, the costs of perceived social isolation are already high. Perceived Social Isolation and Cognition. How Social Isolation Affects the Brain. Daisy Fancourt was at her home in Surrey in southeast England when the UK government formally announced a nationwide lockdown.
Speaking in a televised address on March 23, UK Prime Minister Boris Johnson laid out a suite of measures designed to curb the spread of COVID-19, including closing public spaces and requiring people to stay home except for exercise and essential tasks. For Fancourt, an epidemiologist at University College London (UCL), the announcement meant more than just a change to her daily life.
It was the starting gun for a huge study, weeks in the planning, that would investigate the effects of enforced isolation and other pandemic-associated changes on the British public. We're a social species. We really need others to survive. Social isolation results in memory loss in later life. Social isolation contributes to memory loss in older age, research by LSE's Care and Evaluation Centre has shown for the first time.
The paper, published in Journals of Gerontology, found that both men and women were affected, but in different ways. Men with high levels of social isolation experienced subsequent memory decline. For women it was not the overall level of lack of social contact, but rather increases in isolation that were important. Researchers studied data from 6123 women and 5110 men over the age of 50, from the English Longitudinal Study of Ageing (ELSA), over six two year intervals from 2002. By using a statistical model to analyze the changes in levels of social isolation and memory loss over time, they were able to establish that isolation precedes memory loss—rather than the other way around. Dr. For women it was more relative—when isolation increased by two points, women's memory declined, on average by 18 percent over the next two years. Dr. Social isolation impacts late-life socio-cognitive decline in APP/PS1 mice.
Social Isolation and Cognitive Function in Later Life: A Systematic Review and Meta-Analysis.
Related: Does elderly's social isolation matter? - Effects of social isolation on elderly's development - A Guide to the Effects of Social Isolation on Elderly - PSY207 Developmental Psychology - Social Isolation - the Effects on Elderly's Development. - Effects of social isolation on elderly's development - Effects of social isolation on elderly's development - Caregiver's guide to Effects of Social Isolation on Elderly's Development - The effects of social isolation on elderly development - Caregiver's Guide: Effects of Social Isolation on Elderly's Development. - Resource centre for caregivers of the elderly - Effects of social isolation on elderly's development - Caregiver's resource on the effects of social isolation on elderly development - Effects of social isolation on elderly's development - Caregivers Sharing Session: Effects of Social Isolation on Elderly's Development - Guide to Elderly's Development and Social Isolation - Importance, reasons and strategies older adults should stay active in order to maintain their cognitive abilities - Physical Effects - Effects of Social Isolation on Elderly - Effects of Social Isolation on Elderly's Development (PSY207e) - Effects of Social Isolation in Elderlies - Effects of social isolation on elderly's development: Caregivers' edition - Social Isolation and its effects on elderly's development - The effects of social isolation on elderly's development - Effects of social isolation on elderly's development - Cognitive Effects - Social isolation and its influence on elderly's development - Social isolation and elderly's holistic development: Relationships, effects, and ways to combat it - Effects of social isolation on elderly's development: Briefing for caregivers - The Singapore Experience on Social Isolation - Effects of social isolation on elderly's development - A caregivers guide regarding the impacts of social isolation on the elderly. - The risks of social isolation - Loneliness and Social Isolation Linked to Serious Health Conditions - Social isolation, loneliness in older people pose health risks - How Social Isolation Affects the Brain - Education - Enterprise


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Attach to an enemy. Limit 1 per enemy. Attached enemy gets -1 Threat. Response: When attached enemy is destroyed, reduce each player's threat by the attached enemy's printed Threat.
Reduce the cost to play Keen as Lances by 1 for each card worth no victory points in the victory display. Action: Add Keen as Lances to the victory display. Then, choose one: add 2 resources to a hero's resource pool, draw 3 cards, or reduce your threat by 4.
Attach to a player's threat dial. Limit 1 per player. Forced: When you would be eliminated by reaching your threat elimination level, instead discard Favor of the Valar and reduce your threat to 5 less than your threat elimination level. You are not eliminated.
Action: Raise your threat by 1 to look at the top card of the encounter deck. Then, reduce your threat by X, where X is the threat of that card.
Attach to a location. Limit 1 per location. Response: When attached location leaves play as an explored location, the first player reduces his threat by 3.
Response: After Frodo commits to a quest, spend 1 resource from his resource pool to ready another questing unique character. If you quest successfully, reduce your threat by 1.
Play only if each of your heroes is a Hobbit. Action: Reduce your threat by 4.
Setup: Attach to a hero in play. Action: Add Three Golden Hairs to the victory display and remove it from the campaign pool, to lower each player's threat by 3. Then, each player draws 3 cards.
Play only if you control the Ring-bearer. Planning Action: Add Brace of Coneys to the victory display to (choose 2): heal up to 2 damage from a hero, add 2 resources to a hero's pool, draw 2 cards, or reduce a player's threat by 2.


everyone seems to be trying to distract you from something over there.
gloomy rows of graves but something there seems to haunt your mind.
to the ground. Crying, Why'd she leave?
who's that person down the hall?
staring at the sullen wall.
with a doleful person's brand?


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Sprinkler malfunction damages gym floors at middle school
by Thomas Vogel January 24, 2017 February 8, 2021
A fire sprinkler at George Washington Middle School mysteriously activated Sunday Jan. 8 around 3:30 p.m., damaging a custodial office and ruining the wood floor in the school's smaller gymnasium.
The weekend timing caused the incident to go unnoticed longer than if school was in session but the fire department, which receives a notification once the sprinkler system is activated, was quick to respond, according to Superintendent Carol Baker. She estimated repairs to the 3,750-square foot gym will cost around $250,000 but added the school's insurance policy will cover the bill entirely.
"Within 24 hours the floor was warped," Baker said. "Unfortunately, you can't replace half a gym floor."
Tyco SimplexGrinnel, the fire sprinkler company, sent employees to inspect the school's sprinklers last week. They were unable to determine the cause but told school officials they didn't expect any more issues.
Baker said a replacement floor, as well as some repairs to the subfloor, should be finished within the next few weeks and, for now, the school is making the best of the reduced space by reworking class locations. George Washington also has a large gym and big events, such as basketball games, which will not be interrupted.
"It affects our kids a little bit," Baker said of the repairs. "We have to crunch some classes down a bit."
Installation of the new subfloor will take place over a weekend to avoid disrupting classes as much as possible.
No damage to the sprinkler was found and no fire or smoke was reported.
"This is why school districts carry insurance policies," Baker said.


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WEDDING WEDNESDAY - Nellie M Shackford marries Capt William Ellingwood Bunton six days before he heads off to war (Blog 101)
We learn from this marriage record that the chaplain of the 14 N. H. Regiment married William E Bunten and Nellie M Shackford on October 12, 1862, six days before his regiment mustered out.
Nellie (Mary Ellen) Shackford was the daughter of William Shackford and Mary Ellen Dame and probably met Capt Bunten in Concord where he had arrived to set up a recruiting station after graduating from Dartmouth. Because a few gentlemen involved in recruitment wanted the role of Captain, an election was held and William was chosen September 22 to become the captain of Company H, 14th Regiment Infantry. He was formally enlisted on October 2, 1862.
Nellie probably watched the departure of her new husband as the 14th Regiment marched to and boarded the train.
Their departure is described by Frances Buffum, author of A Memorial of The Great Rebellion; Being a History of the Fourteenth Regiment New-Hampshire Volunteers, Covering Its Three Years of Service, with Original Sketches of Army Life. 1862-1865 : "
"At seven o'clock, on the morning of Saturday, October 18, the last line was formed in Camp Cheshire. The great battalion stretched out, in a magnificent formation; and "by the right flank, file right," the column moved out to the music, first of drum corp, then of the band. It was an interesting spectacle - to see a thousand soldiers, but yesterday in civil life, arching away from their homes and the dearest objects of existence, to venture every hardship that a colossal struggle, covering a thousand battle-fields, involved, with a risk of life that amounted to a certainty of death for a large number."
Nellie's husband served with the 14th for a year, mustered out and then served as a clerk for the US Quartermaster in Washington DC 1863-1868 before becoming a teacher and then a principal for multiple schools in Marblehead, Massachusetts, Waltham, Massachusetts, Woodstock, Connecticut and Kingston, New York. He died in 1897 in Kingston, Ulster, New York where he was working as the Principal of Ulster Academy.
Buffum Francis Henry, A Memorial of The Great Rebellion; Being a History of the Fourteenth Regiment New-Hampshire Volunteers, Covering Its Three Years of Service, with Original Sketches of Army Life. 1862-1865. (Boston: Franklin Press: Rand, Avery, & Company, 1882), page 29, 30,; digital images, Google eBooks (http://books.google.com : accessed 1 January 2014
The General Catalogue and a Brief History of Kimball Union Academy, Plainfield, (Meriden P. O.) N. H. Also a Biographical Sketch of Hon. Daniel Kimball, Its Founder 1813-1880 (Claremont, N. H.: The Claremont Manufacturing Co, 1880), page 191; digital images, Google Books (http://books.google.com : accessed 1 January 2013
E-mail, Lee Ann Lawler, [rclaw@mc.net], 17 Feb 1999
New Hampshire, New Hampshire, Marriage Records, 1637-1947, , William Bunton m Nellie M Shackford, 12 October 1862; digital records, Family Search (http://familysearch.org : accessed 13 June 2013).
"United States Civil War Soldiers Index," database, FamilySearch (http://familysearch.org : accessed 1 January 2013), William E. Bunten
Copywrite 2014, Joanne Shackford Parkes


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The British government has extended an emergency visa program for truck drivers as fuel shortages frustrate motorists lining up at empty pumps
By PAN PYLAS Associated PressOctober 2, 2021, 4:01 PM• 4 min readShare to FacebookShare to TwitterEmail this article The Associated PressAn employee cleans fuel pumps at a closed petrol station in London, Thursday, Sept. 30, 2021. Many gas stations around Britain have shut down in the past five days after running out of fuel, a situation exacerbated by panic buying among some motorists. Long lines of vehicles formed at pumps that were still open, blocking roads and causing traffic chaos. Some drivers have had to endure hourslong waits to fill up. (AP Photo/Frank Augstein)
LONDON — The British government has extended an emergency visa program for truck drivers as fuel shortages showed few signs of abating Saturday, particularly in London and the southeast of England.
In an announcement late Friday, the Conservative government said temporary visas for nearly 5,000 foreign truck drivers it hopes to recruit would run into 2022 instead of expiring on Christmas Eve as originally planned.
The short duration of the program announced last week drew widespread criticism for not being attractive enough to entice foreign drivers.
The government said 300 fuel drivers would be able to come to the U.K. from overseas "immediately" and stay through March. Some 4,700 other visas for foreign food truck drivers will last from late October to the end of February.
In another move intended to ease the pressure at Britain's pumps, around 200 military personnel, including 100 drivers, will be deployed beginning Monday to help to relieve fuel supply shortages that have caused empty pumps and long lines at filling stations.
The government says the situation is already improving.
"U.K. forecourt stock levels are trending up, deliveries of fuel to forecourts are above normal levels, and fuel demand is stabilizing," Business Secretary Kwasi Kwarteng said. "It's important to stress there is no national shortage of fuel in the U.K., and people should continue to buy fuel as normal."
However, the Petrol Retailers Association, which represents independent filling stations, warned that fuel supplies remain a problem and could be getting worse in places.
"In London and the southeast, and possibly parts of eastern England, if anything, it had got worse," the group's chairman, Brian Madderson, told BBC radio.
Madderson welcomed the deployment of military drivers next week but warned it would have a limited impact.
"This isn't going to be the major panacea," he said. "It's a large help, but in terms of the volume, they are not going to be able to carry that much."
Opposition parties are urging Prime Minister Boris Johnson to recall parliament next week to address the wider situation of labor shortages and disruptions to supply chains. In recent months, many companies have reported shortages, including fast-food chains KFC, McDonald's and Nando's. Supermarket shelves have also looked barren, and fears have grown that they will not be stocked as usual in the run-up to Christmas.
In an attempt to stave off a shortage of Christmas turkeys, the government also announced that 5,500 foreign poultry workers will be allowed into the U.K. beginning in late October and can stay until the end of the year.
Johnson's pro-Brexit government is keen to downplay talk that the driver shortage is a result of Britain's departure from the European Union.
However, when the country left the economic orbit of the EU at the start of this year, one of the bloc's main tenets ceased to apply — the freedom of people to move within the EU to find work. With Brexit, tens of thousands of truck drivers left the U.K. to go back to their homes in the EU, further pressuring an industry already facing long-term staffing issues.
The coronavirus pandemic has exacerbated the problem, prompting thousands of EU drivers to return to their home countries. The U.K.'s tough lockdowns also led to difficulties in training and testing new domestic drivers to replace those who left.
In addition, the pandemic accelerated the number of British truck drivers choosing to retire. Relatively low pay, changes in the way that truck drivers' incomes are taxed and a paucity of facilities — toilets and showers, for example — have also diminished the job's appeal to younger workers.
Follow all AP stories on post-Brexit developments at https://apnews.com/hub/Brexit.
Follow all AP stories on the coronavirus pandemic at https://apnews.com/hub/coronavirus-pandemic.
Tags: AP, England, London, uk
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Ah.. now some of the nerves are settled.
Yaar frankly, Fakhar played himself into pressure.
Babar just oozes class, such a good player to watch.
Partnership is 52 off 59. Didnt look like they are playing hard. They have played very smartly. Please continue the same.
How do you lot reckon the match will turn out at this stage?
Lol what a garbage team selected. Once again - Rayudu , DK, Dhoni not gonna last against Pakistani bowling. Pakistan just need to score 220 and watch us capitulate. If you can't beat this indian team 3 times, you should just quit cricket.
Should send in Asif next.
Brilliant running by the batsmen.
No it's not lol. We haven't even selected a deserving squad. We are playing 2 wicketkeeper s lmao.
As much as I usually dislike Pakistan playing in the UAE, the atmosphere has been great. UAE is no doubt a far superior venue to Bangladesh and Sri Lanka, both venues pretty much killed the previous few Asia Cups and World T20s.
Hard to tell, this pitch will get slower as the game progresses. 270-280 is a good score.
What is India's first eleven then?
Yar let them wail man, these are the same people who will mock us. Awein dramay kar rahay hain. Kohli keya chala gaya, village team ban gayi. Thats their strategy to save face if they lose. Idiots.
once again Indians have stopped caring about this match.
A little reduction in run rate in the last two overs... need to keep the momentum going.
No one says it, but Dhoni's keeping has declined sharply in recent years.
That was a rare drop!
Malik dropped by Dhoni, can;t score a run, can;t take catches, He's there just for showbaazi and acting as a pseudo captain and hog credit when we do well.
This atmosphere was only because it's an India/Pak match and in the first game because Bangladesh were playing in Dubai for the first time.
How many times has Malik tried to dab to third man against pace and gotten OUT?
Kohli ho to game mein energy hotee he..
We are not only missing Kohli but Sehwag, Sachin, Dravid, Ganguly, Yuvi, Zaheer and RP Singh also.
Both batting at 72 SR and decently set.
Should look to play smart and have a RR of 6 until they reach 50+.
Really enjoying watching Malik bat. Especially against spin, he's the best.
Babar just being the GOAT that he is.
Pak. need to pick up the rate a bit. Still under 4 and plenty of batting left.
Yes, but even Bangladesh and Sri Lanka pulled crowds for India vs Pakistan. The venues themselves simply have a very boring vibe.
I think this might be FZ's first duck in ODI cricket. What a time to get it.
Grandpa Dhoni drops a sitter. Plus grandpa is a useless batsman. He will cost us dearly at the world cup. But grandpa will shamelessly refuse to retire until 2023 World cup.
India are just too liberal when Malik is batting, lol.
I thought you meant crowds. Love the stadiums in the UAE. it's the pitches and lack of crowds that I don't like.
Need to get going but now.
Fantastic shot and once again good running.
Wonder if @GoUgandaCares got a leave from his work.
Dhoni has just dropped the match. Congrats ppers.
BVB bhai, appka honsla hai wesay. My respect for you just grows.
Yar hes a legend, have some sharm.
If he has any shame left he would retire and make way for pant. I'll respect whoever I want to. Put a sock on it.


4 days of uninterrupted music and festivities: that's what the Red Bull Music Academy Weekender is all about. The brief was to create a vibrant visual style, linking the RBMA Weekender to Dubai, the first city to host an event of its kind. The style of music you like defines you as an individual, becoming a representation, an expression, or an extension of your identity.
We approached the project from this angle, using the Guthra (a scarf and icon of Arab identity) as a graphical device. By mixing the Guthra patterns with simple geometric shapes, we created different bandana styles used to cover people faces, as is the traditional style.
The visual communicated the Weekender movement with personalised geometric figures that changed with the music, colour, and personality traits of each 'Weekender'.


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