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Islet transplantation has considerable potential as a cure for diabetes. However, the difficulties that arise from inflammation and the immunological rejection of transplants must be addressed for islet transplantation to be successful. Alpha 1-antitrypsin (AAT) inhibits the damage on β cells caused by inflammatory reactions and promotes β-cell survival and proliferation. This protein also induces specific immune tolerance to transplanted β cells. However, whether the expression of AAT in β cells themselves could eliminate or decrease immunological rejection of transplants is not clear. Therefore, we established a β cell line (NIT-hAAT) that stably expresses human AAT. Interestingly, in a cytotoxic T lymphocyte (CTL)-killing assay, we found that hAAT reduced apoptosis and inflammatory cytokine production in NIT-1 cells and regulated the Th1/Th2 cytokine balance in vitro. In vivo transplantation of NIT-hAAT cells into mice with diabetes showed hAAT inhibited immunological rejection for a short period of time and increased the survival of transplanted β cells. This study demonstrated that hAAT generated remarkable immunoprotective and immunoregulation effects in a model of β cell islet transplantation for diabetes model.
Citation: Wang Y, Yan H-J, Zhou S-Y, Wang Y-S, Qi H, Deng C-Y, et al. (2014) The Immunoregulation Effect of Alpha 1-Antitrypsin Prolong β-Cell Survival after Transplantation. PLoS ONE 9(4): e94548. https://doi.org/10.1371/journal.pone.0094548
Editor: Massimo Pietropaolo, University of Michigan Medical School, United States of America
Received: August 21, 2013; Accepted: March 18, 2014; Published: April 10, 2014
Copyright: © 2014 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National 973 Program of China (No. 2007CB516811), The National Natural Science Foundation of China (No. 30772042 and No. 81270857), and The Science and Technology Project of Shenzhen (No. JCYJ20120618153743791, GJHZ20120618153934353). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Type 1 diabetes results from autoimmune destruction of insulin-producing pancreatic β cells, and is characterized by hyperglycaemia due to reduced insulin secretion. Apoptosis is the main mode of pancreatic β cell death in the development of diabetes [1]. Since the implementation of the Edmonton protocol in 2000 [2], islet transplantation has become one of the most promising options to cure Type 1 diabetes. Islet transplantation has been evaluated as a procedure that could enable patients to regain physiological glucose control, yet the immunologic tolerance protocol that accompanies this procedure excludes diabetogenic corticosteroids, resulting in the exposure of grafted cells to an unopposed inflammatory environment [3]. Similar to the process of islet injury during transplantation, the autoimmune response that is directed toward islets in a type 1 diabetic individual appears to overlap with several immune processes that occur during allograft rejection [4]. Autoimmunity and immunological rejection are the two major side effects resulting from islet transplantation. Thus, there is increasing motivation to identify an islet-protective antiinflammatory immune-modulating agent that is safe for use.
Alpha 1-antitrypsin (AAT) is a key serine protease inhibitor [5]. The protein has anti-inflammatory, anti-leukocyte migratory, anti-thrombotic, and anti-apoptotic effects [6]–[9], and also exerts cytoprotective effects upon islets in vitro [8], [10]. As expression of AAT sharply rises in response to inflammation, AAT may function to limit the duration and magnitude of inflammation [11]. Furthermore, short-term AAT treatment restores euglycemia and self-tolerance to islets in overtly T1D nonobese diabetic (NOD) mice [12]. In addition, AAT promotes insulin secretion of islet cells in mice [13]. Therefore, we hypothesized that a transplant of β cells expressing AAT would have a low chance of immunological rejection due to the anti-inflammatory and anti-apoptotic functions of AAT. Essentially, these AAT-expressing cells could induce specific immune tolerance to the transplant.
In the present study, pDsRed–hAAT was transfected into NIT-1 cells, and a stable cell line was generated. By conducting cytotoxic T lymphocyte (CTL)-killing assays and cell transplantations into diabetic mice, we found that hAAT expression reduced immunological rejection of the inflammatory reactions against the β-cell transplantation. Our results indicate that hAAT can exhibit an immune protective effect on transplanted β cells.
Materials and Methods
Plasmid construction
The pBS–RSV–hAAT plasmid was donated by Prof. Andre Lieber (University of Washington, U.S.A). The region encoding hAAT was amplified and subcloned into the eukaryotic expression vector pDsRed-N111 (donated by Prof. Lu Zhigang, Peking University Shenzhen Graduate School, China) to generate the pDsRed–hAAT vector.
Construction of the stable hAAT-NIT-1 cell line
NIT-1 cells (a kind gift from Prof. Li Fangping, Sun Yat-Sen University, China), an insulin-producing insulinoma cell line, derived from non-obese diabetic (NOD) mice prone to autoimmune diabetes [14] were used as a cell model system. These cells were expanded in 24-well tissue culture plates in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO, USA) with 10% FCS (Gibco, CA, USA). Liposome 2000 (Invitrogen, Carlsbad, CA, USA) was utilized to transfect pDsRed–hAAT or pDsRed mock-vector into the NIT-1 cells respectively. Seventy-two hours after the transfection, G418 (350 µg/mL, Sigma) was added to the medium for selection. Low-dose G418 (175 µg/mL) was subsequently applied to generate cells stably expressing the construct. The 10th and the 40th generation of the NIT-hAAT cell line were collected and lysed. Western blotting was conducted to validate that hAAT was expressed at the protein level. The hAAT levels from cultured supernatant samples (72 h) at passage 10 and passage 40 (5×105 cells/mL)were determined by Human Alpha 1-Antitrypsin ELISA(Immunology Consultants Laboratory, Newberg,OR, USA).
BALB/c mice (8 weeks old) were provided by the Experimental Animal Center of Guangdong Province. The mice were kept in the SPF Animal Laboratory (Experimental Animal Center of the Second Clinical Medicine College, Jinan University). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Second Clinical Medical College (Shenzhen People's Hospital), Jinan University. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Second Clinical Medical College, Jinan University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
Collection of lymphocytes
Sixty BALB/c mice, divided into three groups with 20 animals per group, were injected intraperitoneally with 5×106 NIT-1 cells, 5×106 NIT-vector cells and 5×106 NIT-hAAT cells respectively at the first day and the tenth day. Twenty BALB/c mice were injected intraperitoneally with 5% Physiological saline (control group). At day 20, all mice from each group were sacrificed. The spleens were mashed to release single cells, and then red blood cells were lysed. The cells were later washed twice with RPMI 1640. The cells were filtered through a 200-mesh nylon net, and the lymphocytes concentration was adjusted to 1×106 cells/mL.
One-way mixed lymphocyte multiplication experiment
The lymphocytes from each group described above were utilized as the responder cells in a mixed lymphocyte reaction (MLR). NIT-1, NIT-Vector and NIT-hAAT cells were plated on a 12-well culture plate. When the cell concentration reached approximately 5×105 cells/well, 1 mL of 30 mg/mL mitomycin C was added to each well, and the cells were incubated in the dark for 30 min. These cells were later used as the stimulator cells for the MLR. The ratios between the responder cells and the stimulator cells were 5∶1, 10∶1, 20∶1, and 50∶1. The cells were cultured in 5% CO2 at 37°C for 72 h. The cell counting kit-8 (CCK-8, Dojindo,Japan) was utilized according to the manufacturer's instructions to determine the number of cells in each well. The stimulation index (SI) of the responder cells was calculated according to the following equation: SI = (OD value of experimental well – OD value of blank well)/(OD value of negative control well – OD value of blank well). Three holes were analyzed for each group based on the negative control and blank wells.
Measurement of inflammatory cytokines
After seven days of mixing the culture at the ratio of 10∶1 of 5×106 lymphocytes and mitomycin-based NIT-1,NIT-vector and NIT–hAAT cells in each group, the cultured supernatant was obtained and preserved at −80°C for later analysis. Mouse IFN-γ and IL-4 ELISA kits (Neobioscience, China) were utilized to determine the cytokine content of the supernatants. The absorbance was measured at 450 nm, and the cytokine levels were calculated based on the standard curve. Three different supernatants were analyzed for each sample.
Determination of apoptosis
MLR with a ratio of 10∶1 of lymphocytes to NIT-1, NIT-vector and NIT–hAAT cells were allowed to proceed for seven days, and then the lymphocytes were removed. The NIT-1, NIT-vector and NIT–hAAT cells were collected and digested with pancreatin without EDTA. The cells concentration was adjusted to 1×106 cells/mL, and 100 µL of the cells suspension were subsequently mixed with 5 µL of Annexin V-FITC and 1 µL of PI. The cells were incubated in the dark for 30 min and were washed twice with PBS. The number of cells undergoing apoptosis was determined by flow cytometry(BD Biosciences,Franklin Lakes, NJ, USA).
Real-time quantitative PCR for CRP, IL-1β, IL-6 and IL10 mRNA
MLR with a ratio of 10∶1 of lymphocytes to NIT-1,NIT-vector and NIT-hAAT cells were allowed to proceed for seven days, and mRNA was extracted from the lymphocytes and reverse transcription was carried out with 1 µg of RNA. Real-time quantitative PCR was performed as previously described [15]. The following sequence-specific primers were used: CRP Forward: 5′-GGATTGTAGAGTTCTGGATTG-3′; CRP Reverse: 5′-TCACCCTGTGCTTTATAGTTC-3′; IL-1β Forward: 5′-GACGTTCCCATTAGACAACTGC-3′; IL-1β Reverse: 5′-GGTATAGATTCTTTCCTTTGAGGC-3′; IL-6 Forward: 5′-GACTTCCATCCAGTTGCCTTCT-3′; IL-6 Reverse: 5′-CCAGTTTGGTAGCATCCATCAT-3′; IL-10 Forward: 5′-TCCTTGGAAAACCTCGTTTG-3; IL-10 Reverse: 5′-CTTCAATTGCTTCCCAAGGA-3′; β-actin Forward: 5′-TACACCCAGGAAAGACAGCAACC-3′; and β-actin Reverse: 5′-CTCCTCGAAGACCTTCTCACAACCA-3′.
Induction of diabetes in mice
Thirty BALB/c mice were injected intraperitoneally with a 2% STZ solution (170 mg/kg). The mice were later fed a high-sugar and high-fat diet. Diabetes was monitored by measurement of blood glucose concentration from the tail vein using a blood glucose device (Roche Accu Check III, Roche, Basel, Switzerland). Mice with non-fasting blood glucose 300 mg/dl for 3 consecutive days were considered as onset of diabetes.
β Cell transplantation
There was no difference of immune response between NIT-1 and NIT-vector cells in vitro, therefore, the diabetic BALB/c mice were randomly divided into three groups: (1) NIT-hAAT Group (n = 15), in which 1.5×107 NIT-hAAT cells were injected under the left kidney capsule; (2) NIT-1 Group (n = 15), in which 1.5×107 NIT-1 cells were injected under the left kidney capsule; and (3) Diabetes group (n = 15), where 0.1 mL of PBS was injected under the left kidney capsule. In addition, 10 normal BALB/c mice were used as control group.
β-cell functionality after the transplantation
The tails of the BALB/c mice were cut for blood collection at 4:00 p.m. every day after the transplantation. A MediSense Optium blood glucometer and the paper disk method were utilized to test the non-fasting blood glucose concentration for 36 consecutive days. At the 30th day, the left kidney was removed. A glucose tolerance test was conducted on the 14th and 28th days after transplantation. Briefly, the mice that underwent fasting for 6 h to 8 h were given an intraperitoneal injection of 2 g/kg glucose in saline. Blood was collected at 0, 15, 30, 60, 90 and 120 min to measure the blood sugar concentration. Additionally, on the 7th, 14th, 21st, and 28th days after the transplantation, 0.5 mL of blood was collected from each mouse periorbitally. The serum was preserved at −20°C after centrifugal separation. ELISA (Mercodia, Sweden) were performed to determine the insulin and C-peptide levels in the serum.
Pathological observation
At 14 and 28 days after the NIT-hAAT and NIT-1 cells were transplanted, three mice from each group were selected, and their left kidneys were collected. The tissue was fixed with 10% formaldehyde and embedded in paraffin. The tissue was cut into 5-µm sections and stained with HE. Under a 400× optical microscope, ten fields of visions were randomly chosen to record the number of infiltrating white blood cells in transplantation site.
Fluorescence immunocytochemistry
At 28 days after the NIT-hAAT and NIT-1 cells were transplanted. The left kidneys tissue was collected, fixed with 10% formaldehyde and embedded in paraffin. The tissue was cut into 5-µm sections and incubated with rat anti-mouse insulin antibody (Zymed Laboratories), rabbit anti-human AAT(Abcam, Cambridge,UK) antibody overnight at 4°C. Tissue were further incubated with FITC-labeled goat anti-rat polyclonal antibody (Jackson ImmunoResearch) and PE-labeled donkey anti-rabbit (Rockland) at 37°C for 1 hour. Tissues were washed with 1×PBS three times. The tissues were observed under fluorescent microscope (ECLIPSE TE2000-U,Nike, Germany).
Generation of the NIT–hAAT cell line
In vitro expression of hAAT was examined by western blot. Untransfected NIT-1 cells and NIT-vector cells did not express hAAT; however, NIT-hAAT cells did express hAAT. No significant difference exists in protein expression between cells at passage 10 and passage 40. NIT-hAAT cells maintained stable expression of hAAT in vitro (Figure S1). The hAAT levels from cultured 72 h supernatant concentration at passage 10 and passage 40 (5×105 cells/mL) were 355.33±22.79 µg/mL and 340.33±20.55 µg/mL, respectively.
Immunoregulation effects of hAAT
One-way mixed lymphocyte multiplication experiment.
NIT-1,NIT-vector and NIT-hAAT cells were treated with mitomycin C and used as stimulator cells in an MLR. Proliferation of lymphocytes co-cultured with the NIT-hAAT cells was greatly inhibited compared with those cultured with NIT-1 cells or NIT-vector (P<0.05). The inhibitory effects were enhanced as the ratio of NIT–hAAT to lymphocytes increased, demonstrating a dose–dependent effect (Figure 1). At the ratio of 10∶1, the effects presented a significant difference (P<0.01). The lymphocyte amplification ability of the NIT-1 group and NIT-vector group were remarkably better (P<0.01, P<0.05) compared with that of the control group and the NIT–hAAT group.
The effects of hAAT on a β islet cell line were examined via an extended CTL assay in vitro (n = 3). BALB/c mice were immunized twice with NIT-1 or NIT-vector or NIT–hAAT cells, and the splenocytes were collected. These splenoctyes were used as responder cells in a 7 day MLR, while NIT-1 or NIT-vector or NIT–hAAT cells pretreated with mitomycin C were used as stimulator cells. An in vitro CTL assay was later performed using NIT-1 or NIT-vector or NIT–hAAT cells as target cells. Target cells cultured with unprimed splenocytes were utilized as control cells. The stimulation index (SI) of NIT-1 primed lymphocytes co-cultured with NIT-1 or NIT-vector or NIT–hAAT cells (A).The stimulation index (SI) of NIT-vector primed lymphocytes co-cultured with NIT-1 or NIT-vector or NIT–hAAT cells (B), The stimulation index (SI) of NIT-hAAT primed lymphocytes co-cultured with NIT-1 or NIT-vector or NIT–hAAT cells (C). *P<0.05.
Th1 and Th2 cytokine levels.
The Th1 cytokine (IFN-γ) and Th2 cytokine (IL-4) levels in the supernatants of the lymphocyte-NIT-1 cell, NIT-vector, NIT-hAAT co-cultures were determined by ELISA. The results demonstrate that the levels of IFN-γ were significantly reduced and the levels of IL-4 were significantly elevated in cultures containing NIT-hAAT cells compared with those containing NIT-1 cells and NIT-vector cells (P<0.05). However, NIT-1-primed lymphocytes or NIT-vector-primed lymphocytes co-cultured with NIT-1 cells or NIT-vector did not exhibit significantly different levels of cytokines compared with NIT-hAAT-primed lymphocytes co-cultured with NIT-hAAT cells. (P>0.05; Table 1).
Expression of CRP, IL-1β,IL-6 and IL-10 mRNA.
The IL-10 mRNA levels in the splenocytes collected from mice that received a NIT–hAAT cell transplant, as detected by real-time quantitative PCR, were significantly enhanced compared with that from mice that received NIT-1 cells or NIT-vector (P<0.05). However, the expressions of CRP,IL-1β and IL-6 mRNA of the inflammatory factors were decreased (P<0.05) (Figure 2). CRP, IL-1β, IL-6 and IL-10 mRNA levels were not significantly different between the NIT-1 and NIT-vector groups, indicating that hAAT inhibits the expression of the inflammatory gene through in vivo adjustment of related cytokine levels.
Mice were immunized twice with NIT-1 or NIT-vector or NIT–hAAT cells, and the splenocytes were collected and co-cultured for 7 days with NIT-1 or NIT-vector or NIT–hAAT cells pretreated with mitomycin C. CTL assay was subsequently performed using NIT-1 or NIT-vector or NIT-hAAT cells as target cells in vitro. CRP mRNA levels(A), IL-1β mRNA levels (B), IL-6 mRNA levels (C), IL-10 mRNA levels (D). *P<0.05,**P<0.01.
Detection of apoptosis.
Seven days after the mixed culturing of NIT-1-primed or NIT-vector-primed or NIT–hAAT -primed lymphocytes and NIT-1 or NIT-vector or NIT–hAAT cells treated with mitomycin, flow cytometry was performed to analyze the mixed culture cells. The apoptosis of the NIT-1 cells and NIT-vector cells in the mixed culture containing NIT–1-primed or NIT-vector primed lymphocytes showed a significant increase (37.6±1.1%,32.5±2.1%)(Figure 3A,B). The apoptosis of the NIT-1 cells and NIT-vector cells in the mixed culture containing NIT–hAAT-primed lymphocytes was lower (12.9±0.4%, 14.3%±1.1%)(Figure 3C,D) (P<0.01). The apoptosis of the NIT–hAAT cells in the mixed culture containing NIT-1-primed and NIT-vector primed lymphocytes was lower (10.1±0.9%, 9.5±0.9%)(Figure 3E,F). However, the apoptosis of the NIT–hAAT cells was the lowest in the NIT–hAAT -primed lymphocytes (8.6%±1.3%) (Figure 3G)(P<0.01).
Apoptosis of NIT-1 and NIT-vector cells (A) mediated by NIT-1 primed lymphocytesp; Apoptosis of NIT-1 and NIT-vector cells (B) mediated by NIT-vector-primed lymphocytes; Apoptosis of NIT-1(C),NIT-vector(D) mediated by NIT-hAAT primed lymphocytes. Apoptosis of the NIT–hAAT cells mediated by NIT-1-primed(E) and NIT-vector (F)primed lymphocytes;.Apoptosis of the NIT–hAAT cells(G)mediated by NIT–hAAT -primed lymphocytes.
Pancreatic functionality after the β-cell transplantation
Blood glucose levels and weight changes of the diabetic mice.
NIT-1 or NIT–hAAT cells were transplanted to under the left kidney capsule of the diabetic mice. The blood sugar levels of the mice in each group were tested every three days (Figure 4A). On the 3rd day after transplantation, the blood sugar levels of the NIT-1 and NIT–hAAT groups mice decreased sharply. The blood sugar concentration of the mice in the NIT-1 group increased at the 15th day, the blood sugar concentration increased to 300 mg/dL. At the 30th day after the removal of the left kidney, the blood sugar concentration did not exhibit obvious changes. The mice in the NIT–hAAT group maintained a normal blood sugar level up to 30 days. On the 30th day after transplantation, upon the removal of the left kidney, the blood sugar level of the mice increased significantly and reached the standard level of diabetes. It showed that the NIT–hAAT cells could secrete insulin. The weights of the mice in the NIT–hAAT group steadily increased over time, while the weights of the mice in the NIT-1 group and the untreated diabetic group gradually decreased (Figure 4B).
NIT-1 or NIT–hAAT cells were transplanted into diabetic mice. After transplantation, the blood sugar level, body weight, glucose tolerance, and insulin and C-peptide concentrations were monitored. (A) Blood sugar levels of the recipients (n = 10). (B) Changes in the body weight of the recipients (n = 10). (C,D) GTT at 14 d (c) or 28 d (d) after transplantation (n = 5). (E) In vivo glucose-stimulated insulin secretion in the recipients (n = 5). (F) In vivo C-peptide levels in the recipients (n = 5). *P-value <0.05,**P-value <0.01 compared with the diabetic group.
NIT-1 group at 14 d and 28 d after transplantation. Fourteen days after the transplantation, only a small number of transplanted NIT-1 cells remained in the kidneys. No transplanted NIT-1 cells were observed after 28 d, showing that the cells occurred fibrosis(A, B). NIT–hAAT group at 14 d and 28 d after transplantation, Fourteen days after transplantation, most of transplanted NIT–hAAT cells were still visible in renal capsule. After 28 days, there were no significant changes in the numbers of infiltrating inflammatory cells and a number of the transplanted NIT–hAAT cells were still present in the renal capsule(C,D). (scale bars, 200 µm). Immunostaining was performed at 28 d after transplantation NIT-hAAT cells under the kidney capsule. After rehydration, sections were incubated with anti-insulin and anti-hAAT antibodies. Insulin and hAAT at 28 d after transplantation are shown(E,F,G). (scale bars, 50 µm).
Sugar tolerance test.
At 14 days after transplantation, the mice were given an injection of glucose, and blood sugar levels were tested at several time points after the injection. Fifteen minutes after the glucose injection, the blood sugar levels of the mice in the NIT–hAAT group decreased quickly, whereas that of the NIT-1 and the diabetic group declined slightly (Figure 4C). When the glucose tolerance test was conducted 28 d after the transplantation, the mice in the NIT–hAAT group controlled their blood sugar levels, as we noted on the 14th day after transplantation. However, the blood sugar levels in the NIT-1 and untreated diabetic groups mice did not decrease significantly in the times tested after the glucose injection (Figure 4D). These data indicated that the NIT-hAAT transplantation resulted in the presence of living β cells that were capable of regulating blood sugar levels.
Insulin and C-peptide analysis.
The insulin and C-peptide levels in the serum were increased three days after transplantation from the NIT-hAAT group mice and remained elevated throughout the periods tested. These levels were significantly higher compared with those from the untreated diabetic group (P<0.01). Three days after the transplantation, the insulin and C-peptide levels in the serum from the NIT-1 group mice were increased but stabilized after 14 days(P<0.05). Thereafter, the insulin and C-peptide levels gradually declined. On the 21st day after transplantation, no significant difference was observed in the diabetic group (P>0.05). (Figure 4E and 4F).
At 14 d and 28 d after cell transplantation, the left kidneys from mice in each group were stained with HE, and the pathological changes in the transplant sites were analyzed (Figure 5A–D). Fourteen days after the transplantation, only a small number of transplanted NIT-1cells remained in the renal capsule. No transplanted cells were observed after 28 d, showing that the cells occurring fibrosis. In contrast, fourteen days after transplantation, most of transplanted cells were still visible in renal capsule from mice in the NIT–hAAT group. There were significantly fewer infiltrating inflammatory cells in the NIT-hAAT group (14±2.9 cells) compared with the NIT-1 group (31±10.98 cells; P<0.01). After 28 days, there were no significant changes in the numbers of infiltrating inflammatory cells in the NIT-hAAT group (16±3.6 cells), and a number of the transplanted cells were still present in the renal capsule. Immunostaining was performed at 28 d after transplanted NIT–hAAT cells under the kidney capsule. The expression of transplanted NIT–hAAT group like insulin (green) and hAAT (red) in tissuse was investigated by immunofluorescence(Figure 5E,F,G). The NIT-1 group was not found to have positive expression of these proteins (data not shown).
NIT-1 is a NOD-derived β cell line and maintains the characteristics of primary β cells, such as the capability for secretion of insulin, NIT-1 cells are a well-validated cell model for the study of type 1 diabetes [14]. Recently, various studies suggested that AAT possesses qualities that are favorable for islet graft survival [8], [13], [16]. In particular, our group studied the improvement attributes of AAT for autoimmunity and allograft rejection in beta cell transplantation.In present study, It is the first in the field to explore AAT-expressing beta-cells as targets of an immune response. NIT-1 cells were transfected with a vector encoding hAAT to generate a NIT–hAAT stable cell line. pDsRed–hAAT is a 55-kb plasmid that holds the entire human AAT sequence, including exons and introns, and the ability to self-replicate without integrating into the genome [17]. The expression of hAAT was verified by western blot. The NIT–hAAT cell line continued proliferating and maintained hAAT secretion up to the 40th passage of the cells.
In Cytotoxic T lymphocyte (CTL) experiment in vitro, NIT–hAAT,NIT-vector and NIT-1 cells were co-cultured with CTLs from the same mice that had previously been treated with NIT–hAAT, NIT-vector and NIT-1 cells. The CTLs that were co-cultured with NIT–hAAT cells proliferated significantly less than those co-cultured with NIT-1 cells and NIT-vector. Furthermore, compared with the NIT-1 group and NIT-vector group, the CTLs co-cultured with NIT-hAAT cells secreted significantly lower levels of Th1 cytokines (IFN-γ) (P<0.05), and significantly higher levels of Th2 cytokines (IL-4). Compared with the cells in the NIT-1 and NIT-vector cultures, the IL-10 mRNA levels in CTLs co-cultured with NIT-hAAT cells were significantly elevated (P<0.05), while the levels of CRP, IL-1β and IL-6 mRNA were decreased (P<0.05). These mediators were reported to cause islet graft failure via antigen-independent inflammation and correlate with poor transplant outcome [18]–[20].Thus, the ability of hAAT to diminish inflammatory mediators in the graft tissue may provide the basis for the diminished alloimmune response and prolonged islet graft survival. C reactive protein (CRP) has been proposed as a marker of systemic inflammation [21]. CRP and other inflammatory factors are closely related, once the inflammation reaction initiated, CRP level increased. Furthermore, IL-10 is a pleiotrophic immunomodulatory cytokine that functions at different levels of the immune response [22]. Especially, IL-10 induces anergy of T cells by efficiently inhibiting their proliferation and cytokine production [23], [24]. The expression of IL-10 mRNA plays a key role in anti-inflammatory event, so the expression of inflammatory factors CRP, IL-1β and IL-6 mRNA were decreased.
The percentage of NIT-hAAT cells undergoing apoptosis was at its lowest level when NIT-hAAT-primed CTLs were co-cultured with NIT-hAAT cells (P<0.05), indicating that hAAT protected the NIT-1 cells from apoptosis. In agreement with our data, it has been demonstrated that hAAT inhibited damage to β cells by decreasing the levels of inflammatory cytokines, such as IFN-γ and IL-1β [25]. Recent study demonstrated that treatment with AAT protects β-cells against apoptosis through inhibition of caspase-3 activity in a dose-dependent manner [26]. Moreover, hAAT can directly inhibit caspase-3 activation in β cells and thus can function as an anti-apoptotic agent [27]. IL-4 is Th2-type cytokine that promotes Th0 to Th2 differentiation and inhibits Th0 to Th1 differentiation, thereby adjusting the Th1/Th2 cytokine balance. In our study, the NIT-hAAT cells promoted the production of IL-4; therefore, AAT could induce the production of Th2 cells, which would reduce the amount of Th1 cytokines produced. This protein also inhibits the expression of several inflammatory factors. The findings of this study also indicate that co-culture of NIT-hAAT- or NIT-vector or NIT-1-primed CTLs with NIT-hAAT cells caused reduced apoptosis of the NIT-hAAT cells. Taken together, these findings indicate that AAT has a role in immunosuppression [28].
There was no difference of immune response between NIT-1 and NIT-vector cells in vitro.To examine the effect of AAT in vivo, NIT-1 or NIT-hAAT cells were transplanted onto the left kidney of diabetic mice. In mice that received NIT-hAAT cells, the blood sugar levels were significantly reduced three days after the transplantation, and these low levels were maintained until the 30th day. After the removal of the left kidney, the blood sugar concentration rapidly increased and the mice developed hyperglycemia. In the NIT-1 transplantation group, the blood sugar levels of the mice were decreased three days after the transplantation, but after twelve days, the levels increased significantly. We observed that on the 14th, 21st, and 28th days after transplantation, the insulin and C-peptide levels of the mice in the NIT–hAAT group were higher than those in the untreated diabetic and NIT-1 groups (P<0.05). Moreover, the weights of the mice in the NIT–hAAT group increased compared with those of the mice in the untreated diabetic and NIT-1 groups. The glucose tolerance tests and pathology also suggest that hAAT improves the survival of NIT-1 cells and lessens inflammatory infiltrates. In another islet transplantation study, Lewis et al. found that AAT hinders the infiltration of neutrophils, granulocytes, and macrophages into the transplanted tissue [8]. The cuff-shaped Treg cellular layer was formed around the transplant; These cells expressed Treg-related genes, such as Foxp3, TGF-β, and CTLA-4. Walters et al. discovered a similar protective cellular layer around the transplant that reduced inflammatory infiltration and extended the lifespan of the transplant [29]. Given that the beta cell line used in this study originates from the NOD/LT mice, immunological rejection was observed after the transplantation. Forced expression of AAT by transplanted NIT-1 cells demonstrated that islet cells can exhibit immunoprotective abilities and inhibit immunological rejection for a short time. By increasing IL-4 production, the Th1 and Th2 cell balance was skewed toward Th2, which inhibited inflammation and reduced the immunological rejection of the islet β cell transplantation. Thus, the lifespan of these transplanted β cells was extended. However, the in vivo mechanisms by which hAAT causes immunosuppression, induces immune tolerance, and maintains the survival of the islet cells need further investigation [30].
In summary, an AAT-expressing β cell line was established and used for transplantations in diabetic mice. In vitro MLR experiments demonstrated that AAT can regulate immune functions and exhibits immunoprotective effects on islet cells. When NIT-hAAT cells were transplanted into diabetic mice, immunoregulation effect was observed, and transplant rejection was partially inhibited.
Supporting Information
Figure S1.
hAAT expression was confirmed by Western blot assay. 1. NIT-1 cells transfected with the vector containing the hAAT gene, 2. NIT-1 cells transfected with the empty vector.
Author Contributions
Conceived and designed the experiments: FRL. Performed the experiments: YW HJY SYZ YSW. Analyzed the data: CYD. Contributed reagents/materials/analysis tools: HQ. Wrote the paper: FRL YW.
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- 5. Brantly ML, Wittes JT, Vogelmeier CF, Hubbard RC, Fells GA, et al. (1991) Use of a highly purified alpha 1-antitrypsin standard to establish ranges for the common normal and deficient alpha 1-antitrypsin phenotypes. Chest 100: 703–8.
- 6. Churg A, Dai J, Zay K, Karsan A, Hendricks R, et al. (2001) Alpha-1-antitrypsin and a broad spectrum metalloprotease inhibitor, RS113456, have similar acute anti-inflammatory effects. Lab Invest 81: 1119–31.
- 7. Jie Z, Cai Y, Yang W, Jin M, Zhu W, et al. (2003) Protective effects of alpha 1-antitrypsin on acute lung injury in rabbits induced by endotoxin. Chin Med J (Engl) 116: 1678–82.
- 8. Lewis EC, Shapiro L, Bowers OJ, Dinarello CA (2005) Alpha1-antitrypsin monotherapy prolongs islet allograft survival in mice. Proc Natl Acad Sci USA 102: 12153–12158.
- 9. Petrache I, Fijalkowska I, Zhen L, Medler TR, Brown E, et al. (2006) A novel antiapoptotic role for alpha1-antitrypsin in the prevention of pulmonary emphysema. Am J Respir Crit Care Med 173: 1222–8.
- 10. Zhang B, Lu Y, Campbell-Thompson M, Spencer T, Wasserfall C, et al. (2007) Alpha1-antitrypsin protects beta-cells from apoptosis. Diabetes 56: 1316–23.
- 11. Brantly M (2002) Alpha1-antitrypsin: Not just an antiprotease: extending the half-life of a natural anti-inflammatory molecule by conjugation with polyethylene glycol. Am J Respir Cell Mol Biol 27: 652–654.
- 12. Lu Y1, Tang M, Wasserfall C, Kou Z, Campbell-Thompson M, et al. (2006) Alpha1- antitrypsin gene therapy modulates cellular immunity and efficiently prevents type 1 diabetes in nonobese diabetic mice. Hum Gene Ther 17: 625–34.
- 13. Lewis EC1, Mizrahi M, Toledano M, Defelice N, Wright JL, et al. (2008) alpha1- Antitrypsin monotherapy induces immune tolerance during islet allograft transplantation in mice. Proc Natl Acad Sci U S A 105: 16236–41.
- 14. Hamaguchi K, Gaskins H, Leiter E (1991) NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse. Diabetes 40: 842–849.
- 15. Wei P, Li L, Qi H, Zhou HX, Deng CY, et al. (2012) Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells. Biochem Biophys Res Commun 418: 330–5.
- 16. Weir GC, Koulamnda M (2009) Control of inflammation with alpha1-antitrypsin: a potential treatment for islet transplantation and new-onset type 1 diabetes. Curr Diab Rep 9: 100–2.
- 17. Ye J, Liao YT, Jian YQ, Zhang XD, Wei P, et al. (2013) Alpha-1-antitrypsin for the improvement of autoimmunity and allograft rejection in beta cell transplantation. Immunology Letters 150: 61–8.
- 18. Bertuzzi F, Marzorati S, Maffi P, Piemonti L, Melzi R, et al. (2004) Tissue factor and CCL2/monocyte chemoattractant protein-1 released by human islets affect islet engraftment in type 1 diabetic recipients. J Clin Endocrinol Metab 89: 5724–8.
- 19. Abdi R, Means TK, Ito T, Smith RN, Najafian N, et al. (2004) Differential role of CCR2 in islet and heart allograft rejection: tissue specificity of chemokine/chemokine receptor function in vivo. J Immunol 172: 767–75.
- 20. Böni-Schnetzler M, Thorne J, Parnaud G, Marselli L, Ehses JA, et al. (2008) Increased interleukin (IL)-1beta messenger ribonucleic acid expression in beta -cells of individuals with type 2 diabetes and regulation of IL-1beta in human islets by glucose and autostimulation. J Clin Endocrinol Metab 93: 4065–74.
- 21. Ottaviani S, Gorrini M, Scabini R, Kadija Z, Paracchini E, et al. (2011) C reactive protein and alpha1-antitrypsin: relationship between levels and gene variants. Transl Res 157: 332–8.
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- 23. Ozeri E, Mizrahi M, Shahaf G, Lewis EC (2012) á-1 antitrypsin promotes semimature, IL-10-producing and readily migrating tolerogenic dendritic cells. J Immunol 189: 146–53.
- 24. Tawara I, Sun Y, Lewis EC, Toubai T, Evers R, et al. (2012) Alpha-1-antitrypsin monotherapy reduces graft-versus-host disease after experimental allogeneic bone marrow transplantation. Proc Natl Acad Sci U S A 109: 564–9.
- 25. Koulmanda M, Bhasin M, Fan Z, Hanidziar D, Goel N, et al. (2012) Alpha 1-antitrypsin reduces inflammation and enhances mouse pancreatic islet transplant survival. Proc Natl Acad Sci U S A 109: 15443–8.
- 26. Zhang B, Lu Y, Campbell-Thompson M, Spencer T, Wasserfall C, et al. (2007) Alpha1-antitrypsin protects beta-cells from apoptosis. Diabetes. 56: 1316–23.
- 27. Kalis M, Kumar R, Janciauskiene S, Salehi A, Cilio CM (2010) α 1-antitrypsin enhances insulin secretion and prevents cytokine-mediated apoptosis in pancreatic β-cells. Islets. 2: 185–9.
- 28. Shahaf G, Moser H, Ozeri E, Mizrahi M, Abecassis A, et al. (2011) α-1-antitrypsin gene delivery reduces inflammation, increases T-regulatory cell population size and prevents islet allograft rejection. Mol Med 17: 1000–11.
- 29. Walters S, Webster KE, Sutherland A, Gardam S, Groom J, et al. (2009) Increased CD4+Foxp3+ T cells in BAFF-transgenic mice suppress T cell effector responses. J Immunol 182: 793–801.
- 30. Pott GB, Chan ED, Dinarello CA, Shapiro L (2009) Alpha-1-antitrypsin is an endogenous inhibitor of proinflammatory cytokine production in whole blood. J Leukoc Biol 85: 886–95.


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How to get there
Chateau de Bertichères is part of the Golf Country Club of Chaument-en-Vexin. The grounds include an 18-hole golf course, 2 tennis courts, a volleyball court and an equestrian club.
The guest rooms are spacious and are equipped with satellite TV and free WiFi internet access. Each one has a private bathroom with a bath or shower, and a hairdryer is provided.
The restaurant is open for lunch only and and guests can eat on the terrace or in the comfort of their rooms.
From May to September you can enjoy the property's swimming pool. Chateau De Bertichères is situated 50 minutes from Paris, 45 minutes from Charles de Gaulle Airport and just 25 minutes from Beauvais Airport.

subset: commoncrawl


text: Neesha Hathi, senior vice president, Advisor Technology Solutions for Schwab Advisor Services (Photo: Business Wire)"Looking to the future, it is clear that the RIA office must evolve to keep up with emerging client needs and competitive forces," said Neesha Hathi, senior vice president, Advisor Technology Solutions, Schwab Advisor Services. "Mobile technology is a must-have for advisor firms looking to grow and build relationships - both with today's clients and with the next generation - because it allows the advisor to deliver the 'know me', anywhere, anytime service clients expect." Schwab OpenView Mobile was first announced at IMPACT 2013, where Hathi identified mobile, cloud-computing, big data and integration as four key trends that will drive the success of an advisor's business in the future. "Technology is playing an increasingly important role in helping advisors scale their business, enhance the experience of their clients and differentiate their brand," said Hathi. According to Schwab's 2013 RIA Benchmarking Study, 73% of advisors currently invest or plan to invest in mobile technologies, and the most recent Schwab Independent Advisor Outlook Study found that seven in ten advisors are focusing on differentiating their firm to stand out from the pack. Schwab OpenView Mobile - How it Works SPT will work with advisors to customize their app to reflect their own brand, including adding their firm logo, contact details and choosing icons and colors. Once the app has been customized, the advisor will post it on Google Play and iTunes App Stores where their clients can find it by searching for the firm's name. An RIA can customize the experience they want to create for their clients by including advisor news, promotional content, contact information and a website link, as well as real-time data on their Schwab accounts.


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Looking back, I don't know how I thought my meeting with Jacqui Ainsley, the model who recently became the new Mrs Guy Ritchie, would go. What I certainly didn't expect was this: to be driven by a groundsman across their country estate in Wiltshire to find Jacqui's entire clan holed up in a log cabin. Guy Ritchie is in an armchair, as relaxed as any man has ever been, handing me a glass of damson gin, finding out I'm from York and launching straight into a conversation about its medieval massacre of Jews — 'the darkest day for British Jewry,' quoting a history book he's just read.
His wife is outside, standing topless in a lake in the pouring rain for our photo shoot, speaking Hebrew to the make-up artist, while her mum, who clearly runs things, is looking after the small Ritchie children, despite having her arm in a sling from dancing too hard at the wedding (I thought she said driving accident — 'No, jiving accident'). Their posh drunk neighbour is hanging out, too, making repeated attempts to dive into my midriff for a cuddle, with Jacqui, 33, and Guy, 47, shouting and laughing, 'Get off her, she's come to do an interview,' before we all pile into Land Rovers, with Jacqui 'doing a Britney', driving with a toddler on her lap. Back at the main house, Guy's older two boys, Rocco and David Banda, from his marriage to Madonna, are hiding around corners to attack us with Nerf guns.
I'm not going to lie to you — I've had duller days. A character from Game of Thrones could come through on horseback and nobody would bat an eyelid before handing him a drink. 'I am never, ever in a hurry to leave this place and get back to f***ing London,' says Guy, and his wife agrees — though they spend most of their time at their townhouse in Marylebone. The older boys spend their summer holidays here. 'Guy recently asked David if he could remember a time before me, and he said he couldn't,' she says. There is even a huge photo of Lourdes, Guy's former stepdaughter, on the wall.
So how did all this happen? Who is this woman and how has she managed to produce three babies and a wedding in four years, like some kind of Richard Curtis film? Before all this, she dated the Pop Idol runner-up Darius for two years, living with him in Fulham, and did various commercial campaigns, including playing the silhouetted woman who dances through Madonna's song 'Die Another Day' in the opening credits of the James Bond film. She met Guy through mutual friends at The Langham hotel. 'Guy said: "That's a pretty dress," and I thought, "Well, he could just be a bit of a charmer." So I said, "Are you auditioning for a new wife?" And he went, "Oo-ooh, cocky," and we laughed.'
That is quite cocky. 'It was and it wasn't even the headspace that I was in — I was dating a French guy at the time, I had nothing to lose. But it was the first thing I ever said to Guy and we remembered it before the wedding. He said, "So maybe I was auditioning for a new wife!" We got goosebumps talking about it.' Still, after the initial cockiness, she says, 'I realised, "He's fascinating — there's so much more." '
Guy started sending texts asking, 'Are you still with the Frog?' Months later, both single, they finally went on their first date, by which point they had been emailing and texting '30 to 40 times a day'. Sorry, what? 'Yeah, I'd be in South Africa doing a hair commercial and we'd be on Skype, quoting poems to each other, talking about members of our families. Immediate chemistry. When someone responds like that' — she clicks her fingers — 'you know they're not playing a game. But then the first date is even more daunting, after we'd had all that, what if it was a massive letdown? Or he was like, er, "See you later"? So I was nervous and when I'm nervous I do like to have a drink,' she laughs.
He'd asked her if she fancied posh or earthy and she'd replied earthy, not entirely sure what he was talking about. So he took her to the pub. 'And he kept going, "One more, just one more," and I kept saying I had to get my last train back to Southend.' (She owned a sea-facing flat in her Essex hometown.) She missed the train and he said she could stay in his spare room. 'And I thought, "Oh, that old chestnut." Anyway I went' — she laughs again — 'and the next morning I had a cover shoot. It's the first time in my life I'd drunk before work, I'm always very professional. And he rang me at work and said, "You are coming back tonight, aren't you?" And I said, "I'm exhausted, hungover and I haven't got all my stuff." He goes, "OK, you can get your stuff, just bring it here." So that was it. I never stayed at my place again.'
Guy and Jacqui Ritchie's wedding
Guy and Jacqui Ritchie's wedding
1/5 All eyes on Jacqui
The blushing bride on her wedding day
Jacqui Ritchie/ Instagram
2/5 Take my hand
The couple shared shots from their special day with their Instagram followers
Jacqui Ritchie/ Instagram
3/5 Family photo
The newlyweds pose with their children
Jacqui Ritchie/ Instagram
4/5 Guy's got the moves
Jacqui and Guy enjoy their first dance as husband and wife
Jacqui Ritchie/ Instagram
5/5 Here comes the bride
Jacqui is photograhed en route to her festival inspired wedding
Luca Calvani/ Instagram
That was it? 'That was it. Nine months later we were talking about babies. I mean, I knew in the pub. I looked at him and thought, "You're the father of my kids." I had an absolute certainty that I've never had about anything before. Do you know how scary that is?'
She goes quiet. I ask if he felt the same way. 'He thinks I'm more intuitive than him, but he says that from the moment we started texting and emailing, he was saying to all his friends, "I think I've found her." '
They were both right. First there was Rafael, who's just turned four and looks like his dad, then there's their daughter Rivka, two, and now there is baby Levi, born in June 2014. They are looked after by French nannies and will be educated half in French, as Jacqui speaks it fluently and so do the older boys, since Madonna raised them that way, too. Jacqui wanted the kids to have something in common. As for her stepsons, she says, 'You know, David's a dream-boat, and so's Rocco — they're both fabulous human beings. Their parents have done a good job on them. Guy's divorce was two years before I even arrived on the scene, so I think I came in at the right time of their lives, very lovely ages — Rocco must have been eight and David four. And the miniature three, the pickles [she means her own kids], they really benefit from being around their big brothers. We love to have them as much as possible, the house seems so quiet after they go. And my socks don't go missing — David's got the same size feet as me. I've been teaching myself guitar from YouTube and me and Rocco are about the same level now. We both just play 'Wonderwall' a hundred times a day and that's it. But Guy is pretty amazing on the guitar.' At Christmas they had a singalong with the kids and a friend came round with a mandolin and someone else was on the ukelele: 'And my nan was here, singing all the old songs.' Jacqui's own childhood was far more modest — parents Bob and Janet, both secondary school English teachers, raised their family in a small semi — and she says she doesn't want her kids to lose their 'hunger' in life by having wealth.
She is learning Hebrew with the help of a teacher in Israel via Skype, after Guy got her into it. She was always a bit of a linguist and fancies doing Arabic next. 'He got me into studying the Kabbalah and the Torah and the ancient texts, but I was more interested in reading the original text than a translation. Then I found out what a complex language it was. I go into my lessons like I'm going into a meditation, like I'm not anxious about anything else that's going on in my world.'
As for having spent the past three years pregnant, how does that feel? 'Well, there's nine months of pregnancy every time, then three months when you're nursing and not really yourself, another year to really recover and I'm not very good on no sleep, but I'll do it for them. I was so used to being selfish, doing everything that I wanted to, when I wanted to. It was quite a big learning curve, but I think that's the biggest blessing. I remember with number two, worrying if I was going to be able to split my love. And then I held her and realised there was an infinite world of love. And then you go, "Ah!" ' She gasps with delight. 'We could do this over and over ten times! So then I became addicted to that feeling of being a mum and we wanted to go for number three. If I'd started younger, I think I could have gone for ten.' I ask if she has stopped. She answers carefully, 'For me, the shop is not shut.'
After the first two, she worked on regaining her figure. 'Not while I was breastfeeding, because I was in a lovely place, but after that.' It sounds like she was pretty strict. 'But after Levi everything shifted and, honestly and truthfully, I didn't care about the weight, I just wanted to feel strong. I became obsessed with watching YouTube videos with kettlebells and chin-up bars, Pilates. Built it up to five days a week training, then pole-dancing.'
At home? 'No! I'm not sure I warrant a pole at home yet, I need to practise a bit more. It's unbelievably hard — I nearly cried on my second lesson. You're up there, upside down, and you're coming down — it's either concussion or Chinese burns. There's no graceful at the beginning. Guy kept saying, "Bring back a video! Get someone to take one!" I kept saying, "Er, I forgot today…" '
And so they decided to hold the wedding after all of that. They do seem well-suited; they are making a documentary together, but can't tell me what it is. Jacqui stresses she will never become an actress, despite having a cameo in Guy's new movie The Man From U.N.C.L.E. 'I need to get one in King Arthur, too. I'm like, "Surely you need a mermaid? I could rock up with a tail?" ' She loves talking to Guy about work and advising him on casting. 'I made Guy watch Alicia Vikander in a Swedish film called A Royal Affair because I thought she'd been so good in it, then he put her in U.N.C.L.E.'
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I ask if she turned into a Bridezilla, but she says she was very calm. 'The night before, Rocco came to me and said he needed help writing his speech — he was pacing up and down. So I felt like I was calming everyone down.' Her only anxiety was the weather, as it was an outdoor wedding with 150 guests staying in tents for three days, but the sun came out. They built a mini Glastonbury with yurts and food stalls; even David Beckham camped.
The second night was 'a bit nippy' and they held an outdoor screening of The Man From U.N.C.L.E. 'We had to bring in onesies for everyone. So many people brought their kids. Oh, it was just the most magical time ever. I'm so glad I paid attention to someone's tip not to get too drunk, as I spent eight hours on the dancefloor anyway. I sprained both my ankles and my mum broke her arm. The dancefloor was a dangerous place.' Brad Pitt was there, too. 'But I loved everyone who was there.' I wonder if, amid all this madness, she still sees her old mates from Southend? 'They were my bridesmaids!' she says.
When I arrived here, many hours ago, Madonna felt like the elephant in the room. But clearly that was just in my room, because the new Ritchie gang is so much stronger and more fun than I could have foreseen. No tails. No trunks.
Jacqui Ritchie (Instagram @jacquiritchie) is represented by Premier Model Management
- More about:
- Jacqui Ritchie
- Guy Ritchie


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The 16GB SD memory card is specifically developed in response to demands for better security in the field of digital audio and video. It includes copyright protection to the SDMI (Secure Digital Music Initiative) standard. The system to protect and encrypt files (CPRM: Content Protection for Recordable Media) in integrated into the SD card.
Electronics, Computer & Photo > Camera & Photo > Accessories > Digital Camera Accessories > Memory Cards


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supporting Healthy Eating and active living in vermont communities
Healthy community design means planning and designing communities to make it easier for people to live healthy lives. Researchers and community members recognize that an environment that supports active living and expands access to healthy and affordable foods is essential for good health. Healthy community design changes the physical environment, community infrastructure and local policy to create such an environment.
The Centers for Disease Control & Prevention's Recommended Community Strategies and Measurements to Prevent Obesity describes a range of strategies that communities can use to support physical activity and healthy eating.
Vermont partners from planning, transportation, recreation and agriculture identified the following four strategies to be most relevant for Vermont's population, rural character, and framework for local planning:
The Vermont Healthy Community Design Resource: Active Living and Healthy Eating was created around the four strategies described above for health professionals, planners, and anyone seeking to make their community a place that supports active living and healthy eating. The information, tools and resources in this guide may be used to successfully navigate the worlds of land use planning and public health.
The Vermont Healthy Community Design Resource, Examples for Creating Healthy Communites: Physical Activity, Healthy Eating, Tobacco, Alchol & Drug Abuse Prevention provides examples of policies, plans, and strategies that Vermont communities have implemented to create places that support healthy lifestyles. It also includes sources to find "best practice" examples of zoning, ordinance and policy language municipalities could adopt to support healthy lifestyle choices."
3-4-50 offers examples of Healthy Community Design action that is happening across Vermont and can be modeled in other communities.
Streets should safely accommodate all transportation system users, regardless of age, ability, or their preferred mode of transportation, including walking, biking, driving, or the use of transit. Vermont took these principles and put them into policy by passing a Complete Streets law that supports the state's goal of increasing the number of Vermonters who engage in regular physical activity, by creating communities where walking and bicycling are made safe and accessible.
Complete Streets: Guide for Vermont Communities and slides were developed to assist towns with understanding the law and providing examples of how Complete Streets can be applied in Vermont communities.
Health Impact Assessment, or HIA is a process that helps evaluate the potential health effects of a plan, project or policy before it is built or implemented. A HIA can provide recommendations to increase positive health outcomes and minimize adverse health outcomes. HIA brings potential public health impacts and considerations to the decision-making process for plans, projects, and policies that fall outside the traditional public health arenas, such as transportation and land use. – Centers for Disease Control & Prevention
Health Department staff have been trained and may be able to provide support to communities interested in conducting health impact assessments.
Learn more about the Health Department's HIA efforts.
Aspects of the community environment such as the availability and accessibility of bicycle or walking paths, exercise facilities or farmer's markets influence a person's health behaviors such as level and amount of physical activity or consumption of fresh fruits and vegetables. For these reasons the Health Department, in collaboration with the UVM Center for Rural Studies, conducted a survey of public resources related to physical activity and nutrition in Vermont's cities and towns in 2005.
Survey results are presented, and data compared from each participating town and city. The survey was redone in 2016. The results will be updated when available. See the 2005 survey results
Creating healthy communities is a team effort. Here are a few of our partners:
- Vermont Agency of Transportation Bicycle and Pedestrian Program
- Vermont Agency of Commerce & Community Development
- Vermont Association of Planning and Development Agencies
- Local Motion
- AARP Vermont, Livable Communities Initiative
- Vermont Department of Forest Parks and Recreation
- Vermont Recreation and Parks Association
- Vermont Agency of Agriculture


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When you create an account with us, you'll be able to save your favourite books, make a wishlist of upcoming titles, receive newsletters about books you'll love, get recommendations tailored to you and order our books directly. Join us by creating an account and start getting the best experience from our website!
Date published: New > Old
"1001 Escapes To Make Before You Die" is the essential guide for all discerning travelers seeking an escape from the madding crowd. Every experience described in this book represents a genuine respite from the drudgery of daily life and guarantees a boost to your general well-being. Contemplate the sunset from a minimalist mountain retreat, cruise the Nile on a nineteenth-century cabin cruiser, hire a fort with its own private beach, chill out in an ice hotel, or wallow in a mud bath. The choice is yours. This book is quite simply a compilation of some of the most amazing escapes from the everyday that you can experience in the world - however much time you have to spare and whatever your budget. Within these pages you will find classic escapes that offer away-from-it-all tranquility, such as a sun-drenched private Caribbean island, complete with butler; a converted monastery in Peru, at such high altitude that oxygen has to be pumped into the bedrooms; a lighthouse in Newfoundland; and, that most classic of great train journeys, the Trans-Siberian railway.Beautifully illustrated, "1001 Escapes To Make Before You Die" will also show you some hidden gems located in some of the world's biggest and busiest cities that offer the perfect retreat from the frenzy of city life. Discover an oasis of greenery just a stone's throw from Djemma El Fna, the pulsating center of Marrakech; a tranquil walled garden in the heart of London's Chelsea; and, a hidden treehouse deep within the Amazon rain forest.


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New Delhi, July 12 (ANI): The much awaited full night Yakshagana, a theatre form that combines dance, music, dialogue, costume, make-up and stage techniques with a unique style and form which is popular in South Kanara and adjacent areas of coastal Karnataka, came to an end on Sunday morning at Delhi's Karnataka Sangha Auditorium.
Yakshagana lovers came and watched the performance whole night despite the continuous rain.
The performance, which began at 9 p.m. yesterday, concluded at 6 a.m. today. This nine-hour long performance was witnessed by jam packed audience. 36 eminent professional artists selected from various troupes made this event a grand success.
The episode began with creation of three supreme gods 'Brahma, Vishnu and Maheshwara' by supreme goddess. After that the story narrates the victory of demons over gods including supreme vedic god Indra, but finally the goddess Durga wins over all demons, including Chanda, Munda, Mahishasura Rakthabija and Shumbha Nishumba.
This narrative is seen as an attempt to unify the vedic male pantheon with the pre-existing mother goddess cult possibly dating to the 9th century BC and an attempt to define divinity as a female principle.
The performance synthesized a number of pre-existing mother goddess (Chamundi, Kali, Durga, Sapta Matrikas, Raktheswari etc) myths into a single narrative which has been crystalized and beautifully presented by the artists. Various colors, headgears, costumes and dancing styles made this episode very unique. Especially, the entry of Mahishasura with fireworks and burning torches made this event memorable.
The young kids watched this performance with scary eyes, holding their mother's hands.
"I don't think, I will watch this episode again during my remaining life time, hence decided to watch till morning," said the oldest spectator, a woman nearing her 90s.
Free tea and eatables were distributed during the performance.
Delhi-based two organizations, South Canara Club and Academy of Tenku Tittu Yakshagana, in association with Delhi Karnataka Sangha organized this grand event. Many banks and local organizations supported this event. (ANI)


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With First Tutors: Music you can find a Spalding composition teacher. Composition tuition are an excellent method to begin a stimulating new hobby, pick up a new talent and gain confidence with composition. Try our service to find Spalding composition tutors and select which matches your needs so you can begin your composition lessons in Spalding immediately!
First Tutors: Music is the fastest way to contact Spalding composition tutors in the UK, helping you find Spalding composition lessons close to you and Spalding tutors to assist with any instrument like piano, violin or guitar. We can help you find Spalding composition lessons or a Spalding composition teacher to prepare for composition exams and develop your composition skills! We order Spalding composition tutors near you by their hourly prices and distance from you.
If you are a composition teacher and would like to offer your composition tuition services in Spalding or throughout the UK, please register with First Tutors: Music or find out more about us here. Becoming a Spalding composition tutor is a fantastic way to re-affirm your own skills through giving composition tuition while assisting others, all in Spalding!


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Great Expectations: Can SOA Deliver? Part III: Front End Driven SOA
AbstractBangalore, India 14 January 2009
Great Expectations: Can SOA Deliver? Part III: Front End Driven SOA
SOA is both an overused buzzword and a real solution to banking problems. Celent demystifies SOA, explaining what it is, how it helps banks, and how banks can deploy SOA.
In a new report, Great Expectations: Can SOA Deliver?, Celent explains the various components of service-oriented architecture (SOA) and how they apply to banking. There is a great deal of ambiguity around SOA. It is both a buzzword and a real architecture designed to help banks abstract and expose their core systems. Celent defines SOA to be a set of loosely coupled modular services to support both business and IT requirements. In attempting to clarify what SOA is and how the pieces work together, Celent has created a map of SOA and charted how various front end vendors provide solutions to this map. Celent then ranks these vendors on its ABCD analysis.
"Front end vendors have primarily focused on providing multichannel support for banking services and developing solutions which increases the customer interaction capabilities of banks," says Rajesh M R, Celent analyst and co-author of the report.
"Depending on the requirements and existing infrastructure of the financial institution, different front end SOA solutions would be preferred by each firm," says Bart Narter, senior vice president of Celent's Banking Group and co-author of the report.
The 72-page report contains 44 figures and 59 tables.
A table of contents is available online.
of Celent's Retail and Business Banking and Corporate Banking research services can download the report electronically by clicking on the icon to the left. Non-members should contact email@example.com for more information.
Celent is a research and advisory firm dedicated to helping financial institutions formulate comprehensive business and technology strategies. Celent publishes reports identifying trends and best practices in financial services technology and conducts consulting engagements for financial institutions looking to use technology to enhance existing business processes or launch new business strategies. With a team of internationally based analysts, Celent is uniquely positioned to offer strategic advice and market insights on a global basis. Celent is a member of the Oliver Wyman Group, which is a wholly-owned operating unit of Marsh & McLennan Companies [NYSE: MMC].
Media Contacts
North America
Michele Pace
Tel: +1 212 345 1366
Europe (London)
Chris Williams
Tel: +44 (0)782 448 3336
Asia (Tokyo)
Yumi Nagaoka
Tel.: +81 3 3500 3023
Table of ContentsBangalore, India 14 January 2009
Great Expectations: Can SOA Deliver? Part III: Front End Driven SOA
|Executive Summary
|ABCD Analysis
|Appendix A -- An SOA Primer


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|[jira] Updated: (HADOOP-3098) dfs -chown does not like "_" underscore in user name
|Thu, 27 Mar 2008 20:07:24 GMT
[ https://issues.apache.org/jira/browse/HADOOP-3098?page=com.atlassian.jira.plugin.system.issuetabpanels:all-tabpanel ] Raghu Angadi updated HADOOP-3098: --------------------------------- Attachment: HADOOP-3098.patch Updated the patch to allow '@' and '/'. Documentation includes an explicit warning for Linux users (may be 90+% of hadoop users?) : {noformat} $ bin/hadoop fs -Dfs.default.name=file:/// -help chown -chown [-R] [OWNER][:[GROUP]] PATH... Changes owner and group of a file. This is similar to shell's chown with a few exceptions. -R modifies the files recursively. This is the only option currently supported. If only owner or group is specified then only owner or group is modified. The owner and group names may only cosists of digits, alphabet, and any of '-_.@/' i.e. [-_.@/a-zA-Z0-9]. The names are case sensitive. WARNING: Avoid using '.' to separate user name and group though Linux allows it. If user names have dots in them and you are using local file system, you might see surprising results since shell command 'chown' is used for local files. {noformat} > dfs -chown does not like "_" underscore in user name > ---------------------------------------------------- > > Key: HADOOP-3098 > URL: https://issues.apache.org/jira/browse/HADOOP-3098 > Project: Hadoop Core > Issue Type: Bug > Components: fs > Affects Versions: 0.16.0, 0.16.1 > Reporter: Koji Noguchi > Assignee: Raghu Angadi > Priority: Blocker > Fix For: 0.16.2 > > Attachments: HADOOP-3098.patch, HADOOP-3098.patch > > > :~$ hadoop dfs -chown aaa_bbb /user/knoguchi/test.txt > chown: 'aaa_bbb' does not match expected pattern for [owner][:group]. > in 0.16.1, only alphabets and numbers are allowed. Shouldn't '_' be allowed? > I couldn't find any standard, but in Solaris10, it's defined as > http://docs.sun.com/app/docs/doc/816-5174/6mbb98uhg?a=view > bq. The login (login) and role (role) fields accept a string of no more than eight bytes consisting of characters from the set of alphabetic characters, numeric characters, period (.), underscore (_), and hyphen (-). The first character should be alphabetic and the field should contain at least one lower case alphabetic character. A warning message is displayed if these restrictions are not met. -- This message is automatically generated by JIRA. - You can reply to this email to add a comment to the issue online.
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About the Book
Street Photography, abandoned ruins turned into art, walls made colourful, trash made beautiful.
The cover of the book is blank so you can create your own art work on a blank space.
Features & Details
- Category Arts & Photography
Standard Landscape, 10×8 in, 25×20 cm
20 Pages
- Publish Date Jun 04, 2014
- Language English
- Tags photography, street, art, graffiti
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Tesco Viagra Sales Set to Beginposted by Paul Fiddian | 20.09.2010
Viagra drugs will soon start to be sold by supermarket chain Tesco: a British High Street first
Viagra drugs will soon start to be sold by British supermarket chain Tesco, it emerged on 20 September 2010.
From 27 September onwards, males suffering from ED – erectile dysfunction – will be able to purchase the infamous blue-coloured treatment drugs at 300 Tesco stores across the UK.
Based on the figures that have been released, it seems that Tesco intends to undercut the only other high street Viagra-provider at present – pharmaceutical chain, Boots. Tesco is set to price eight Viagra pills at £55 – almost the same price that Boots charges for half this amount.
Tesco Viagra
No other supermarket has done anything like this before and, according to the firm itself, there will be controls in place to ensure that Tesco Viagra sales don't involve everyone that wants them. Rather, customers will be vetted, and only pharmacists will be able to dispense them.
Furthermore, the Viagra will only be supplied to males between 40 and 65 years of age. The checks to be performed on them to ensure that they are suitable Viagra candidates will include diabetes tests, and blood and cholesterol assessments.
"Provided that the men are suitable, we will discuss their options and sell them an effective treatment", Tesco Pharmacy Services commercial manager, Shona Scott, stated.
Approximately 2.3 million males in the UK alone suffer from erectile dysfunction on a regular basis, while 50 per cent of men aged forty-plus experience it less frequently.
Viagra Sales: Tesco
Speaking to the BBC, a Sexual Advice Association representative welcomed the Viagra sales at Tesco news, especially given the safety implications it carries. Embarrassed by their predicament, Doctor Nick Tomlinson explained, some ED sufferers were resorting to purchasing Viagra online, but this opened up the potential for fake versions to be supplied in place of the real thing.
He added, however, that GP prescriptions still provided the cheapest way to obtain Viagra in many instances.
Pfizer's Viagra is a global phenomenon. In circulation for over ten years now, the familiar blue pill has been prescribed to over 25 million male patients to date.
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Defensive Buildings
In Clash of Clans, having a strong defense is more Important.Defenses serve to defend trophies and secure assets from foe troops. Every safeguard has its qualities and shortcomings, and its area in your town may as well reflect that. For example, the compelling Mortar is moderate terminating, yet bargains mind boggling sprinkle harm to ground troops.
A downside is that it can't attack troops inside four tiles of itself (or air troops whatsoever) - so it ought to be set in the inside of your town with sufficient assurance from Air Defenses. On the other hand, Cannons and Archer Towers bargain lesser measures of harm to gatherings (as they can target and hit stand out unit at once) however attack faster also fill in as a successful first line of safeguard. The Wizard Tower bargains unbelievable sprinkle harm to both ground and air units, however has a generally short extend.
|Archer Tower
|Air Defense
|Wizard Tower
|Hidden Tesla
|Inferno Tower
Resource Buildings
Resources are the currencies used to purchase and upgrade assets. The game has four different resources; gold, elixir, dark elixir, and gems.
Gold, elixir and dark elixir all have buildings that are used for storing and generating them.
|Gold Mine
|Gold Storage
|Elixir Collector
|Elixir Storage
|Dark Elixir Drill
|Dark Elixir Storage
|Builder's Hut
Source: Clash of Clans Wikia
Army Buildings
The Army section of the in-game shop that will impact the strength of your attacking army, allowing you to win more attacks.
All buildings from the Army section require Elixir or Dark Elixir to build.
|Army Camp
|Dark Barracks
|Spell Factory
|Barbarian King Altar
|Archer Queen Altar
Other Buildings
These buildings are those that are none, of these hostile, preventive, or asset giving. Be that as it may, they are all in all as basically significant. The Town Hall, case in point, is the heart of the town and directs what structures you can have, what number of them you can have, and how far you can redesign them. The Clan Castle, then again gives you a chance to unite with different players in a tribe. Enhancements are there to beautify your town. Obstructions are trees and rocks which could be found in your town. Clearing them will off and on again provides for them you Gems.
|Town Hall
|Clan Castle
To know more about Troops in Clash of Clans, Click Here.
You can Unlock all these Buildings easily with the Hack Tool from our site.


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Climate change may be one of the most complex and fundamental drivers of future change. Not only are we seeing changes in the climate itself - the opening up of the Northwest Passage, local floods and droughts - it is exacerbating the rapidly emerging food crisis.
Australia's worst drought in 100 years has halved its wheat crop. An unusually cold year has damaged Ukraine's crops. Booming Chinese demand has pushed up prices while severe droughts have cut its own wheat production. In search of clean fuel, farmers have converted to biofuel crops, with at least one third of the US maize crop going to ethanol in 2006 even though some biofuels are dirtier than fossil fuels. Longer run, however, warming may allow Siberia and Northern Canada to become the new bread baskets of the world. The race for minerals and energy in the North Pole has already begun. While global carbon emission negotiations slowly edge forward, businesses, cities, citizens, regional governments and geo-engineers are rushing ahead. Examples of the green innovation range from carbon neutral cities such as China's Dongtan Eco-city, to the more radical technology solutions such as carbon absorbing synthetic trees.
This is part of Outsights 21 Drivers for the 21st Century ™, a future-orientated scan of the 21 key forces shaping this century.
- Plain sailing on the Northwest Passage
- Fresh records for price of wheat
- Drought grows slightly in E. Australian farmlands
- Biofuel demand to push up food prices
- Top scientists warn against rush to biofuel
- Global Warming: Who Loses—and Who Wins?
- Russia Claims the North Pole
- Dongtan Eco-city
- Can science really save the world?
- Outsights


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Puffs & Fryums Foods India Pvt. Ltd. a Leading Manufacturer and Trader of Puffs, Fryums and Extruded Sticks(kurkure) is Looking for Potential Candidates for the Job of Sales Officer. Sales Officer Will Develop the Business of Company in Retail Ma...
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Dear Candidate:- We are offering work in Realestate as Freelancer on very good comission.you can do work as full time. can earn 50000- 100000 rs/month The main job purpose of a real estate appraiser is shaping the worth of a property or structur...
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Survey Field Executive ( 0 - 4 yrs)
Hiwa Healthcare
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Need for Sales Manager ( 1 yrs)
Shree Sati Consultant
RS 3,00,000 To 4,25,000 Faridabad, Gurgaon, Aligarh, Mathura, Noida
We are Looking For
Key Skills : sales manager,sales executive,sales,sales head,sales officer
Business Development Manager ( 2 - 5 yrs)
Drs Consultants
RS 1,75,000 To 3,00,000 Aligarh, Bijnor, Bulandshahr, Ghaziabad, Greater Noida, Hapur, Mathura, Moradabad, Muzaffarnagar, Noida, Saharanpur
Position : Business Development Manager Experience: 2-4 Years Location : West U.P. Sal : 1.8-3.0 LPA Job Responsibilities : * Take care of western U.P. market for any given location. ^ Have experience of sales / marketing in FNCG /...
Key Skills : sales,marketing,business development,business development manager,business development officer,business development head,business developer
Future Cell
RS 1,25,000 To 1,75,000 Agra, Kanpur, Lucknow, Mathura
• Responsible for selling overall products to customers by assessing their requirements. • Generate new customers and growing business from existing customers. • Maintaining database of qualified leads through referrals and cold calling. ...
Key Skills : direct sales,business development,sales,marketing,sales officer,telemarketing,telesales,direct...
Sales Development Manager- Agency ( 1 - 5 yrs)
Skywings Advisors Private Limited
RS 1,25,000 To 2,00,000 Aligarh, Ghaziabad, Gorakhpur, Lucknow, Mathura, Meerut, Mirzapur-cum-Vindhyachal, Renukoot, Saharanpur, Varanasi
Hiring for the position of Agency Manager for Birla Sun Life Insurance Company Ltd. Location: uttar pradesh Salary- 1.8L to 2.2L per annum - Candidate should be graduate. - Candidate should have at-least 6 months of sales experience. - Candid...
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Sales Manager Life Insurance L10 ( 3 - 12 yrs)
Satvam Consulting Private Limited
RS 2,00,000 To 3,00,000 Mehsana, Karnal, Mangalore, Calicut, Kottayam, Malappuram, Ludhiana, Ajmer, Kota-Kota, Madurai, Lucknow, Mathura, Kurukshetra
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Om Consultancy
Agra, Mathura
- Position - Area Sales Manager - Company - Telecom - Location - Mathura / Agra - Exp. - min 3 yrs in channel sales in telecom sector - Qualification - Min graduate in any stream - Salary Range - Rs. 2.5 to 3 LPA Job Description - - Channel...
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Sales Officer for Agra and Mathura ( 1 - 4 yrs)
Future Cell
RS 1,50,000 To 2,00,000 Agra, Mathura
Job Description- • Responsible for selling products to customers by assessing their requirements. • Generate new customers and growing business from existing customers. • Maintaining database of qualified leads through referrals and cold...
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Assistant Sales Manager ( 10 yrs)
U. D. Food Products
RS 3,00,000 To 6,00,000 Ajmer, Jaipur, Agra, Ghaziabad, Lucknow, Mathura, Noida, Delhi
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Marimallapa's High School, Mysore.
Photographer: Unknown
Medium: Photographic print
Date: 1890
This photograph of the Marimallappa's High School, Mysore taken in the 1890s by an unknown photographer, is from the Curzon Collection's 'Souvenir of Mysore Album'. The view shows pupils and staff posed in front of the school, built to European designs. A caption note gives the following details, "Marimallappa was a Bakshi, or head of a department of the palace under Krishna Raja Wodeyar III, who, having no heirs, left his wealth for the promotion of education. The building was erected in 1886 and educated boys up to the Matriculation standard."