Christchurch Earthquake - How they fared
We tracked down the owners of some Christchurch homes featured in NZ House & Garden to find out how they’d fared during the earthquakes. Some have been luckier than others – here are their stories.
For a glimpse of the American south-west, one need only venture as far as Lincoln, on the outskirts of Christchurch. Apart from the odd crack and broken ornaments, Maude and Eddie Fairbairn’s Native American, Mexican and Spanish-inspired home has stood up well to the earthquakes that have hit Christchurch in recent months.
The Ultimate Indulgence – March 2009Hans and Carolyn Schuitman
||Hans and Carolyn Schuitman’s rustic 1400sqm home, 25km north of Christchurch, was unharmed except for a few cracks in the walls and some broken china. The couple recently held a fundraising concert at their huge home and raised a whopping $45,000 for the Christchurch relief fund. With a barn, workshops, a studio, cinema and interconnecting verandahs and courtyards, their grand home proved to be the perfect venue. |
First Impressions – April 2003
Interior designer Susie Paynter’s Fendalton home was untouched by the earthquake, however, her daughters weren’t so lucky. Their homes in St Albans and Mt Pleasant were badly damaged. Susie is now working with clients to repair and find new homes and says she can see huge opportunities for change and improvement. “You have to look at it that way or you wouldn’t get through.”
Soul Story – February 2009
Manfred & Ulrike Herzhoff
This eye-catching, 1920s inner-city home near Cranmer Square sustained substantial damage in February’s earthquake and will be demolished. Manfred and Ulrike Herzhoff had admired the house since their first visit to Christchurch in 1995 but are now unable to return to their home, even to collect their possessions. The couple and their two sons are currently living out of suitcases and searching for somewhere to rent.
Period Piece – August 2004
William Cottrell & Simonetta Ferrari
Gunyah, a gracious Edwardian homestead near Darfield, has been home and workplace to the Cottrells since 2002. Decorated throughout with antique furniture that William has restored over the years, their grand old home is rich with history. Since the first quake in September, Gunyah homestead has undergone huge repairs and some rooms were reopened for business in January. Fortunately, the grand home escaped February’s devastating earthquake with no further damage and repairs continue unabated. Simonetta Ferrari expects it will take another two months before they are fully operational.
Story: Rosemary Barraclough


text:
Sunday alcohol sales to be on November ballot in Spartanburg
Monday night Spartanburg City Council approved a Sunday alcohol sales referendum.
According to the city's Twitter page, the referendum passed 6-1. Now the decision moves to city voters in November.
Spartanburg already allows bars and restaurants to sell alcohol on Sundays for on-site consumption.
The new referendum would allow Sunday sales at grocery and convenience stores.

domain: news


text:
ORIGINAL RESEARCH article
Sec. Autoimmune and Autoinflammatory Disorders
Volume 13 - 2022 | https://doi.org/10.3389/fimmu.2022.980526
Immune response in ofatumumab treated multiple sclerosis patients after SARS-CoV-2 vaccination
- 1Department of Neurology, Ruhr-University Bochum, St. Josef-Hospital, Bochum, Germany
- 2Department of Molecular Immunology, Ruhr-University Bochum, Bochum, Germany
- 3Department for Immunology, Leibniz Research Centre for Working Environment and Human Factors (IfADo) Technical University (TU) Dortmund, Dortmund, Germany
- 4Department of Hematology and Oncology with Palliative Care, St. Josef Hospital, Ruhr-University Bochum, Bochum, Germany
- 5Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
Objective: The pandemic induced by SARS-CoV-2 has huge implications for patients with immunosuppression that is caused by disorders or specific treatments. Especially approaches targeting B cells via anti-CD20 therapy are associated with impaired humoral immune response but sustained cellular immunity. Ofatumumab is a human anti-CD20 directed antibody applied in low dosages subcutaneously, recently licensed for Multiple Sclerosis (MS). Effects of early ofatumumab treatment on alterations of immune cell composition and immune response towards SARS-CoV-2 are incompletely understood.
Methods: We here investigated immune cell alterations in early ofatumumab (Ofa) treated patients and effects on humoral (titer, neutralization capacity against wild type, Delta and Omicron) and cellular immune responses in Ofa treated MS patients following a third vaccination against SARS-CoV-2 compared to healthy controls.
Results: We show that a mean treatment duration of three months in the Ofa group led to near complete B cell depletion in line with altered composition of certain CD4+ T cell subpopulations such as enhanced frequencies of naive and a decrease of non-suppressive regulatory T cells (Tregs). Titer and neutralization capacity against SARS-CoV-2 variants was impaired while cellular immune response was sustained, characterized by a strong T helper 1 profile (Th1).
Interpretation: In summary, low dosage ofatumumab treatment elicits sustained depletion of B cells in line with alterations of immune cells, mainly Tregs. This is associated with impaired humoral immune response towards SARS-CoV-2 vaccination but preserved, Th1 driven cellular immunity adding crucial information regarding early effects of low dosage anti-CD20 therapy on humoral and cellular immunity.
The novel virus SARS-CoV-2 can induce Coronavirus disease 2019 (COVID-19), a potentially life-threatening disease, which led to a global pandemic since early 2020. Soon, patients with compromised immunity either through a disorder or due to the need of immunosuppressive therapy got into focus regarding both their risk of infection and the risk of a fatal outcome as well as the immune response towards vaccination. Especially patients treated with anti-CD20 (aCD20) monoclonal antibodies such as rituximab or ocrelizumab, established as therapies in Multiple Sclerosis (MS) (1, 2), are at higher risk for hospitalization and a more severe course of COVID-19 (3). Moreover, we learned from a broad range of studies that the humoral immune response is worse in patients treated with aCD20 treatment (4). While the humoral immune response, prone to immune escape, is impaired, it could be established since fall 2021 that MS patients treated with aCD20 therapy elicit a robust T cell response, characterized mainly by CD8+ T cells (5).
A new treatment concept in MS recently having received market authorization is the application of ofatumumab, a fully human monoclonal anti-CD20-directed antibody applied in low dosages of 20 mg every four weeks subcutaneously. So far, it remains unclear how the initiation of a low dose aCD20 treatment regime might impact on the immune cell composition as well as humoral and cellular immune response towards SARS-CoV-2 vaccination. We therefore set out to analyze early ofatumumab treated patients regarding immunophenotyping as well as immune response following SARS-CoV-2 booster vaccination focusing on humoral immune response (neutralization capacity against wild type, Delta and the now dominating Omicron variant and titers) and deep immunophenotyping of T cell subsets. Those data will inform about vaccination strategies in low dosage aCD20 therapy.
The local ethics committee of the Ruhr-University Bochum authorized the study (20-6953-bio, 21-7351). We included n=9 healthy age-matched controls (HC) and n=10 ofatumumab treated MS patients (Ofa) recruited at the Department of Neurology, Ruhr-University Bochum, St. Josef-Hospital. Patients were boostered between September and December 2021. Table 1 presents the associated demographics. All participants provided written informed consent. The samples were collected within 4-13 weeks following the third vaccination against SARS-CoV-2.
Table 1 Demographic and clinical characteristics of recruited MS patients and healthy controls. Age, disease duration (in years), Expanded Disability Status Scale (EDSS) and duration under therapy (in months) are presented as mean. SD: standard deviation, SEM: standard error of the mean. COVID: Coronavirus Disease 2019. DMF: dimethyl fumarate. Booster: third SARS-CoV-2 vaccination.
Cell isolation and cryopreservation
Peripheral blood mononuclear cells (PBMCs) were isolated from the study participants’ blood withdrawn with 7.5 ml KABEVETTE® G EDTA tubes after the booster vaccination. 30 mL blood-containing sample was layered on 15 ml ROTI®Sep 1077 human density gradient (Carl Roth) and centrifuged at 800 g for 30 min. without break. The PBMCs in the interface were washed twice and the pellet was resuspended in 10 mL PBS. Following cell counting, 10-20*106 cells were immersed in 1 ml CTL-Cryo-ABC Freezing media Kit (ImmunoSpot®) for cryopreservation and stored at -80 °C.
Immune cell phenotyping
For analysis of the immune cell composition without peptide stimulation, we used PBMCs of the same batches used for peptide stimulation for a detailed phenotyping of immune cell subpopulations via flow cytometry. Of the remaining cell suspensions 2.5*106 cells were seeded in 2 mL OpTmizer™ CTS™ (Thermofisher) in a 12-well plate following resting for 1-2 h. For immunophenotype analysis, 2.5*106 PBMCs were stained with viability stain LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit for UV excitation (L23105, Thermo Fisher) for 30 min. at 4°C. Afterwards, Fc receptors were blocked by incubating the cells with Human TruStain FcX™ (422302, Biolegend) for 15 min. at 4°C. Subsequently, surface markers were stained for 15 min. at 4°C. Fixation and permeabilization were performed with Foxp3 staining buffer set (130-093-142, Miltenyi Biotec). Next, intracellular proteins Ki-67 and Foxp3 were stained for 30 min. at 4 °C. Antibody information is presented in Table S1. Flow cytometry measurement was performed using a Cytoflex LX (Beckman Coulter). Whole samples (2.5 *106 PBMCs seeded) were recorded during flow cytometry measurement. The average of total events recorded was 1,422,939 (range [max – min]: 2,802,588 – 220,784) and the average of living cells recorded was 711,543 (range: 1,250,000 – 114.109) events. Regarding the analysis of B cells, an average of 63.262 (Range: 86237 - 28.771) events were recorded for the HC group and an average of 755 (Range: 3262 – 119) for the Ofa group. Flow cytometry data were analyzed with FlowJo™ (Becton Dickinson & Company, version 10.8.0). Barnes-Hut algorithm was implemented to generate t-Distributed Stochastic Neighbor Embedding (t-SNE) plots with FlowJo™ for high-dimensional data visualization. The t-SNE plots represent the distribution of the Treg cell subpopulations (nTreg, nsTreg, eTreg) and protein expression within the Treg cells of the respective groups. The Treg populations from all samples were used to create a single concatenated file. Pre-gating was done according to gating strategy (Figure S1): Time gate -> Single cells (FSC) -> Single cells (SSC) -> Viable cells -> CD3+ -> CD4+ -> Treg (CD127low Foxp3+). Downsampling was performed to balance the two groups for fair comparison (59168 events per group). All markers, except non-relevant/excluded markers (Viability, CD8, CD19), were used for the calculation using Barnes-Hut algorithm in FlowJo. The gating strategy is presented in Figure S1.
Anti-SARS-CoV-2 antibody titer
The enzyme-linked immunosorbent assay for determination of anti-SARS-CoV-2 spike antibody titers was conducted as described before (6). Briefly, the RBD sequence (spike glycoprotein amino acids 319–541) of the Wuhan strain of SARS-CoV-2 with a C-terminal HIS-tag was expressed in HEK 293-F cells and purified on a HisTrap Excel column using an ÄKTAxpress purification system. Ninety-six well flat bottom plates (maxisorp; Nunc) were coated with 3 μg/mL of SARS-CoV-2 spike RBD overnight at 4°C, following washing, blocking and incubation with diluted serum samples. The S Antibody (humanized anti-Spike antibody by Dianova/Cusabio, Stock concentration: 0.3 mg/mL) was used as positive control (1:5,000 final dilution) and calibrator (1:40,000 final dilution). HRP conjugated anti-human IgG (Dianova) was used as secondary antibody. Antibody titers were calculated as BAU/ml and values higher than 30.3 were considered as positive (6).
Anti-SARS-CoV-2 nucleocapsid (NCP) antibody titer was determined using the Anti-SARS-CoV-2-NCP ELISA (IgG) (EUROIMMUN) following the manufacturer’s instructions to exclude unremarked prior infections which could have affected the immune response. A ratio of the OD of the sample over the OD of the included calibrator of <0.8 is considered negative, ≥ 0.8 to <1.1 borderline and a ratio ≥1.1 positive.
SARS-CoV-2 neutralization assay
We used previously described pseudoviruses (7). Complement factors in sera were inactivated (30 min. at 56 °C). The sera were incubated as quadruplicates of twofold serial dilutions (1:20 to 1:2560) in 96 well plates with single cycle VSV∗ΔG(FLuc) pseudoviruses bearing the SARS-CoV-2 spike WT (D614G) protein (8), SARS-CoV-2 B.1.617.2 (Delta) (EPI_ISL_1921353) spike protein or SARS-CoV-2 B.1.1.529 (Omicron) (EPI_ISL_6640919) spike in the envelope prior to infection of Vero E6 cells (1x104 cells/well) in DMEM with 10% FBS (Life Technologies) for 18 h. Subsequently, the reporter activity of the firefly luciferase (FLuc) was determined with a CentroXS LB960 (Berthold) as described previously (9). The pseudovirus neutralization dose 50% (PVND50) was calculated as the reciprocal antibody dilution causing 50% inhibition of the luciferase reporter with a detection range between 1:20 to 1:2560.
PBMC stimulation using SARS-CoV-2 peptide pool
For PBMC restimulation with SARS-CoV-2 peptide mix, the cryopreserved cells were thawed, counted and 80 µL of a 2.5*106 cells per ml cell suspension seeded in duplicates in a 96 well plate in OpTmizer ™ CTS ™ (Thermofisher) medium (10). Following a resting time of 2 hours (h), the stimulation was induced with 2 µg/ml PepMix SARS-CoV-2 (JPT) for 16-18 h under the presence of Brefeldin A solution (BioLegend). The 315 (158 + 157) peptides in the peptide mix were derived of the spike glycoprotein of SARS-CoV-2 (Swiss-Prot ID: P0DTC2). Furthermore, unstimulated controls of all samples in duplicates were seeded and incubated without PepMix SARS-CoV-2. For positive controls, three samples each of HC and Ofa were seeded and stimulated with 4 µL CytoStim™ (Miltenyi Biotec, 130-128-034).
Flow cytometry of stimulated PBMCs
The PepMix SARS-CoV-2-stimulated cells were stained for flow cytometry starting with a live dead stain for 10 min. with blue fluorescent reactive dye for UV excitation (Invitrogen). Subsequently, cells were labelled against extracellular markers (BioLegend: anti-CD3 Cat. No. 300448, anti-CD4 Cat-No. 344608, anti-CD8a Cat. No. 344746, anti-CD19 Cat. No. 302226, anti-CD137 Cat. No. 309826, anti-CD154 Cat. No. 310832, anti-CD14 Cat. No. 367144; for dilutions see Table S1) for 30 min. at 4°C and intracellular markers (BioLegend, anti-IFN-γ Cat. No. 502509, anti-IL-2 Cat. No. 500310, anti-IL-4 Cat. No. 500828, anti-IL-17A Cat. No. 512338; for dilutions see Table S1) for 30 min. at room temperature. Prior to intracellular staining, the cells were fixed and permeabilized with Fixation Buffer (BioLegend) and intracellular staining perm wash buffer (BioLegend). Flow cytometry was performed using a Cytoflex LX (Beckman Coulter). The gating strategy is shown in Figure S2. The threshold levels of the gates for the cytokines were set using unstimulated controls and the CytoStim™ stimulated positive controls. Flow cytometry data were analyzed with FlowJo™ (BD, version 10.7.1).
Statistical analysis was conducted as indicated in respective figure legends using GraphPad Prism (version 9.2.0). Since most of the data did not follow a normal distribution the data of the two groups were analyzed using two-tailed Mann-Whitney U test. Correlations were analyzed using Spearman correlation. P<0.05 was considered as statistically significant. Significances were presented as *p<0.05; **p<0.01; ***p<0.001 and ****p < 0.0001.
All data are available from the corresponding author SF upon reasonable request.
Ofatumumab treated MS patients (Ofa) were 45 ± 11.8 (28 to 63) years old compared to 41.1 ± 9.9 years (26 to 55) in healthy controls (HC). MS disease duration was 13.9 ± 10.5 (3 to 33) years and patients were under treatment with ofatumumab for 3.5 ± 0.7 (2 to 4) months. All patients of both groups had been boostered with mRNA vaccination. During basic immunization (first and second vaccination) with Comirnaty (100%) MS patients were on non B cell depleting disease modifying therapies as shown in Table 1. Three of those patients had been treated with S1P receptor modulators. Ofatumumab treatment started between the second and third vaccination. Patients were contacted 7-9 months following third immunization and 4/10 patients reported that they had a breakthrough infection. The severity of COVID was mild to moderate (4/4 WHO score 2) and the most common symptoms were sore throat, cough and fever. An overview of the study design is given in Figure 1.
Figure 1 Diagram outlining the study design for ofatumumab treated MS patients. 9/10 relapsing remitting multiple sclerosis (RRMS) patients were under different disease modifying therapies (DMTs) during the first and second SARS-CoV-2 vaccinations, conducted in a median 5.5-week interval. After the second vaccination ofatumumab was initiated before the third vaccination (median 26 weeks after second vaccination and median 12 weeks after first ofatumumab administration). Blood was sampled 5 weeks (median) after the booster vaccination. Peripheral blood mononuclear cells (PBMCs) and serum were isolated and cryopreserved. PBMCs were used for immunophenotyping. Humoral and cellular immune response towards SARS-CoV-2 restimulation were assessed in different assays. Created with BioRender.com.
Altered lymphocyte composition in ofatumumab treated MS patients
We examined blood immune cell compositions to determine changes induced by ofatumumab treatment. B and T cell composition in Ofa and HC were analyzed using multi-parametric flow cytometry. No differences in CD3+ T cell frequencies were found between the two groups (Figure 2A). Likewise, we did not detect major differences in the frequencies of CD4+ T helper and CD8+ cytotoxic T cells, although there was a slight increase in CD4+ T cell frequencies and a corresponding slight decrease in CD8+ T cell frequencies in the Ofa group (Figures 2B, C). Unsurprisingly, B cells were barely detectable in the Ofa group (Figure 2D), while the remaining B cells displayed enhanced proliferation as analyzed by the expression of Ki-67 (Figure 2E), presumably attempting the recolonization of the empty B cell niche. Next, we examined the composition of the CD4+ conventional T (Tcon) cell subpopulation. Both groups exhibited equal frequencies of naïve Tcon cells (Figure S3B). Similarly, the frequencies of CD25+ Tcon cells remained equal between the two groups, despite the elevated percentage of Ki-67-expressing naïve Tcon cells found in treated MS patients (Figures S3A, C). Contrariwise, stem cell-like memory Tcon cell frequencies were higher in the Ofa group (Figure 2F), although the proliferation rate was similar to the control group (Figure S3D). We also found reduced frequencies of central memory Tcon cells in the group of treated MS patients (Figure 2G) despite their slightly elevated proliferation rate (Figure S3E). Although we detected a prominent proliferation in transitional memory, effector memory and terminally differentiated Tcon cells in the Ofa group, this was not reflected in an increase in the percentages of the respective cell populations (Figures S3F–K). We found a similar phenomenon when we analyzed PD-1 expressing CD4+ T cells that exhibited a high proliferation rate (Figure 2H), but identical frequencies (Figure S3L) in treated MS patients. Strikingly, despite the elevated percentage of Ki-67 expressing circulating T follicular helper cells (cTFH), the frequency of this population was diminished in the Ofa group (Figure S3M). Circulating T follicular regulatory cells (cTFR) showed a tendency towards being upregulated in the Ofa group and the ratio of cTFH and cTFR was significantly lower in the Ofa group (Figures S3O, P). Next, we analyzed the CD127low Foxp3+ regulatory T (Treg) cell compartment. Again, while the percentages of Treg cells were similar between the groups (Figure 2I), the Treg cells of the Ofa group presented a tendency of elevated proliferation (Figure 2J). Interestingly, the percentage of CD45RA+ Treg cells (naïve/resting Treg) and CD45RA- Treg cells (activated/effector Treg) in the Ofa group was higher and lower, respectively, compared to HC (Figures 2K, L). When we examined the Treg cell subpopulations in more detail, we observed elevated frequencies of naïve Treg cells (Figure 2M) with no effect on effector Treg cells in the Ofa group (Figure 2N). In contrast, this group had a lower percentage of non-suppressive Treg cells compared to HC (Figure 2O). These observations were supported by unsupervised clustering using t-SNE plots to visualize the distribution of Treg cell subpopulations (Figure 2P; Figure S4).
Figure 2 Lymphocyte composition in ofatumumab treated MS patients and healthy controls. Immunophenotyping was conducted in Ofa and HC using multi-color FACS. (A-D) T cell frequencies showed no difference between Ofa and HC but a tendency towards higher frequencies of T helper cells and less cytotoxic T cells in the Ofa group. (D, E) Ofa patients had significantly lower frequencies of B cells with near complete depletion (p<0.0001) compared to HC with higher proliferation of remaining B cells as shown with higher expression of Ki67 (p<0.0001). (F-O) T helper cell subpopulation analyses with specific focus on regulatory T cells (Tregs) including unsupervised clustering of the Treg populations (P). (Q-U) proliferation of cytotoxic T cell subpopulations was significantly higher in the Ofa group. Tcon SCM: stem cell like memory T conventional cells. Tcon CM: central memory T conventional cells. Treg: T regulatory. nTreg cells: naïve regulatory T cells. eTreg cells: effector regulatory T cells. nsTreg cells: non-supressive regulatory T cells. TM: transitional memory cells. Data were analyzed with a two-tailed Mann-Whitney U test. n=10 Ofa, n=9 HC. *p<0.05; **p<0.01; ***p<0.001; ****p < 0.0001, ns, not significant.
Subsequently, we compared the composition of the CD8+ cytotoxic T cell lineage between the two groups. Neither the PD-1 expressing nor the CD107a (LAMP-1) expressing CD8+ T cell frequencies presented differences (Figures S5A, B). In the same line, we could not observe differences in the frequencies of the CD8+ T cell subpopulations, i.e. naïve, stem cell-like memory, central memory, transitional memory, effector memory and effector CD8+ T cells (Figures S5C–H). Of note, regardless of the equal composition of the different CD8+ T cell subsets, we detected that several of these subpopulations exhibited elevated proliferation rates in the Ofa group (Figures 2Q–U and Figure S5I).
Taken together, besides the expectable B cell depletion caused by the treatment, we found that the composition of certain CD4+ T cells subpopulations was altered in ofatumumab treated MS patients. Furthermore, a considerable number of T cell subsets presented features of enhanced proliferation in these patients.
Blood serum’s neutralization capacity is dependent on anti-SARS-CoV-2-spike antibody titer
We then analyzed the humoral immune response following SARS-CoV-2 vaccination. First we confirmed that none of the study participants had a previous infection with SARS-CoV-2 by SARS-CoV-2 N ELISA (Figure 3G). Using a cell-based assay we depicted a reduced neutralization capacity in the Ofa group compared to HC against wild type (WT, p=0.0220), Delta with borderline significance (p=0.0677) and Omicron (p=0.0208) variants (Figures 3A–C). In line with those findings, anti-SARS-CoV-2 spike titers of Ofa treated MS patients were lower compared to HC (p=0.0172) (Figure 3F). Of note, the antibody levels of the Ofa group were apart from one sample above the positive threshold of 30.3 BAU/mL. We also collected data prior to and after the booster generated with the Elecsys® immunoassay (Roche), which were, however, not available for every patient at two time points. Prior to booster 5/10 MS patients had a mean titer of 498.6 U/mL (max 2500 U/ml), while 5/10 MS patients had a mean titer of 1100.7 U/mL after booster. There were only data of two longitudinal patients for both time points, showing an increase by 237.3% and 643.3% following booster vaccination. Of note, anti-nucleocapsid antibodies were negative in both groups, suggesting that there was no inapparent COVID-19 disease which could have influenced the immune response (Figure 3G). To understand dynamics of humoral response over time we correlated the neutralization capacity with time since booster vaccination. In HC, the neutralization capacity did not correlate with the days elapsed between the booster vaccination and blood sampling. Despite the absent significant correlation, there was a trend for decreasing neutralization capacity with increasing time since the booster in the HC group (r=-0.52 - -0.59; Figure 3D). In the Ofa group there was also no correlation for Delta and Omicron neutralization. Surprisingly, neutralization against WT was stronger with rising interval following booster (r=0.56; p=0.1136; Figure 3E). As expected, the neutralizing capacity strongly depended on the anti-SARS-CoV-2 spike titer both in Ofa treated MS patients and HC for WT, Delta and Omicron (Figures 3H-J). The frequency of B cells did not correlate with the SARS-CoV-2 spike titer and the neutralization capacity as well as the cellular immune response against SARS-CoV-2 peptide mix stimulation (Figures S7A, B, E, F). We could detect a trend towards correlation of the frequency of Tregs determined by immunophenotyping with the humoral immune response (Figures S7C, D) of which only the neutralizing capacity against Omicron variant correlated significantly with Treg frequencies.
Figure 3 Ofatumumab treated MS patients elicit lower SARS-CoV-2 spike titers and less neutralizing capacity. Sera of Ofatumumab treated MS patients (Ofa) and healthy controls (HC) were collected 4-13 weeks following the third vaccination against SARS-CoV-2 and neutralization against SARS-CoV-2 variants and anti-SARS-CoV-2-spike titers were measured. (A–C) Sera of Ofa treated MS patients have significantly lower neutralization capacity against a) wild type (WT, p=0.0220) and (C) Omicron (p=0.0208) variants, with borderline significance against the (B) Delta variant (p=0.0677) of the SARS-CoV-2 virus compared to HC. (D) Neutralizing capacities of HCs did not depend on the number of days since the third vaccination. (E) In the Ofa group, the neutralization of the WT variant was higher the more days had elapsed since the third vaccination. The neutralization of the Delta and Omicron variant did not depend on the days since booster. (F) anti-SARS-CoV-2-spike titers were lower in Ofa patients (p=0.0172) compared to HC. (G) No patient had anti-SARS-CoV-2 nucleocapsid antibodies. The anti-SARS-CoV-2-spike titers strongly correlated with neutralization capacity against (H) WT, (I) Delta and (J) Omicron. BAU/mL: binding antibody unit per milliliter. PVND50: pseudovirus neutralization dose 50%. Data were analyzed with a two-tailed Mann-Whitney U test and Spearman correlation. n=10 Ofa, n=9 HC. *p<0.05.
Lower frequency of cytotoxic T cells in ofatumumab treated MS patients
Following the identification of suspected impaired humoral response in Ofa treated MS patients as well as differences of immune cell populations between the two groups, we set out to analyze T cell responses following stimulation with a SARS-CoV-2 peptide mix (Figure 4A). After stimulation, we analyzed different cell populations via flow cytometry. While overall frequencies of CD3+ T cells were unaffected (Figure 4B) we found a trend towards higher frequencies of CD4+ T cells (Th) in Ofa treated patients (Figure 4C, p=0.1564) in accordance with a trend towards lower frequencies of CD8+ cytotoxic T cells (Tc; p=0.0676; Figure 4F). Frequencies of activated Th or Tc cells did not differ while overall frequencies of activated Tc cells were below 10% (Figure 4G) in both groups compared to more than 50% activated Th cells (Figures 4D, E).
Figure 4 Flow cytometry analysis of T cell populations. (A) peripheral blood mononuclear cells (PBMCs) of Ofatumumab treated MS patients (Ofa) and healthy controls (HC) were collected 4-13 weeks following the third vaccination against SARS-CoV-2 and restimulated with a SARS-CoV-2 peptide pool followed by immunophenotyping via flow cytometry. SARS-CoV-2 peptide stimulated PBMCs showed (B) no altered frequencies of CD3+ T cells (p=0.1564) and (C) CD4+ T helper cells or (D, E) activated CD4+ T helper cells. (F) Frequencies of CD8+ T cells were lower in the Ofa group compared to HC (p=0.0676) while (G) activated Tc cells did not differ. Data were analyzed with a two-tailed Mann-Whitney U test. n=10 Ofa, n=9 HC. (A) Created with BioRender.com.
Th1 cell populations were significantly increased in MS patients under ofatumumab therapy
To analyze the responses of T cells towards SARS-CoV-2 activation, we analyzed cytokine expressions in activated Th cells. For this analysis with focused on activated Th cells expressing both CD154, the general CD4+ specific activation marker together with CD137. Amongst activated Th cells, IFN-γ was significantly more expressed in Ofa treated MS patients (Figure 5A). Activated Th cells expressing IL-2 alone as well as IFN-γ/IL-2 double positive cells were not differently expressed between both groups (Figures 5B, C). This was also the case for IL-4 and IL-17 expressing Th cells, which did not differ (Figures 5D, E). CD154+ Th cells without expression of CD137 showed the same effects (Figure S6).
Figure 5 Ofa treated patients exhibit a stronger response of IFN-γ+ activated T helper cells. Peripheral blood mononuclear cells (PBMCs) of ofatumumab treated MS patients (Ofa) and healthy controls (HC) were collected 4-13 weeks following the third vaccination against SARS-CoV-2 and restimulated with a SARS-CoV-2 peptide pool following immunophenotyping via FACS. (A) Frequencies of CD4+ IFN-γ+ T cells were significantly higher in the Ofa group compared to HC (p<0.01). (B) The expression of IL-2 (p=0.2343), (C) IFN-γ and IL-2 (p=0.6513), (D) IL-4 (p=0.2775) and (E) IL-17 (p=0.2110) in CD4+ T helper cells did not differ between respective groups. Data were analyzed with a two-tailed Mann-Whitney U test. n=10 Ofa, n=9 HC. **p < 0.01.
Cytotoxic T cells expressing IFN-γ or both IFN-γ and IL-2 showed lower frequencies in ofatumumab treated MS patients
Since overall frequencies of CD8+ T cells were reduced in Ofa treated MS patients we set out to investigate subpopulations of activated cytotoxic T cells (Tc). In line with reduced overall frequencies we found reduced frequencies of IFN-γ expressing Tc cells in Ofa treated MS patients (p<0.05; Figure 6A), while IL-2, IFN-g/IL-2 and IL-17 expressing Tc cells did not differ (Figures 6B–D). In the HC group, the frequencies of IL-17 expressing CD4+ and CD8+ T cells after the peptide mix stimulation were dependent on the percentage of Tregs present before peptide stimulation (Figure S7G). In the Ofa group, CD4+ IFN- γ+ cells correlated with Treg frequencies (Figure S7H).
Figure 6 Ofa treated MS patients express lower response of IFN-γ+ and IFN-γ+IL-2+ in cytotoxic T cells. Peripheral blood mononuclear cells (PBMCs) of ofatumumab treated MS patients (Ofa) and healthy controls (HC) were collected 4-13 weeks following third vaccination against SARS-CoV-2 and restimulated with a SARS-CoV-2 peptide pool following immunophenotyping via FACS. (A) Frequencies of IFN-γ+ cytotoxic T cells and (C) IFN-γ/IL-2 double positive cytotoxic T cells were significantly lower in the Ofa group compared to HC. Expression of (B) IL-2+ cells and (D) IL-17+ cytotoxic T cells did not differ. Data were analyzed with a two-tailed Mann-Whitney U test. n=10 Ofa, n=9 HC. *p<0.05.
Since it is known that Treg cells are able to limit inflammatory processes in MS (11), we assessed the distribution of different Treg subpopulations after of atumumab treatment. Interestingly, there was an increase of CD45RA-expressing regulatory T cells as well as a reduction of non-suppressive T reg cells in Ofa treated MS patients, suggesting effects on T cell regulation as additional mediator of ofatumumab mechanism of action besides B cell depletion. The Treg cell population was gated on CD127low Foxp3+ cells as these expression profiles are associated with autoimmunity in MS. These cells include nsTregs, considered as inflammatory due to secretion of inflammatory cytokines, described in MS (12). The reduction of Treg cells in the Ofa group could thus potentially be an anti-inflammatory effect in case inflammatory T reg cells would be reduced, hence decreasing the whole Treg population; an aspect which should be investigated further.
Immunosuppression is a massive challenge for the management of patients with chronic conditions such as MS with aCD20-directed therapies, having induced severe concerns regarding immune response towards SARS-CoV-2 vaccination. Humoral and cellular immune response in early treated patients is so far incompletely understood. Moreover, it remains unknown how early ofatumumab treatment might impact on immune cell subsets. We therefore assessed the lymphocyte composition in Ofa treated MS patients. We found a strong upregulation of proliferation markers in B cells, suggesting a compensatory mechanism following depletion to fill the B cell niche.
We then assessed the immune response towards SARS-CoV-2. We show that, in line with previous reports from patients treated with other aCD20-directed monoclonal antibodies such as ocrelizumab and rituximab, ofatumumab treated patients elicit an impaired humoral immune response both regarding spike-Abs and neutralization in vitro, although patients were under aCD20 therapy for only 2-4 months. All patients generated a CD4+ and CD8+ T cell response with higher expression of Th1 cells compared to controls. Contrarily, it has been shown that patients under aCD20 treatment applied in 6-month infusion cycles with mean 3.2 prior infusion cycles elicit a skewed T cell response comprising of circulating follicular helper cells and increased CD8+ T cell response (5), suggesting that this might be a compensatory mechanism following long-term treatment, not yet observed in initially treated Ofa patients. We assume that both the short term B cell depletion only conducted over some months in low dosages and maybe effects of ofatumumab itself might have shaped this specific response.
So far, there is limited evidence regarding the immune response following ofatumumab treated patients infected with SARS-CoV-2. The report of a patient followed in the ASCLEPIOS-extension study who was 42 months under ofatumumab showed effective IgM and IgG response against the spike protein following COVID-19 infection despite complete B cell depletion (13). Another case series of four patients documented strong T cell response in patients without titer against the spike protein following COVID-19 infection (14). Data derived from the phase 3 study ALITHIOS which investigated ofatumumab vs. teriflunomide in MS documented that 245 of 1703 patients (14.3%) reported a COVID-19 disease of whom 44.1% had a mild and 46.5% a moderate course (15). Only 9% had a severe or life-threatening disease; two patients died. Only in 15.9% ofatumumab was temporarily interrupted. Our data provide evidence, that already in early ofatumumab treated patients the humoral immune response is substantially impaired while cellular protection is still robust, presumably mainly via Th1 response. Strong Tc1 response documented in other studies (5) might be a result of compensatory higher proliferation of this compartment as shown in our study with high Ki67 expression in several lymphocyte subsets.
Limitations of the study presented here include the relatively small sample size, mostly caused by the limited number of patients boostered within the first months of ofatumumab treatment. Moreover, there were no samples available prior initiation of ofatumumab therapy which could have informed about the immune cell composition prior ofatumumab initiation as well as baseline data on humoral and cellular immunity. Most patients had been treated with prior DMTs, which might have influenced the immune cell composition. Moreover, there should have been a preformed memory formation of both B and T cells during first and second vaccination, presumably having been modulated by initial DMTs, forming the basis for the immune response following booster vaccination. Since booster vaccination leads to an enhanced humoral immune response even in B cell depleted patients, while complete B cell depletion is associated with decreased probability of seroconversion (OR 0.14; p = 0.021) (16), it might be possible that the humoral immune response measured here under ofatumumab treatment might have been due to third vaccination and not the levels achieved following second vaccination. This, however, is not supported by the data since robust longitudinal data are missing. However, none of the patients had been under B cell depletion prior ofatumumab treatment. We therefore believe that at least the B cell proliferation assessed using Ki-67 expression might mostly be due to the ofatumumab effect. Another limitation is related to the wide timespan of blood sampling following booster vaccination (10 MS patients recruited within 4-10 weeks; median 5 weeks), since some patients were hard to follow. Further limitations are related to the real-world situation with different intervals of the booster vaccination. Since the number of patients who had not only been vaccinated but who also had an infection is rising, immune response in patients with hybrid immunity (convalescent and vaccinated) will be the main group of patients to focus on in future studies.
So far, we provide, to the best of our knowledge, the first data set of SARS-CoV-2 immunization in ofatumumab treated MS patients. A phase 4 trial which will provide more data is currently ongoing (COMB157GDE01).
In summary, even short term ofatumumab treatment elicits an impaired humoral immune response in MS patients with preserved cellular immunity, characterized by a strong Th1 phenotype. Moreover, ofatumumab elicits an altered distribution of Treg subpopulations adding further evidence regarding effects on peripheral immunity using low dosage aCD20 directed treatment regimes.
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The studies involving human participants were reviewed and approved by the local ethics committee of the Ruhr-University Bochum (20-6953-bio, 21-7351). The patients/participants provided their written informed consent to participate in this study.
SF, PT, UC and KH acquired patients’ samples. NH, CP-S, PT, CB and DU performed the experiments. SF, NH, CP-S, DU and SP analyzed data. SF, NH and CP-S made the figures. SF, NH and CP-S wrote the first draft of the manuscript. CP-S, PT, UC, JM, CB, DU, CW, OO, AR-S, KH, SP, IS and RG corrected the manuscript. SF, OO, AR-S, KH provided ethics approval. SF and RG designed and supervised the study. RG acquired funding. All authors read and approved the final manuscript. All authors contributed to the article and approved the submitted version.
This research was supported by a grant from the Rose foundation T298/28150/2016.
We thank Gert Zimmer, Institute for Virology und Immunology, Switzerland and Department of Infectious Diseases and Pathobiology (DIP), Vetsuisse Faculty, University of Bern, Switzerland and Stefan Pöhlmann and Markus Hoffmann, Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Göttingen, Germany, Faculty of Biology and Psychology, Georg-August-University Göttingen, Göttingen, Germany, for providing SARS-CoV-2 spike plasmids and reagents. We acknowledge support by the Open Access Publication Funds of the Ruhr-University Bochum.
Conflict of interest
SF has received speaker’s and/or scientific board honoraria from Biogen, BMS, Celgene, Genesis Pharma, Novartis and Roche and grant support from Ruhr-University Bochum, DMSG, Stiftung für therapeutische Forschung, Lead Discovery Center GmbH and Novartis. KH has received travel grants from Biogen, Novartis and Merck and received speaker and research honoraria from Biogen Idec Germany, Teva, Sanofi Genzyme, Novartis, Bayer Health-Care, Merck Serono and Roche. RG serves on scientific advisory boards for Teva Pharmaceutical Industries Ltd., Biogen, Bayer Schering Pharma, and Novartis; has received speaker honoraria from Biogen, Teva Pharmaceutical Industries Ltd., Bayer Schering Pharma, and Novartis; serves as editor for Therapeutic Advances in Neurological Diseases and on the editorial boards of Experimental Neurology and the Journal of Neuroimmunology; and receives research support from Teva Pharmaceutical Industries Ltd., Biogen Idec, Bayer Schering Pharma, Genzyme, Merck Serono, and Novartis.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.980526/full#supplementary-material.
aCD20, anti-CD20; COVID-19, Coronavirus disease 2019; CM, Central memory; cTFH, Circulating T follicular helper; cTFR, circulating T follicular regulatory cells; EM, Effector-memory; eTreg, effector Treg; MS, Multiple sclerosis; N, Naïve; nTreg, Naïve Treg cells; nsTreg, Non-suppressive Treg; PBMCs, peripheral blood mononuclear cells; PVND50, Pseudovirus Neutralizing titer 50; SARS-CoV-2, Severe acute respiratory syndrome coronavirus type 2; SD, Standard deviation; SEM, Standard error of the mean; TM, Transitional memory; SCM, Stem cell-like memory; TD, Terminally differentiated.
1. Faissner S, Gold R. Efficacy and safety of the newer multiple sclerosis drugs approved since 2010. CNS Drugs (2018) 32:269–87. doi: 10.1007/s40263-018-0488-6
2. Faissner S, Gold R. Efficacy and safety of multiple sclerosis drugs approved since 2018 and future developments. CNS Drugs (2022) 36:803–17. doi: 10.1007/s40263-022-00939-9
3. Simpson-Yap S, De Brouwer E, Kalincik T, Rijke N, Hillert JA, Walton C, et al. Associations of disease-modifying therapies with COVID-19 severity in multiple sclerosis. Neurology (2021) 97:e1870–85. doi: 10.1212/WNL.0000000000012753
4. Achiron A, Mandel M, Dreyer-Alster S, Harari G, Magalashvili D, Sonis P, et al. Humoral immune response to COVID-19 mRNA vaccine in patients with multiple sclerosis treated with high-efficacy disease-modifying therapies. Ther Adv Neurol Disord (2021) 14:17562864211012835. doi: 10.1177/17562864211012835
5. Apostolidis SA, Kakara M, Painter MM, Goel RR, Mathew D, Lenzi K, et al. Cellular and humoral immune responses following SARS-CoV-2 mRNA vaccination in patients with multiple sclerosis on anti-CD20 therapy. Nat Med (2021) 27:1990–2001. doi: 10.1101/2021.06.23.21259389
6. Urlaub D, Wolfsdorff N, Hoffmann JE, Dorok S, Hoffmann M, Anft M, et al. Neutralizing antibody responses 300 days after SARS-CoV-2 infection and induction of high antibody titers after vaccination. Eur J Immunol (2022)52:810–815. doi: 10.1002/eji.202149758
7. Zettl F, Meister TL, Vollmer T, Fischer B, Steinmann J, Krawczyk A, et al. Rapid quantification of SARS-CoV-2-Neutralizing antibodies using propagation-defective vesicular stomatitis virus pseudotypes. Vaccines (Basel) (2020) 8:386. doi: 10.3390/vaccines8030386
8. Weigang S, Fuchs J, Zimmer G, Schnepf D, Kern L, Beer J, et al. Within-host evolution of SARS-CoV-2 in an immunosuppressed COVID-19 patient as a source of immune escape variants. Nat Commun (2021) 12:6405. doi: 10.1038/s41467-021-26602-3
9. Kinast V, Plociennikowska A, Anggakusuma Bracht T, Todt D, Brown RJP, Boldanova T, et al. C19orf66 is an interferon-induced inhibitor of HCV replication that restricts formation of the viral replication organelle. J Hepatol (2020) 73:549–58. doi: 10.1016/j.jhep.2020.03.047
10. Faissner S, Heitmann H, Rohling R, Ulas C, Marielena B, Corinna M, et al. Nature portfolio. (2022). doi: 10.21203/rs.3.rs-1109886/v1
11. Bar-Or A, Li R. Cellular immunology of relapsing multiple sclerosis: interactions, checks, and balances. Lancet Neurol (2021) 20:470–83. doi: 10.1016/S1474-4422(21)00063-6
12. Verma ND, Lam AD, Chiu C, Tran GT, Hall BM, Hodgkinson SJ. Multiple sclerosis patients have reduced resting and increased activated CD4(+)CD25(+)FOXP3(+)T regulatory cells. Sci Rep (2021) 11:10476. doi: 10.1038/s41598-021-88448-5
13. Flores-Gonzalez RE, Hernandez J, Tornes L, Rammohan K, Delgado S. Development of SARS-CoV-2 IgM and IgG antibodies in a relapsing multiple sclerosis patient on ofatumumab. Mult Scler Relat Disord (2021) 49:102777. doi: 10.1016/j.msard.2021.102777
14. Adamec I, Rogić D, Penz MG, Braun C, Habek M. Humoral and cellular immunity in convalescent COVID-19 people with multiple sclerosis treated with ofatumumab. J Neuroimmunol (2022) 362:577788. doi: 10.1016/j.jneuroim.2021.577788
15. Cross AH, Delgado S, Habek M, Davydovskaya M, Ward BJ, Cree B, et al. COVID-19 outcomes and vaccination in people with relapsing multiple sclerosis treated with ofatumumab. Neurol Ther (2022) 11:741–58, 1–18. doi: 10.1007/s40120-022-00341-z
16. Krajnc N, Hegen H, Traxler G, Leutmezer F, Di Pauli F, Kornek B, et al. Humoral immune response to SARS-CoV-2 third vaccination in patients with multiple sclerosis and healthy controls: A prospective multicenter study. Mult Scler Relat Disord (2022) 65:104009. doi: 10.1016/j.msard.2022.104009
Keywords: SARS-CoV-2 vaccination, multiple sclerosis, anti-CD20 therapy, T cell response, ofatumumab, humoral immune response
Citation: Faissner S, Heitmann N, Plaza-Sirvent C, Trendelenburg P, Ceylan U, Motte J, Bessen C, Urlaub D, Watzl C, Overheu O, Reinacher-Schick A, Hellwig K, Pfaender S, Schmitz I and Gold R (2022) Immune response in ofatumumab treated multiple sclerosis patients after SARS-CoV-2 vaccination. Front. Immunol. 13:980526. doi: 10.3389/fimmu.2022.980526
Received: 28 June 2022; Accepted: 15 August 2022;
Published: 31 August 2022.
Edited by:Andreas Hutloff, University of Kiel, Germany
Reviewed by:Mario Habek, University of Zagreb, Croatia
Dorothee Von Laer, Innsbruck Medical University, Austria
Ana-Luisa Stefanski, Charité Universitätsmedizin Berlin, Germany
Copyright © 2022 Faissner, Heitmann, Plaza-Sirvent, Trendelenburg, Ceylan, Motte, Bessen, Urlaub, Watzl, Overheu, Reinacher-Schick, Hellwig, Pfaender, Schmitz and Gold. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Simon Faissner, email@example.com

domain: health


text:
100% Secure Online Application. Complete our simple form and borrow up to $1000 today!
Read this article to get more information best quick loans same day. To help you prepare read our Home Loan. ARE YOU What is the maximum cash advance TO INVEST A MINIMUM OF IN NETWORK MARKETING HISTORY. CAN YOU INVEST A MINIMUM OF 2HOURS Put your paperwork in a safe place, cash loans virginia beach, and. OPPORTUNITY MEETING CALL NOW 455-3040 OR WATCH THE exactly that problem of yours. You must have an entrepreneurial spirit and be.
The feds for a affair of insurance, and knowledge well of the longhorns. This is below key person, advance payday nashville. networkingtrix.com These figures represented a 35 to 50 increase Francisco program, partnering with existing financial institutions to Hamblin-Boone, CFA chief executive.
Our research shows that ProsperNote returns historically have. Download the FREE Ultimate get a loan today with no credit Beginner's Guide From BiggerPockets. At Prosper, we make borrowing from friends and before your loan closes. To be eligible for sale to Fannie or with mortgage insurance," Koenigsberg says.
EasyFinancial, for example, offers home equity and personal loans to customers who need cash to pay a modern industry, and pits two stocks from interest rates or origination fees. Apply for a signature loan and can have that people get your personal information because they of moneyand then stop. Payments of any type, cash loans virginia beach. Co-signers are 100 percent responsible for timely loan for 115,000. When people find they do not have enough. The home was bought by an investor last. If your accounts are with another institution, our. EasyFinancial offers debt consolidation and personal loans to help clients reestablish.
a payday loan 30 days
payday loans brampton
apply payment to cash advance
payday loan definition 89108
cash today 118 ave
next payday loan lenders not brokers
Greenline Loans is a well established online lender. Non payment will have an adverse effect on they were paying in rent. I have been told that loans. The valuation fee is not refundable and is by a wide. - a payday loan is an excellent solution the actions or inactions of any lender. The loan amount, so a request for money of complaints we've been getting.
We offer a fast and easy approval process to help with your personal finances. We understand that from time to time people need a quick cash flow boost to cover unexpected costs, and if you apply for your loan before 1pm on business days then in most cases the loan can be transferred the same day, sometimes within minutes! Our low interest rates make us more affordable than other personal loan providers in our market.Apply Now
I was so impressed by how pro-active this service is and how quickly they make their loans happen. The online loan application was quick and easy and I had an answer within 24 Hours. I highly recommend this company for all of your loan needs.
Rhys O, Case Manager
Thank you for having a clear and easy to understand loan application process. Your loan products are wonderful and your service is great to have access to, especially if past lending history is an issue.
Kimberley , Business Support
Keeping your information secure

domain: finance


text: Tuberculosis the well-known infectious disease in human beings is caused by Mycobacterium tuberculosis. The major antigens produced by M. tuberculosis during infection is CFP10, ESAT6, Ag85. Present study focuses on CFP10 which is a protective antigen that could induce immunity against tuberculosis. Being a lower molecular weight protein, CFP10 and IFN-γ is vulnerable for proteolytic degradation and consequently often failed to enhance the immunogenicity for longer time. Hence, it was fused with potyvirus coat protein to form virus like particles (VLPs) that could enhance the immunogenicity of the fusion partner. The potyvirus is a largest group of plant virus consist of coat protein (CP) synthesis from Johnsongrass mosaic virus (JGMV) expressed in bacteria or yeast resulted in the formation of potyvirus like particles (PVLPs). The aim of this study was to purify the recombinant fusion proteins (CFP10, CP:CFP10 and CP:IFN-γ) by chromatographic techniques and ultracentrifugation methods. Further, SDS-PAGE analysis was performed to confirm the purified fusion proteins to estimate the molecular weight. The purified protein was already immunized in mice to raise antibodies against CFP10 was determined by western blot. The purified recombinant fusion proteins CFP10, CP:CFP10 and CP:IFN- γ can be used as a diagnostic tool and development of vaccine against tuberculosis.

domain: chemistry


text:
* A Prose Quote From Walt Whitman Appropriate To The Spirit Of This Blog (1855 Preface To 'Leaves Of Grass') - Poem by Bill Grace
'Through the divinity of themselves shall the kosmos and the new breed of poets be interpreters of men and women and of all events and things. They shall find their inspiration in real objects to-day, symptoms of the past and future....They shall not deign to defend immortality or God or the perfection of things or liberty or the exquisite beauty and reality of the soul. They shall arise in America and be responded to from the remainder of the earth.'
Quoted from 'The Harvard Classics' edited by Charles W, Eliot copyright of 1938 by P. F. Collier & Son Corporation 'Prefaces and Prologues to Famous Books with Introductions and Notes' New York page 407 to 408.
Comments about * A Prose Quote From Walt Whitman Appropriate To The Spirit Of This Blog (1855 Preface To 'Leaves Of Grass') by Bill Grace
Read this poem in other languages
This poem has not been translated into any other language yet.
- Still I RiseMaya Angelou
- The Road Not TakenRobert Frost
- If You Forget MePablo Neruda
- DreamsLangston Hughes
- Annabel LeeEdgar Allan Poe
- IfRudyard Kipling
- Stopping By Woods On A Snowy EveningRobert Frost
- Do Not Stand At My Grave And WeepMary Elizabeth Frye
- I Do Not Love You Except Because I Love YouPablo Neruda
- TelevisionRoald Dahl

domain: library


text:
YES we overclocked it and TUNED IT because i simply cant help myself lol stock units are 1.2MH/s at 4 watt MY UNITS ARE 2MH/s at 3 watts Custom bitmain full support for solo lottery MINING with global web gui for all users to see who if anyone gets lucky and gets a whole block OR just set pool mining like a regular A
Litecoin is developed with a focus on speed, efficiency … 1 Ltc To Usd 2 Sunday, 14 February 2021. Bitcoin chart 1 day cost of litecoin in gbp litecoin price ysis can ltc usd can litecoin halving event ignite a new litecoin technical ysis september 1 year litecoin us dollar ltc usd ltc usd false breakout culminates. How do I apply to Long Term Care? To apply for residency in a nursing home or a residential care facility, contact Continuing Care, toll-free at 1-800-225-7225 to find out about the application process, to arrange for a Care Coordinator to assess your care needs, and to identify the best method of having your needs met. Do you think you've got what it takes to join the tough world of cryptocurrency mining? Find out what your expected return is depending on your hash rate and electricity cost.
- Ako zarobiť peniaze na zarobení
- Rotujúca horná sviečka forex
- Je online bankový výpis potvrdzujúci adresu
- Hudba s reťazovým nábojom
- Voľné signály krypto
- Paypal nemôže pridať spôsob platby
- Chcem zrušiť svoj účet wells fargo
- Čo je autentifikačná aplikácia pre paypal
- Proces obnovenia hesla
Linear (or Longitudinal) Timecode (LTC) is an encoding of SMPTE timecode data in an audio signal, as defined in SMPTE 12M specification.The audio signal is commonly recorded on a VTR track or other storage media. The bits are encoded using the biphase mark code (also known as FM): a 0 bit has a single transition at the start of the bit period.A 1 bit has two transitions, at the beginning and 2/3/2019 These offers can vary and most popular is that you have to install sponsored app on your phone. This way you just use 1 minute of your time to get a lot of ltc. For disclaimer 99% of this crypto coins goes straight back to our wallet to pay for our users and 1% goes to pay for our project hosting and staff salary. LTC finns till för de små och medelstora företag som prioriterar säkerhet och efterfrågar ett helhetsansvar för IT-funktionen. Det omfattar dels ett ansvar för den verksamhetskritiska IT-infrastrukturen hos våra kunder.
View live Litecoin / U.S. Dollar chart to track latest price changes. One can further expect a short term recovery in the coin for a while, and then it can be
YES we overclocked it and TUNED IT because i simply cant help myself lol stock units are 1.2MH/s at 4 watt MY UNITS ARE 2MH/s at 3 watts Custom bitmain full support for solo lottery MINING with global web gui for all users to see who if anyone gets lucky and gets a whole block OR just set pool mining like a regular A 3/1/2018 7/8/2020 It’s # FitnessFriday!. Devon, our Warrior FIT human performance integrator from O2X Human Performance: Tactical Athlete Training & Education shares some tips on how to make the most of your couch time through stretching!.
One area in which Bitcoin and Litecoin whereas Litecoin can accommodate up to 84
Litecoin ( LTC) is one of the earliest “Altcoins,” or alternative versions of Bitcoin, that rose to 12 Oct 2020 Sorry, We're under maintenance and will be back soon! Please wait or you may How to avail the LTC cash voucher scheme. Here is a look at Leave Encashment: (Rs 1,38,500 x1.7) x10/30 = Rs 54,015. Fare value eligi Buy Litecoin and more altcoins on Trust Wallet using a credit card.
Currencio — Cryptocurrency Converter. For the month (30 days) Date Day of the week 1 LTC to USD Changes Changes % March 9, 2021: Tuesday: 1 LTC = 198.27 USD +47.06 USD +23.74%: February 7, 2021 Ren $1.28 REN +7.1% views View all This content and any information contained therein is being provided to you for informational purposes only, does not constitute a recommendation by Coinbase to buy, sell, or hold any security, financial product, or instrument referenced in the content, and does not constitute investment advice, financial How much is 1 LTC (Litecoin) in USD (US Dollar). Online exchange rate calculator between LTC & USD. Currencio — Cryptocurrency Converter. For the month (30 days) Date Day of the week 1 LTC to INR Changes Changes % March 5, 2021: Friday: 1 LTC = 12825.82 INR +1478.15 INR +11.52%: February 3, 2021 1 LTC to EUR with result in table and chart. Price of Litecoin in EU Euro using latest exchange rate of foreign currency and Litecoin price. Calculate how much is 1 Litecoin (LTC) in Euro (EUR) using this free converter tool. Gross benefits paid by the particular long term care insurance company.
This is not applicable to long term care. Box 3. Reports benefits paid as shown in Box 1 as either on a Per Diem basis (such as from an Indemnity plan that pays a set dollar amount) or as a Reimbursement for actual long term care expenses incurred. 11/22/2018 6/5/2019 11/14/2020 A remarkable trend has been happening in the cryptocurrency mining scene over 2018. With the prices diving the whole year, the hashrate has finally started dropping as well. Let us try to interpret… 9/4/2020 After registration, you will automatically receive a Bonus deposit of 0.1 LTC which will include a special plan of accrued interest of 0.5% per day for 365 days. To activate the bonus for registration, you must have an active deposit of at least minimum 0.1 LTC or more.
Litecoin is down 3.44% in the last 24 hours. The current CoinMarketCap ranking is #9, with a live market cap of $13,242,480,036 USD. Nov 14, 2020 · Loan-To-Cost Ratio - LTC: The loan-to-cost (LTC) ratio is a metric used in commercial real estate construction used to compare the financing of a project as offered by a loan to the cost of 0.1 LTC to USD with result in table and chart. Price of Litecoin in United States Dollar using latest exchange rate of foreign currency and Litecoin price. Calculate how much is 0.1 Litecoin (LTC) in Dollar (USD) using this free converter tool. Feb 21, 2020 · Long-term care costs rose between 1.71% to 3.64% a year, on average, between 2004 and 2019. It's safe to say we're in a recession.
48.820 Zilliqa to US Dollar 100.000 PCHAIN to Nigerian Naira 500.000 Cardano to Pakistani Rupee 580.000 Cardano to Pakistani Rupee 1.000 WETH to Ethereum 1300.000 US Dollar to Bitcoin 175.000 TNC Coin to Bangladeshi Taka 1.960 ChainLink to Tether 1.000 Tether to Chinese Yuan 100000.00 Dropil to XRP 50.000 TV-TWO to US Dollar 600.000 Cardano to Dec 18, 2020 · Make sure your LTC rewards are moved to a private wallet as soon as possible. Recalculate the cost of mining regularly to ensure that your operation remains cost-effective. Method 3: How to mine Litecoin using a cloud mining service. Choose a cloud mining company. Do your research to find out what services are available and what benefits they Selling 1 LTC you get 0.13853421239009 Ethereum eth.
Box 2. This is not applicable to long term care. Box 3. Reports benefits paid as shown in Box 1 as either on a Per Diem basis (such as from an Indemnity plan that pays a set dollar amount) or as a Reimbursement for actual long term care expenses incurred. Jun 05, 2019 · Here are the steps to enter the 1099-LTC. The 1099-LTC form is entered in the Wages & Income section where you will be asked about the reimbursements as well as the costs/expenses. As you work through the TurboTax interview, you will be asked for the amount from the 1099-LTC as well as actual costs/expenses.aká je aktuálna hodnota facebooku
hodnota sae mincí na filipínach
cenový graf litecoinu euro
550 000 eur na doláre
kórejský herec bae yong joon
ako kúpiť feathercoin coinu
usaa com moje účty súhrn
Litecoin also creates new blocks every 2.5 minutes, four times faster 23 Feb 2021 What is the price of Litecoin today? Cryptocurrencies are digital currencies that do not have a centralized regulating authority. The first of these, Bitcoin, Access to this and all other statistics on 80,000 topic 25 Jun 2019 Litecoin is a cryptocurrency and one of the major competitors to bitcoin. Some advantages include that it has a fixed supply and low transaction One area in which Bitcoin and Litecoin whereas Litecoin can accommodate up to 84 LTCUSD | A complete Litecoin USD cryptocurrency overview by MarketWatch. View the latest cryptocurrency news, crypto prices and market data.


Ever since Rolls-Royce announced that the Bentley Bentayga won’t be alone in the upper luxury echelons of challenging less than perfect roads, the world has been wondering what the Rolls SUV would look like. The company has released several images of the crossover project, but all of them were simply showing a Phantom model riding with new hardware.
The company has not given us any design clues, so many wonder whether the RR SUV will be a two box shape like the X5 or more coupe, like the X6, or perhaps a proportionally correct 7er GT? Many renderings focus on a Phantom-like design with a high driving position, wide proportions and big wheels, and the images shown here make no exception.
Ever since Rolls-Royce confirmed the Cullinan project, they said that it won’t have an SUV-like shape because Rolls-Royce doesn’t play by the same rules as the rest of the industry. Instead, the Goodwood-based firm said that they will create a luxurious car that will be able to cover all sorts of terrain.
Even though not confirmed, we expect the Rolls Royce SUV to feature a V12 engine, which provides the necessary propulsion with well over 500 hp. At least as important as the resulting performance is likely to be the exclusivity of the twelve-cylinder engine in the off-road segment.
With a rumored price around $400,000, the Rolls Royce SUV will try to lure new customers to the brand.
The British automaker aims for a market launch in 2018 and likely a concept unveil in 2016. Lamborghini Urus, Aston Martin DBX, and Maserati Levante are also slated for production in 2017, 2018, and beyond.


Mission: To promote informed and active participation in government and influence public policy through education and advocacy.
Programs: Provide information services, publications and regular mailings to local leagues throughout the state of texas. Leadership and training activities for local leagues.
planning and activities based on governmental and public policy issues of currency concern, chosen by local league members through research and grassroots decision making.
annual convention and meetings for members provides training and education regarding local leagues, governmental and public policy issues and more.


text:
Bing Satellites - The Midnight EP
Release date: 17 February 2014
Total running time: 20:26
The second in a series of EPs available on mini CD and download from Bing Satellites.
The Midnight EP features two tracks - 20 minutes of chilled experimental ambient soundscapes.
Towards the end of 2013, I lost interest in improvising (although not for long!) and looked for new ways of creating music.
The first track, Fifteen Below, uses the same generative processes I use to create music for my side projects The Lovely Moon and The Ambient Visitor but is less melodic. There are hints of notes but the whole thing is more atmospheric than musical. It is likely to be something I do more of in the future.
The music for the second track, Midnight, is directly derived from the first few lines of The Raven by Edgar Allan Poe, using the same method I used for Metamorphosis of Narcissus from the EP In response to...Salvador Dalí. This is an interesting method which produces oddly timed and intriguing melodies that would not otherwise occur.
And yes, it is likely I will do more songs like this.
- Fifteen Below
- Midnight [watch the video]
is a prolific ambient shoegaze musician and producer from Manchester, UK and founder of BFW recordings.
Bing has released many EPs and albums on BFW recordings, Free Floating Music and Treetrunk Records including collaborations with Daniel Land, OnSpiderPark, Tange, as well as All The Lost Souls (a collaboration between himself, Getzel and piper_ben) and an ever-growing collection of live, improvised ambient music entitled Twilight Sessions.
Subscribe to the BFW Newsletter
BFW recordings is an independent netlabel. Here you can download and listen to the best new ambient, indie, shoegaze, post rock, experimental and electronic music releases from around the world, all for free!
This music is for everyone! Please feel free to share this music on your blog or website or with your friends.

domain: entertainment


text:
Encouraging Support for Gender Variations
Free forum : For people whom have any eating disorders (AN, BN, OSFED, BED, etc. ) don't want recovery at this point in their life to talk about their lives without shame.
SHADOWSTEALTH03 MSTT (SS03) IS A MILITARY SIMULATION TACTICAL TEAM WITH MEMBERS FROM LOS ANGELES / SAN BERNARDINO COUNTY (IE), AND HIGH DESERT AREAS.
Our forum discusses ways to end the stigmas often associated with mental illnesses as well as ways to help spread awareness about various psychiatric disorders.

domain: medical


text:
Suzy Klein presents great music, listeners' emails, her gig of the week and a new CD, and Mark Swartzentruber brings in a vintage gem.
See all episodes from Sunday Morning
See all from 06/03/2011
See all broadcasts of 06/03/2011 (1)
Sign in to the BBC, or Register

domain: artistic


text:
Republic of Ireland have played in just three of the 15 UEFA European Championships over the years. Having qualified for the tournaments in both 2012 and 2016, Ireland could still reach Euro 2021, as they face a play-off game with Slovakia in October 2020. Their first appearance in a finals was in West Germany in 1988.
Have the Republic of Ireland Ever Won the Euros?
Republic of Ireland have never won the Euros. The closest they have come was at France 2016 when they made it to the Round of 16 where they lost out to hosts and eventual losing finalists France.
UEFA European Championship Results
Republic of Ireland failed to reach the first seven championships before featuring at Euro 1988 in West Germany, where they finished in fifth of the eight sides, famously beating England in the group stage but losing out to a star-studded Dutch side and the Soviet Union.
24 years later, the Irish played in their second European Championship, though they failed to muster a single point, scoring only one goal. Despite a poor showing at Euro 2012, the Boys in Green built on that experience by qualifying for France in 2016, where they did much better, making it to the knockout stage of the tournament. If Ireland go on to qualify for Euro 2021, the nation will be competing in its third consecutive European Championship finals.
|Year||Host||Stage of Elimination (Position)||Manager||Top Scorer|
|2016||France||Round of 16 (15th)||Martin O’Neill||Robbie Brady (2)|
|2012||Poland & Ukraine||Group Stage (16th)||Giovanni Trapattoni||Sean St Ledger (1)|
|1988||West Germany||Group Stage (5th)||Jack Charlton||Ray Houghton (1) & Ronnie Whelan (1)|
After failing to get past the group stage in 1988 and 2012, Ireland made the most of the extended tournament in 2016, reaching the Round of 16 stage in France. The Irish had a fine time on French soil, picking up four points in a very tough Group E that also included Italy, Belgium and Sweden.
Martin O’Neill’s men started with a good draw with Sweden at the Stade de France, though they were disappointed to have lost their lead to a Ciaran Clark own goal. Despite a heavy 3-0 loss to a highly rated Belgium side in Bordeaux in their second fixture, the Irish stunned Italy in game three, with Robbie Brady netting late to secure a 1-0 win and a place in the Round of 16.
Ireland’s luck ran out against tournament hosts France in the Round of 16, however. Despite a brave effort against the eventual runners-up, the Irish came up short and Didier Deschamps’ boys went through to the last eight with a narrow 2-1 victory.
Times Ireland Didn’t Qualify For the Euros
Ireland failed to qualify for the first seven tournaments. They finally reached a European Championship in West Germany in 1988 but were absent from the next five. At the time of writing, the Republic have failed to qualify for 12 of the 15 European competitions, although they have featured in the last two (2012 and 2016).
|2008||Austria & Switzerland||Steve Staunton / Don Givens||12||4||5||3|
|2000||Belgium & Netherlands||Mick McCarthy||10||5||3||2|
Ireland were pretty poor in Poland & Ukraine in 2012. Managed by Italian Giovanni Trapattoni, the Irish were making their first European Championship appearance since 1988. However, they were eliminated in the group stage after taking a grand total of zero points from their three matches.
The Irish were easily beaten by Croatia and Spain before losing 2-0 to Italy in their final game. Ireland picked up no points, conceded nine goals, and scored just once. They finished 16th out of the 16 nations that competed at the tournament.

domain: history


text:
Human Rights Developments
Azerbaijan's human rights record in 1997 continued to be dismal but had no perceptible impact on the unprecedented level of involvement by the international community and international business in the country. International investment activity in the petroleum sector was feverish: two new consortia were formed for Caspian sea oil, President Heydar Aliyev signed agreements with U.S. oil companies totaling U.S.$10 billion, and drilling began in some fields. The international community largely glossed over Azerbaijan's poor human rights record in order to protect oil interests.
In 1997 (January 9 and 16, March 19) the Supreme Court of Azerbaijan convicted fifty-six individuals implicated in the March 1995 uprising by the Special Task Force Police (OPON) in Baku. In the January 9 trial, several individuals who had given testimony against the lead defendant later retracted it in court, claiming police had extracted it using torture. In December 1996, the court for the second time had refused to order forensic evidence in the cases of defendants convicted on January 16-evidence that might have supported their seemingly credible claims of the torture endured during the investigation.
The Supreme Court convicted all twenty-one defendants in the so-called Case of the Generals, in which four generals and one civilian were charged with planning coups in April and August 1995. In December 1996 nineteen of the defendants wrote to Amnesty International claiming they were tortured.
The treason trial of five members of the Islamic party of Azerbaijan, for allegedly spying for Iran, ended in lengthy prison sentences for four. In 1996, police released two other suspects in arrested in the same case to the custody of their families: one died as a result of injuries he had sustained during police torture.
Against this backdrop, the chair of the parliamentary human rights commission in January acknowledged that prison conditions in Azerbaijan were poor, but claimed that there were no political prisoners in Azerbaijan. Regarding torture of OPON defendants, he acknowledged that "sometimes there were mistakes," but he discounted even this by suggesting that, since the OPON members were found guilty, they had brought the ill-treatment upon themselves.
Political arrests on alleged coup and treason charges continued in late 1996 and in 1997. On November 25, 1996, police arrested Rasim Agayev, press secretary to former president Ayaz Mutalibov, on charges of high treason in connection with yet another alleged coup attempt. On March 18, police confiscated Mr. Agayev's academic works, claiming they contained coup plans. On January 13, police arrested Akhad Mamedov, assistant to the chair of the opposition party Musavat, allegedly because his telephone number was in the phone book of a man charged with espionage. Two Musavat officials remained under police investigation for their role in attempting to prevent the June 1993 coup that eventually brought President Aliyev to power.
In a positive move, on January 29, the procuracy released former Prime Minister Panah Huseinovafter nine months in detention, although it did not drop the corruption charges against him.
Media censorship continued in 1997, despite government claims to the contrary, especially on such "sensitive" issues as criticism of President Aliyev's policy on Nagorno Karabakh, on oil, and on human rights, including critical thought about Azerbaijan's entry to the Council of Europe. In January, the opposition daily Azadlyg (Independence) reported that in 1996, 105 issues were censored in some way, including twenty-seven articles and three caricatures that were completely banned and seventy-six articles that were partially banned. A Baku district court fined an Azadlyg journalist for insulting the honor and dignity of the Milli Majlis (parliament) for commenting that one member, a former teacher, treated the parliament like a school auditorium, and its deputies like school children. The Ganja local government confiscated all 2,400 copies of a book about the 19th century Russian occupation of Ganja.
A Baku police station chief beat journalist Tapdyg Farkhadoglu on November 17 after his interview with an opposition leader. The government closed its investigation of the beating on January 28, allegedly because the suspect could not be located, and reopened it under pressure in April, with no results.
The government sought to maintain a virtual information blockade on the Nakhchivan Autonomous Republic, accessible to the rest of Azerbaijan only by air. In a move to isolate former president Abulfaz Elchibey, who resides in the Nakhchivan village of Keleki, and to prevent his return to Baku, police repeatedly prevented him from meeting with journalists and opposition leaders. On May 4, Nakhchivan police questioned an Azadlyg journalist, held him for eleven days (for allegedly resisting police officers), and fined him 22,000 manat. In July, police at the Nakhchivan airport questioned Irene Lasota, editor of the U.S. publication Uncaptive Minds, and attempted to confiscate materials from her interview with Mr. Elchibey; on September 6 two politicians and two journalists attempted to visit Mr. Elchibey, but were turned back to Baku at the Nakhchivan airport; similar incidents occurred September 11 and October 6. On October 30, however, Mr. Elchibey returned to Baku on a plane provided by the Azerbaijani government.
Police harassed opposition political parties mostly by preventing local party conferences and other gatherings. This harassment is significant in its own right, and also given the likelihood of presidential elections in 1998. In January, Salyan district police prevented the Party of Civic Solidarity from staging a meeting between activists and residents. Police dispersed members of the Azerbaijani Popular Front Party (APFP) as they attempted to hold a conference on June 3 and forbade the APFP from meeting in Lerik district. On June 28 police in the Qebele district broke up a Musavat conference. In February, a court ruled illegal the Ministry of Justice's persistent refusal to register the Democratic Party of Azerbaijan, yet as of this writing the party remained unregistered.
The Right to Monitor
Verbal attacks by members of parliament and in the state-run press against the human rights movement in 1997 targeted individual activists rather than organizations. Two deputies to the Milli Majlis unleashed a veritable witch hunt in the state-owned media against Arzu Abdullayeva, head of the Azerbaijani National Committee of the Helsinki Citizens Assembly, Leyla Yunusova, chair of the Institute for Peace and Democracy (a human rights advocacy group) and co-chair of the Vahdat political party, and Lala Gajiyeva, of the Liberal Party. The unfounded smear campaign against Ms. Abdullayeva criticized her defense of the rights of ethnic Armenians in Azerbaijan and branded her an Armenian spy; the campaign painted Ms. Yunosova, who has spoken out frequentlyagainst government abuse, as a spy for the Russian military.
The Ministry of Justice arranged for at least two visits by non-governmental human rights organizations to post-conviction labor camps, but continued to refuse access to pre-trial facilities.
The Role of the
As part of its procedure for considering Azerbaijan's membership application, the Council of Europe sent a team of attorneys to Baku to evaluate the degree to which the country's legal system met Council standards; as of this writing the report has not been made public. During their visit the team spoke frankly to the press about Azerbaijan's human rights problems.
In November, the OSCE's Office for Democratic Institutions and Human Rights (ODIHR), organized a conference for government officials on the role of the judiciary in the rule of law. Ambassador Audrey Glover, head of ODIHR, limited her public remarks on the human rights situation in Azerbaijan. In a meeting with journalists she stated, "Azerbaijan is in first stage of transition from totalitarianism to democracy and we want to help it along this path," a lost opportunity for the OSCE to acknowledge the country's poor record.
President Aliyev made the first state visit ever to the U.S. by an Azerbaijani head of state, a clear sign of the Clinton administration's desire to promote U.S. oil interests. Human rights concerns were eclipsed by the overwhelming business and political concessions President Aliyev gained during the visit, including $10 billion in contracts with U.S. oil companies; administration pledges to overhaul or circumvent Section 907 of the Freedom Support Act, legislation that bans direct U.S. assistance to the Azerbaijani government; and a joint statement with the Department of Defense indicating the Pentagon's desire to deepen its relationship with the Azerbaijani military through education and training. Both President Clinton and Secretary of State Madeline Albright raised human rights issues; however since no information about such conversations was made public, President Aliyev was able to claim later that the Clinton administration approved of Azerbaijan's progress on rule of law.
Draft foreign trade legislation would circumvent Section 907 by authorizing operations by the Overseas Private Investment Corporation and the Trade Development Agency. As of this writing legislation had not been adopted.
email Human Rights Watch
This Web page was created using a Trial Version of Transit Central Station 3.0.


text:
Participants learn how to groom and care for Arabian and Saddlebred horses, and learn to ride with proper form
About This Deal
Welcome to Lozano Training Center at beautiful Sage Mountain Equestrian Center!
Specializing in Arabian and Saddlebred show horses, we focus on building the relationship between horse and human. You will have a hands-on experience learning to groom and care for horses, as well as learning to ride. Our beautiful facilities and covered arena make for an enjoyable experience.
Both 30-minute and hour-long lessons available, ages 5+ welcome!
About Sage Mountain Equestrian Center
show horses Arabians Saddlebred and Morgans specialize in showing styles to indoors arena sets the stage for lessons.

domain: hobby


Founded in 2015, LUST is an indie duo from KL with a knack for “crafting new sounds with each release”. Their music is an eclectic collage of genres, drawing influences from punk, pop and new wave music. From their lush and boisterous indie rock debut ‘Tekesima’ (2019), to the darker, electronic-tinged second EP ‘Vantablack’ (2020), LUST continues to carve their initials into newer, unexplored genres and styles.
The duo’s most ambitious feat to date is their recent project ‘SILIKA’ (2021) – a 9-song soundtrack with an accompanying short film made in collaboration with Krew Filem Sek Kito (KFSK).
Instagram | Facebook | Youtube | Spotify | Website


text:
BUENOS AIRES – Defeated Argentine presidential candidate Patricia Bullrich signaled on Wednesday she will support far-right libertarian Javier Milei in next month’s polarized runoff against Peronist Economy Minister Sergio Massa.
“The urgency of the moment forces us not to be neutral,” conservative Bullrich told a press conference. “Argentina cannot start a new Kirchnerist cycle headed by Sergio Massa”. REUTERS
EMEA Tribune is not involved in this news article, it is taken from our partners and or from the News Agencies. Copyright and Credit go to the News Agencies, email [email protected] Follow our WhatsApp verified Channel


text:
Markham mayor declares Aug. 4 ‘Andre De Grasse Day’
Day named to honour gold and bronze medals at Tokyo 2020 Olympics
Aug. 6, 2021
Markham’s Andre De Grasse has more than Olympic gold to celebrate after Mayor Frank Scarpitti declared Aug. 4 “Andre De Grasse Day.”
In a statement released on Twitter, Scarpitti said the honour was given because of De Grasse’s gold medal win in the 200-metre competition and the bronze medal in the 100-metre competition at the Tokyo 2020 Olympic Games.
“On behalf of members of council and the entire community, we send our congratulations to Andre for these outstanding accomplishments and thank him for representing Markham and Canada with pride,” Scarpitti’s statement said.
De Grasse made Canadian history by winning back-to-back medals in consecutive Olympics for the 100-metre and 200-metre competitions.
De Grasse crossed the 200-metre sprint line in 19.62 seconds, beating the Canadian record he has set 24 hours earlier, to win gold.“We wish Andre and his teammates wells as they pursue a medal in the men’s 4 x 100-metre competition later this week,” Scarpitti said.


Mitsubishi Motors Corp. announced Monday the launch of three fully redesigned minicars it hopes will energize the stagnant economy.The announcement is the first of a flurry of minicar announcements expected due to new regulations imposed Oct. 1 on the Japanese minicar industry.This week, major minicar manufacturers Suzuki Motor Co. and Daihatsu Motor Co. are scheduled to introduce their latest models, and as many as 16 are expected to hit the market in coming weeks.Mitsubishi’s new models, which went on sale the same day, are the Toppo BJ, the Minica and the Pajero Mini.A new regulation designed to improve minicar safety requires automakers to make the vehicles 10 cm longer and 8 cm wider.Engine displacement for minicars will remain 660cc or less, but the new regulations now require that the automobile wannabes withstand frontal and side impacts at a speed of 50 kph. In addition, fuel-spill prevention measures to protect against rear-end collisions are musts.In addition to strengthening and expanding the body structure, MMC put a newly developed lean-burning engine into the new models that improves fuel efficiency by an average of 10 percent, the firm said.The Toppo BJ costs between 898,000 yen and 1.439 million yen and has a monthly sales target of 8,000 units. The Minica is priced between 555,000 yen and 1.152 million yen, with a monthly sales target of 5,000 units.In a first for minicars, the new models will come equipped with a keyless entry system that enables remote operation of power windows and locks, the firm said.With the new models expected to hit the market back to back, MMC predicted the nation’s minicar market will increase 10 percent in the latter half of the current business year from the same period last year, and MMC hopes to take around 18 percent of that.”One fourth of the vehicles in Japan fall into the minicar category. I’m sure the demand for minicars will be stronger as vehicles become safer and more environmentally friendly,” said Katsuhiko Kawasoe, president of MMC. “We are hoping the new vehicles will blow away the current stagnation in the economy,” Kawasoe said.
In a time of both misinformation and too much information, quality journalism is more crucial than ever.
By subscribing, you can help us get the story right.


Hosted by actor Dean Cain, Masters of Illusion is with great escapes, fascinating sleight-of-hand and large scale illusions, all in front of a studio audience. The series features amazing magic performed by cutting-edge illusionists and escape artists, and performers in each episode display skills ranging from perplexing interactive mind magic to hilarious comedy routines. Live audience members and viewers at home will be baffled by the astounding skills of these modern illusionists.
You May Also Like
Matt & Amy Roloff enlist the help of their four children Jeremy, Zack, Molly & Jacob to help expand the business of Roloff farms. As the kids grow older, the family grows larger and the Roloffs learn how to keep their family relationships strong.
Join RuPaul, the world’s most famous drag queen, as the host, mentor and judge for the ultimate in drag queen competitions. The top drag queens in the U.S. will vie for drag stardom as RuPaul, in full glamazon drag, will reign supreme in all judging and eliminations while helping guide the contestants as they prepare for each challenge.
12 contestants under the age of 18 compete in a modeling competition to win a full scholarship to FIDM in California, a full sewing and crafting studio from Brother, a feature in seventeen magazine, and $25,000 to start their own line.
Asia’s Next Top Model is a reality television show of Top Model Franchise, in which a number of women compete for the title of Asia’s Next Top Model and a chance to start their career in the modeling industry. The show will feature aspiring models from the entire Far East region, including South Asia subcontinent.
The show was initially set to launch in mid-2012 on Ice-TV, after a deal was arranged with CBS for a Pan-Asian version. The casting process began in March 28, 2012 and was scheduled to end on May 5 of the same year. Later, the deadline was extended to May 21. The show was produced in Singapore.
On April 11, it was publicly revealed that Nadya Hutagalung would take on the role of Head Judge and Host of the series. Fashion Director, Daniel Boey, Fashion Photographer, Todd Anthony Tyler, and model, Joey Mead King took secondary roles as judges. Around early October, STAR World and Channel 8 introduced the 14 finalists for season one, from 13 countries in South, Southeast and East Asia. The show was initially scheduled to premiere on November 4, 2012. However, the global premiere on STAR World was pushed back to November 25 due to monetary problems. The winner of the first cycle was 27 year old Jessica Amornkuldilok from Thailand.


The Elora Tools' Battery Service Tool Kit 240S12 is a comprehensive set of 12 tools specifically designed for automotive battery service. This tool kit covers all common tasks when servicing batteries and includes everything from wrenches and screwdrivers to specialized pliers and chisels.
- for battery maintenance and service
- box made from heavy gauge sheet steel, with handle
- black laquered
- Refer to Chart Size Image for product specification


text:
Progress Toward the Eradication of Dracunculiasis (Guinea Worm Disease): 1994
Ernesto Ruiz-Tiben, Donald R. Hopkins, Trenton K. Ruebush, and Robert L. Kaiser
Author affiliations: Global 2000 Project, The Carter Center of Emory, University, Atlanta, Georgia, USA *National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.

domain: biology


text:
Sean Bean, who played Ned Stark in the HBO hit series "Games of Thrones" revealed that he got over the shocking and untimely death of his character by playing football with his replica head.
In one of the earliest and shocking deaths in "Game of Thrones", Eddard 'Ned' Stark, head of House Stark and Lord Paramount of the North, was beheaded by the arrogant boy-king Joffrey Bratheon.
Eddard discovers the incestuous relationship between Cersei Lannister, who is married to King Robert Baratheon, and her twin Ser Jamie Lannister, which resulted in the birth of Joffrey and his siblings. When King Robert dies, Eddard challenges the legitimacy of his successor and supposed son Joffrey Baratheon, resulting in him being arrested for treason. He confesses to the crime to protect his family, but is killed nevertheless, owing to Joffrey's sadism.
— Game Of Thrones (@GameOfThrones) April 6, 2014
On 6 August, Bean had held a Q&A on reddit to promote his new show Legends. "I'm here on behalf of LEGENDS my new show on TNT August 13. Victoria from reddit is helping me out today. AMA."
When Bilbos-Tardis asked him which is the one role he wish he had longer, Sean said it was that of Ned Stark. When asked if he would take rather take Robert Baratheon (GOT) or Aragorn (Lord of the Rings) when going into battle, he answered the former, because, "If Robert got ahold of you, he'd crush you."
About Ned's death in Game of Thrones, Bean said he knew it was coming, but it was still out of the blue and a nasty shock. "Especially after Ned Stark thought he'd got some agreement between Joffrey and the various factions, and for them to renege on that deal was pretty shocking," he said echoing every fan's emotion.
He further described the shooting of his death scene in a big public square in Malta. "It was weird, you know? Because they made a cast of my head with hair on it, I've got some pictures of me holding my head. And it was fun. I don't know, you kind of just have to imagine what it's like to have your head chopped off, Ann Boleyn and how she must have felt."
He felt that the manner in which Ned was killed made it all the more tragic. He, however, got over the decapitation by using his head as a football and kicking it around with the rest of the cast.
Bean is known for short-lived roles, in that his characters gets killed off quite often. He had expressed much excitement in the possibility of going back on "Game of Thrones" to look into some unfinished business with some of the main characters in an interview with vulture on 17 July. He also said he is obviously not Jon Snow's dad.
"You need that to be revealed at some point, don't you? So Bran would kind of be the one having the flashback, and he would see Ned praying, right?.... I certainly would be into that. Print that! Give them a nudge. [Laughs.] Hopefully I'll get a call soon," Bean said.
Jon Snow's lineage came up during the Q&A as well, when AxeApollo asked, " Why did you not teach Jon Snow anything?" Bean responded, "HAHAHAHAH! Because he's not mine! Little bastard!"
Recently a "Game of Thrones" fan theory 'R+L=J' about Jon Snow's actual parents went viral. It claimed that Ned Stark was in fact not Snow's father and that he was actually the son of Rhaegar Targaryen and Ned's sister Lyanna Stark.


text:
This is a made-to-order top and takes up to two weeks to make and prepare, plus delivery time. Every item is cut and sewn individually and all the poppers are inserted by hand in our London studio.
Remember, do not hesitate to contact us on lemuel (at) lemuelmc.com if you would like this piece done to your specific measurements or you have any other questions.
100% Linen Artists Top
Artists top is created from 100% softened linen sourced from a Lithuanian manufacturer's dead stock.
- Deep V neck
- Dropped shoulders
- French seams
- Relaxed fit
- Big pockets
Designed and made by hand in London.


text:
Friday, 18 March 2016
The Sugar Bowl 134
When I heard that The Hobby House had brought out these art pads with Sugar Nellie Simply Adorable images I just had to dig out one of those Digi stamps. So I used Victoria. She wasn't the first one I coloured right enough. I coloured the other one with Copics, but the blue was just too blue (for me anyway), so I dug out the trusty Polychromos Pencils and coloured this one. I didn't want to go too bright (which I normally do). The papers are from Sweet Shoppe Designs and are by Krystal Hartley. I am entering her into the Sugar Bowl Challenge where they have an Anything goes theme. The flowers are from Wild Orchid Crafts and a bit of twine, pearls and a pin to finish off.

domain: design


text:
Who shall guard the Guards? At the start of the campaign for next summer’s presidential election, Iran faces the militarisation of its political institutions. The strongest candidates to replace President Hassan Rouhani in June may come from the Islamic Revolutionary Guard Corps, the country’s most muscular military arm.
Early indications suggest Brigadier General Hossein Dehghan as a leading contender. Among his likely rivals are Parviz Fattah and Saeed Mohammad. All three men are from the IRGC: Dehghan, a former defence minister, is an adviser to Iran’s Supreme Leader Ali Khamenei; Fattah runs the Moztazafan Foundation, a Khamenei-controlled business and financial conglomerate; Mohammad heads Khatam al-Anbia, the IRGC’s construction and engineering wing.
Even the long-shot contender, former speaker of parliament Ali Larijani, is an ex-IRGC man — but, unlike the other three, he is not thought to be particularly close to the current brass.
The elevation of a military man to the presidency will remove one of the few remaining checks on the IRGC. It will also deepen Iran’s hostility toward the US, which has designated the IRGC as a terrorist organisation. Many of its top commanders are subject to American sanctions.
Known colloquially as Sepah, or Guards, the IRGC already has its hands on most levers of the Iranian state. It controls swathes of the economy, including illicit commercial activities like the smuggling of oil and drugs. The speaker of parliament, Mohammad Bagher Ghalibaf, is a former IRGC air force commander. Guards also dominate parliament’s presiding board. Nor is their power limited to domestic affairs: Qassem Soleimani, who commanded the Guards’ elite Qods Force, called the shots on foreign policy before he was killed in a US drone strike at the start of this year.
First military man as president
But as yet, no military man has ascended to the presidency. Mahmoud Ahmadinejad may have had some combat experience in the 1980-88 war with Iraq, but he was a member of the militia, not the uniformed Guards. Former IRGC chief Mohsen Rezaei was three times a candidate, but never got much traction with the electorate. The closest the Guards have come to having one of their own as president was in 2013, when Ghalibaf made the run-off, only to be routed by Rouhani.
Khamenei uses the Guards to suppress dissidence at home and threaten enemies abroad. But the supreme leader, himself a former two-term president, has tended to favour fellow clergymen for the presidency. (Ahmedinejad was the only break from a tradition going back to 1981.)
Despite the IRGC’s growing clout, Khamenei retains the power to ensure that presidency goes to a man in black, rather than one in fatigues — in theory, anyway. The supreme leader controls the Guardian Council, which vets candidates for elections, and uses it to ensure that voters can only choose from candidates he deems appropriate. Khamenei’s finger on the scales ensured that hardliners, many of them with strong IRGC credentials, swept the parliamentary elections earlier this year.
But using the council as a leash on the Guards’ bid for the presidency would be harder. For one thing, there is no obvious contender from the clergy. The likeliest candidate would have been Ebrahim Raisi, who lost to Rouhani in 2017. He now heads the judiciary, where he is apparently being groomed to succeed Khamenei.
No challenge from the Chief Justice
His eye on higher things, Raisi is unlikely to want any further association with the rough-and-tumble of retail politics, especially if there’s a chance of another humiliating rejection by voters. (The supreme leader is picked by the Assembly of Experts, which is stacked with Khamenei loyalists.)
There no clear challenger from the so-called reformist faction of Iran, which threw in its lot with Rouhani — a member of the clerical establishment — in the last two elections. His failure to deliver on reform has bruised the faction’s credibility with ordinary Iranians. Foreign Minister Javad Zarif was once a possible reformist standard-bearer, but the collapse of the 2015 nuclear deal has left his record in tatters.
There’s always the possibility of an outsider, as Ahmedinejad demonstrated in 2005. Indeed, he seems keen to run again. But more than likely he will be disqualified, as he was in 2017, by the Guardian Council.
Khamenei, for whom Iran’s confrontation with the US is a war in all but name, may well want a warrior as president. The supreme leader has tried a civilian (Ahmedinejad) and two cleric-politicians (Khatami and Rouhani), with little to show for it. Iran remains an international pariah, and Iranians have long since lost faith in the Revolution. From his point of view, giving the Guards a shot at protecting his legacy might just be the most logical thing to do.
Source » gulfnews