text:
PDL241, a novel humanized monoclonal antibody, reveals CD319 as a therapeutic target for rheumatoid arthritis
- Jacky Woo†1, 3,
- Michel PM Vierboom†2,
- Hakju Kwon1Email author,
- Debra Chao1,
- Shiming Ye1,
- Jianmin Li1,
- Karen Lin1,
- Irene Tang1,
- Nicole A Belmar1,
- Taymar Hartman1,
- Elia Breedveld2,
- Vladimir Vexler1, 4,
- Bert A 't Hart2,
- Debbie A Law1, 5 and
- Gary C Starling1, 5
DOI: 10.1186/ar4400
© Woo et al.; licensee BioMed Central Ltd. 2013
Received: 8 February 2013
Accepted: 19 November 2013
Published: 4 December 2013
Targeting the CD20 antigen has been a successful therapeutic intervention in the treatment of rheumatoid arthritis (RA). However, in some patients with an inadequate response to anti-CD20 therapy, a persistence of CD20- plasmablasts is noted. The strong expression of CD319 on CD20- plasmablast and plasma cell populations in RA synovium led to the investigation of the potential of CD319 as a therapeutic target.
PDL241, a novel humanized IgG1 monoclonal antibody (mAb) to CD319, was generated and examined for its ability to inhibit immunoglobulin production from plasmablasts and plasma cells generated from peripheral blood mononuclear cells (PBMC) in the presence and absence of RA synovial fibroblasts (RA-SF). The in vivo activity of PDL241 was determined in a human PBMC transfer into NOD scid IL-2 gamma chain knockout (NSG) mouse model. Finally, the ability of PDL241 to ameliorate experimental arthritis was evaluated in a collagen-induced arthritis (CIA) model in rhesus monkeys.
PDL241 bound to plasmablasts and plasma cells but not naïve B cells. Consistent with the binding profile, PDL241 inhibited the production of IgM from in vitro PBMC cultures by the depletion of CD319+ plasmablasts and plasma cells but not B cells. The activity of PDL241 was dependent on an intact Fc portion of the IgG1 and mediated predominantly by natural killer cells. Inhibition of IgM production was also observed in the human PBMC transfer to NSG mouse model. Treatment of rhesus monkeys in a CIA model with PDL241 led to a significant inhibition of anti-collagen IgG and IgM antibodies. A beneficial effect on joint related parameters, including bone remodeling, histopathology, and joint swelling was also observed.
The activity of PDL241 in both in vitro and in vivo models highlights the potential of CD319 as a therapeutic target in RA.
Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by chronic pain and joint damage characterized by synovial inflammation and hyperplasia. The pathology of RA is complex, with many different cell subsets playing a role in the disease initiation and progression [1]. One of the defining features of the disease is the presence of auto-antibodies in the serum, including rheumatoid factor (RF) and antibodies directed against cyclic citrullinated peptide [2]. Disease modifying anti-rheumatic drugs (DMARDs) include those targeting the underlying immune processes that drive the pathology, including small molecule immunosuppressive agents and biologics. The most widely prescribed biologic agents are blockers of the TNF-α pathway. Patients who become refractory to anti-TNF therapy may be treated with agents that target the IL-6 pathway (tocilizumab, binding the IL-6 receptor), prevent T cell costimulation (abatacept, which binds CD80 and CD86 [3]) or deplete B cells from the circulation (anti-CD20 mAb rituximab [4]). The production of auto-antibodies by cells of the B cell lineage prompted the investigation of anti-B cell therapies for treatment of RA [5]. However, B cell depletion has also been reported to affect other functions, including their ability to stimulate T cell proliferation, produce cytokines and assist in the development of lymphoid tissue architecture [6]. Despite the tremendous progress in the treatment of RA, a substantial group of RA patients have inadequate responses to current therapies or have safety issues. The presence of late stage plasmablasts as a marker of resistance in active RA patients non-responsive to anti-CD20 therapy [7] illustrates the need for therapies targeted against plasmablasts and plasma cells. CD20 is not typically expressed by immunoglobulin (Ig)-producing plasmablasts [8]. To this end, we attempted to identify new targets for development of RA therapeutics that target plasmablasts. Previous studies have demonstrated the expression of the cell surface glycoprotein CD319 on plasma cells [9], which became the focus of the current study.
CD319 (SLAMF7, CS1, 19A24, novel Ly9, CRACC) is a 66 kDa glycoprotein member of the SLAM superfamily [10]. Members of the SLAM superfamily share a common structure consisting of a membrane proximal C-type Ig fold and a membrane distal V-type Ig fold. The cytoplasmic region of CD319 contains two immunoreceptor tyrosine-based switch motifs (ITSM), which bind to SH2-only adapter molecules Src homology 2 domain protein 1A/SLAM-associated protein (SAP) and EWS-activated transcript-2 (EAT-2) [11, 12]. Phosphorylation of the tyrosine motifs leads to activation of downstream molecules including PLCγ1, PLCγ2 and PI3K kinases and modification of a variety of cell functions. As observed with other SLAM family members, CD319 engages in homophilic interactions which may potentiate cell activation [13]. Interestingly in the absence of EAT, CD319-CD319 interactions may exert a negative regulatory effect on natural killer (NK) cells [14]. Two CD319 transcripts have been identified in human NK cells, with a shorter form of CD319 (CD319-S) postulated to have a separate function from the longer form (CD319-L) due to its lack of ITSMs [15].
Expression of CD319 is restricted to cells of hematopoietic origin including plasma cells, resting NK cells, a subset of CD8+ T cells and plasmacytoid dendritic cells (DC), with minimal expression on resting B cells, resting CD4+ T cells and monocytes [9]. Upregulation of CD319 expression has been observed following activation of B cells, CD4+ T cells, monocyte-derived DC and monocytes [16] suggesting that CD319 may play a role in immune regulation. In support of this hypothesis, high CD319 expression has been observed on plasma cells or B cells from several disease indications including systemic lupus erythematosus [17], and the transformed cells in multiple myeloma [16] indicating the potential for CD319 as a therapeutic target for plasmablast and/or plasma cell-driven diseases. In this study, we investigated the expression of CD319 in RA tissues, and generated PDL241, a humanized monoclonal antibody (mAb), to target cells expressing CD319. CD319 was expressed on plasma cells in RA synovial tissues. PDL241 inhibited the production of immunoglobulins in an Fc-dependent manner in vitro by killing plasmablasts and plasma cells. Finally, PDL241 was tested for activity in a human-severe combined immunodeficiency (hu-SCID) mouse model of Ig production and a rhesus macaque model of collagen-induced arthritis (CIA). The data demonstrate the potential of CD319 as a therapeutic target in RA.
Immunohistochemistry analysis
Synovial tissues were obtained from 26 individuals with RA according to the approved protocol (PDL-011-04RA) reviewed by the Mayo Clinic Institutional Review Board. All patients gave their written informed consent after the risks and benefits of the study were explained. The 1G9 mAb, which recognizes an intracellular epitope of CD319 [9], was used to stain formalin fixed paraffin embedded (FFPE) tissues using an automated immunostainer (Dako North America, Carpinteria, CA, USA) with 3,3′-diaminobenzidine detection (Ventana Medical Systems, Tucson, AZ, USA). Double labeling studies were performed using 1G9 in combination with an anti-CD3, anti-CD20, anti-CD56 (LabVision, Fremont, CA, USA), anti-CD68 (Dako) and anti-CD138 (Invitrogen, Camarillo, CA, USA). VS38c mAb (Dako) was also used as a plasma cell marker. PDL241 was used to stain optimal cutting temperature (OCT) embedded frozen tissues from human or rhesus monkeys for immunohistochemistry (IHC) and immunofluorescence studies. AF488-conjugated streptavidin (Invitrogen) was used to detect PDL241 staining after prior incubation with biotinylated donkey anti-human Ab (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). AF555 or AF594- conjugated anti-mouse or rabbit secondary Ab (Invitrogen) were used to detect other cell surface markers in a co-staining study. Slides were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei.
Generation of PDL241
Female BALB/c mice (Taconic, Hudson, NY, USA) were immunized with purified CD319 protein and mAb were generated by fusing spleen cells to the NS0 fusion partner (American Type Culture Collection). Anti-CD319 specific mAb were identified using a variety of selection techniques including ELISA for CD319 protein, immunoblotting and flow cytometry analysis of CD319-expressing and CD319 non-expressing cell lines. The mouse parental mAb of PDL241 (m241) was selected for its ability to bind to CD319 protein from human and non-human primates (NHP). M241, a mouse IgG1, was chimerized to human IgG1 for initial functional characterization prior to humanization. Humanization of m241 was performed by the method of Queen et al. [18] and resulted in PDL241, which was engineered onto an IgG1 κ backbone with T250Q and M428L mutations in the Fc domain designed to extend the in vivo half-life via an enhanced binding to FcRn [19]. A FcR-binding deficient mutant of PDL241 (241-G2M3) was made by fusing the PDL241 variable domains to human IgG2M3 Fc domains [20]. F(ab)'2 fragments were produced by pepsin cleavage and purification on protein A. The negative control IgG1 mAb (cIgG1) for PDL241 used throughout this study was MSL109, a fully human anti-cytomegalovirus mAb [21]. Material for in vivo studies was produced in NS0 cells. All materials were tested for endotoxin (<0.02 EU/mg) and protein aggregation (<1%).
Peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMC) were obtained from the heparinized blood of normal volunteer donors (AbbVie Biotherapeutics or Stanford Blood Center, Palo Alto, CA, USA) by separation on 50 ml Leucosep™ tubes (Greiner Bio-one N.A. Inc., Monroe, NC, USA) or Ficoll-Paque Plus (GE Healthcare Lifesciences, Piscataway, NJ, USA). After two rounds of washes with PBS, PBMC were resuspended at 1 × 106 cells/ml in R-10 media (Roswell Park Memorial Institute (RPMI) containing 10% heat inactivated FBS, 1X Pen/Strep, 20 mM HEPES).
Cell staining and flow cytometry
To determine the phenotype of cells that were bound by PDL241, 2 × 106 PBMC collected from healthy individuals were labeled for analysis of lymphocytes, while 1 × 106 PBMC were used for analysis of plasma cells and dendritic cells. After the addition of Fc block (Miltenyi, Auburn, CA, USA), PBMC were incubated at 4°C for 30 minutes with fluorochrome-conjugated antibodies specific for cell surface markers CD3 (SK7 and SP34-2), CD4 (RPA-T4), CD8 (RPA-T8), CD56 (MY31), CD16 (3G8), CD14 (M5E2), CD19 (SJ25C1), CD27 (M-T271), CD38 (HB7), CD11c (B-ly6), CD123 (7G3), HLA-DR (L243), and lineage cocktail 1 (lin 1) (BD Bioscience, San Jose, CA, USA) and CD138 (B-B4, Miltenyi) with AF488 labeled PDL241. Cells were washed twice with PBS and then analyzed on a BD FACSCanto.novi Luc90 (AbbVie Biotherapeutics) is an anti-CD319 mAb that does not compete for CD319 binding with PDL241.
Pokeweed mitogen-induced IgM production assay
PBMC (2 × 105 in 200 μl) were treated with cIgG1 or PDL241 in the presence of 0.25 μg/ml pokeweed mitogen (PWM) (Sigma, St. Louis, MO, USA) in 96-well round bottom plates. At day 7, supernatants were harvested and the level of secreted IgM was measured using an Easy-Titer IgM assay kit (ICL Inc., Portland, OR, USA) according to the manufacturer's instructions. In some experiments, NK cells or monocytes were depleted from PBMC by positive selection using RoboSep® (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions.
Cell depletion experiments
% live cells were calculated as % of absolute cell number of test mAb over cIgG1.
Co-cultures of RA-synoviofibroblasts with PBMC
RA- synovial fibroblasts (SF) were purchased from Cell Applications, Inc (San Diego, CA, USA), and routinely maintained in synoviocyte growth medium (Cell Applications) at 37°C, 5% CO2. RA-SF (passage number >5) were seeded into 24-well plates at 6 × 104 cells per well and allowed to reach confluence for 24 hours. PBMC (6 × 105/well) were added to the confluent monolayers of RA-SF. The co-cultures were then treated with mAb (10 μg/ml) for seven days. At the end of the culture period, PBMC (suspension and adherent) were harvested by extensive washing followed by trypsin-ethylenediaminetetraacetic acid (EDTA). PBMC were washed once with fluorescence-activated cell sorting (FACS) buffer (PBS + 2% FBS), and subjected to FACS staining in FACS buffer containing cIgG1 or Luc90-FITC and IgD-PE; and CD38-PEcy7, CD27-APC, and CD19-APCcy7.
HuSCID model of IgM production
Human PBMC (2 × 107) were injected into the peritoneal cavity of six to eight week old NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJmice, The Jackson Laboratory, Bar Harbor, ME, USA) in 200 μl of PBS. For histological examination of human leukocyte cell surface markers, mice were sacrificed 14 days post PBMC transfer to assess the cell viability and the expression of CD319 on human T cells, B cells, NK cells and plasma cells in the spleens. Sections (5 μm) were cut from OCT-embedded frozen spleens for immunofluorescence analysis as described above. Plasma was obtained from blood samples by centrifugation, and stored at −20°C until the time of analysis. Human IgM levels were measured as described above. These studies were performed in compliance with the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals under a PDL Biopharma/Facet Biotech IACUC-reviewed and approved protocol.
CpG-driven IgM production in rhesus monkey PBMC
Rhesus PBMCs were isolated by Ficoll gradient separation from freshly drawn rhesus blood (California National Primate Research Center (Davis, CA, USA)). Cells were cultured in 96-well plates in R-10 medium. The TLR-9 agonist CpG-B DNA prototype ODN2006 was obtained from HyCult Biotech (Plymouth Meeting, PA, USA), and added to cultures at a concentration of 2.5 ug/ml. Cells were cultured for thirteen days, and the IgM levels in culture supernatants were determined by ELISA.
Rhesus monkey collagen-induced arthritis study
In accordance with the Dutch law on animal experimentation, the study protocol and experimental procedures were reviewed and approved by the Experimental Animal Care and Use Committee of the Biomedical Primate Research Centre (BPRC) before the experiments started. Animals were purchased from the Animal Science Department of the BPRC in Rijswijk, The Netherlands. CIA-susceptible monkeys were seronegative for the dominant class I major histocompatibility complex resistance marker Mamu-B26[22, 23]. The total study group comprised of 24 young adult, healthy rhesus monkeys (Macaca mulatta). During the study the monkeys were housed socially, where possible, in cages specifically designed to house NHP. The animals were offered a daily diet consisting of monkey food pellets (Hope Farms, Woerden, The Netherlands), fresh fruit and vegetables and bread. Drinking water was available ad libitum. Analgesic medication (Buprecare®, 0.3 mg/ml buprenorfine base; Schering-Plough B.V., Maarssen, The Netherlands) was given based on the assessment of the animal caretakers and BPRC's veterinary staff (start and dosing increased or stopped on the basis of behavioral changes). The ulcerative skin lesions developing at the immunization sites were treated with wound spray (Acederm; Intervet, Boxmeer, The Netherlands) each time that an animal was sedated, in order to prevent infection. The study protocol was reviewed and approved by the BPRC Experimental Animal Care and Use Committee.
For induction of CIA, chicken type II collagen (chCII) was dissolved in 0.1 M acetic acid to a final concentration of 10 mg/ml and mixed with an equal volume of complete Freund's adjuvant (CFA; DIFCO, Detroit, MI, USA). CIA was induced by injection of 1.0 ml emulsion (5 mg chCII/animal) into the dorsal skin distributed over 10 spots of 100 μl (one time only). Clinical signs were recorded by daily cage-side monitoring of behavioral changes (apathy, loss of appetite) or pain (avoidance of limb usage). Monkeys were sedated by intramuscular injection of 0.1 ml/kg of ketamine (10 mg/ml) for determination of bodyweight (an accepted surrogate disease marker for the CIA model), body temperature, blood collection and a physical inspection of the limbs for redness and/or swelling of the joints twice weekly. For the clinical and ethical management of the monkeys, observations were recorded using the integrated discomfort scoring scheme previously described [24–26].
To determine the efficacy of PDL241, groups were treated with either vehicle, PDL241 (30 mg/kg) or PDL241 (100 mg/kg). Eight animals per group ensured sufficient statistical power. The evaluation period was 70 days. Dosing solutions were given as a bolus infusion on days 7, 21 and 35. A planned fourth infusion on day 49 was cancelled due to the development of systemic infusion reactions in eight drug-treated animals (one death) shortly after the third infusion. We hypothesized that this infusion reaction was caused by the development of anti-drug antibodies (ADA) around day 35; therefore, a decision was made to cancel the fourth infusion. ADA were confirmed at the end of study using an ELISA-based method.
Overall clinical score, a composite score ranging from 0 to 5, was the primary endpoint. Serum C-reactive protein (CRP) levels, body weight loss, soft-tissue swelling (STS) count, serum levels of collagen-specific IgG and IgM, urinary excretion of collagen breakdown products hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) (biomarker of bone remodeling), histopathology and time to sacrifice were secondary endpoints. Following immunization, all monkeys developed an acute phase response (serum CRP level >50 mg/L after induction of arthritis), indicating that 100% of all animals showed characteristics of an ongoing severe inflammatory process. Urinary excretion of the collagen crosslinks HP and LP was determined twice weekly, starting from the day of CIA induction, as previously described [24, 27]. The levels of HP and LP were normalized to creatinine levels (nmol levels per mmol creatinine) to compensate for a possible dilution by spilled drinking water. Blood markers of CIA were examined. Blood for hematology and for serum chemistry was collected once a week; CRP analysis was performed twice a week. All hematological and clinical chemistry analyses were performed at the Laboratory for Clinical Chemistry (BPRC) on a Sysmex Sf-3000 (Goffin Meyvis, The Netherlands) and a COBAS INTEGRA-400+ (Roche, Almere, The Netherlands), respectively. Serum samples were collected twice weekly for analyses of rhesus anti-chCII antibody levels of the IgM and IgG isotype as described elsewhere [28].
Histological parameters of the joint were examined. One proximal (PIP) and one distal interphalangeal (DIP) joint of one toe and finger of each foot and hand (two fingers/toes with outward signs of inflammation; two fingers/toes with no visible signs of inflammation) were processed at the BPRC for histopathological examination for synovitis and/or bone/cartilage destruction. After fixation in 4% phosphate-buffered formalin, the bones were decalcified for at least three weeks in Kristensen's solution (17% formic acid in 1 M NaOH, pH 2.2). Decalcified bones were washed in tap water for 16 hours, dehydrated in ethanol/toluene, and embedded in paraffin. Sections of 2 μm thickness were cut and stained with hematoxilin/eosin. Histopathology was analyzed and graded by a pathologist blinded to the study. Histopathology was scored based on a histopathology grading system published by Pettit et al.[29]. This system quantifies the degree of inflammation, cartilage damage and bone damage on an arbitrary scale from 0 to 5.
Statistical analysis
Due to the development of neutralizing ADA prior to day 35, followed by the subsequent loss of drug exposure, the data analysis was limited to the period from day 0 to day 31 post induction. Late responders to CIA induction (CRP levels >100 mg/l after day 21) may have experienced a limited effect of the treatment because of the previously described development of neutralizing antibodies. Analysis was, therefore, performed on 'all animals' (n = 8/group) and on those animals that responded early to the induction (CRP levels >100 mg/l on or before day 21; 'early CRP onset group'; Placebo group n = 5; 30 mg/kg group n = 5; 100 mg/kg group n = 7). Statistical analyses were conducted using Prism 5 software (GraphPad Software, Inc. La Jolla, CA, USA). Statistical differences comparing the placebo-treated group at each time point with either treated group (30 mg/kg or 100 mg/kg) were determined using a two-tailed unpaired t test. Results with P <0.05 (*) or P <0.01 (**) were considered to be statistically significant.
Expression of CD319 in RA synovium
PDL241 binding to leukocytes
Fc-dependent inhibition of Ig production by PDL241
PBMC-RA synovial fibroblast co-culture
CD319 expression and activity of PDL241 in HuSCID mice
The presence of both human NK cells and CD319-expressing plasma cells provided the rationale to test the activity of PDL241 on human IgM production in this model. Detectable levels of human IgM on day 14 in the mouse sera ranged from 0 to 100 μg/ml, and treatment groups were block randomized from mice with serum concentrations of human IgM >10 μg/ml. Experiments in which a majority of mice had <10 μg/ml human IgM on day14 were terminated. Treatment groups were analyzed for the serum levels of human IgM post-treatment twice at approximately two week intervals. An example of a study showing significant activity of PDL241 on reducing human IgM levels is shown (Figure 5B). PDL241 significantly reduced the IgM levels in Hu-SCID sera in 6 of 11 experiments. The reason for the experiment-to-experiment variation in PDL241 activity is unclear, but may reflect the health of human FcR positive cells in the engrafted mice.
Evaluation of PDL241 activity on biomarkers of CIA in rhesus monkeys
Since binding of PDL241 is restricted to human and non-human primate CD319 and does not bind to CD319 from rodent species, including mouse and rat (data not shown), the effect of treatment with PDL241 could not be evaluated in rodent models of arthritis. Therefore, a NHP model of arthritis (collagen-induced arthritis, CIA) was utilized. Disease symptoms in the rhesus monkey CIA model are critically dependent on anti-collagen type II (CII) immunoglobulins [22, 25]. Given the effects of PDL241 on Ig production in vitro and in NSG mice, the hypothesis that PDL241 would reduce the severity of arthritic disease by inhibiting antibody production against CII was tested.
A separate pharmacokinetics/pharmacodynamics (PK/PD) study in rhesus monkeys designed to identify the optimal dosing strategy to ensure sufficient occupancy of CD319 on peripheral lymphocytes by PDL241 over the 70 day study was conducted prior to the efficacy study in the CIA model. Simulation modeling suggested that a regimen of 30 mg/kg every two weeks (q2w) for a total of four doses would maintain a saturating serum concentration of PDL241 for 70 days. A high dose (100 mg/kg) group was included in order to maximize the pharmacological activity of PDL241. This dose was selected as the maximum amount of drug that could be infused based on the formulation. No major toxicities had been observed at this dose level in a separate non-GLP multiple dose-range finding study in cynomolgus monkeys (data not shown). A group of 24 healthy rhesus monkeys (male/female) were immunized with chicken type II collagen (chCII) emulsified in CFA. All 24 animals developed an acute phase response (CRP >50 mg/L after day 7) characteristic of an ongoing severe inflammatory process during the course of the study. In this model, the association between early onset of CRP in immunized monkeys with the rapid loss of body weight (and, therefore, a more rapid and severe form of the disease), has been established [26]. We also observed the development of a strong ADA response in the majority of monkeys treated with PDL241 (data not shown). ADA responses are common in NHP treated with humanized mAb due to the xenogenic nature of the human immunoglobulin. The consequence of ADA in this study was reduced exposure of the mAb, which decreased the ability of PDL241 at the doses administered to be effective at later time points (data not shown). To this end, efficacy data in this study were analyzed from all animals and in a subset of animals with early onset of CRP based on the time the serum CRP reached 100 mg/L after immunization ('early CRP onset'). The subgroup analysis allowed the comparison of treatment versus placebo groups in monkeys that had a more rapid onset and severe disease while removing those monkeys with later disease onset from the analysis.
The treatment paradigm for RA has changed significantly with the advent of biologic therapies, including inhibitors of TNF-α, costimulation blockade, CD20-mediated B cell depletion and modulation of the IL-6 pathway [35]. However, there remains a need to identify safe and effective treatments for patients refractory or intolerant to the current standard of care. In the current study, CD319 was identified as a potential therapeutic target using IHC analysis of RA synovial tissue, where the molecule was expressed at high levels on CD20 negative plasmablasts and plasma cells. The further differentiation of CD319 from CD20-targeted therapies was observed using PDL241, a novel humanized IgG1 mAb. PDL241 inhibited Ig production from PBMC in vitro by specifically depleting plasmablasts and plasma cells that expressed high levels of surface CD319, via antibody-dependent cellular cytotoxicity (ADCC). The activity of PDL241 in a rhesus monkey model of CIA provided further support as to the therapeutic potential of anti-CD319 therapy in RA. An anti-CD319 approach may provide particular benefit to patients who are refractory to anti-CD20 therapy with the presence of late stage plasmablasts in their disease tissue [7]. It is expected that PDL241 will show a different safety profile than anti-CD20 therapy due to the targeting of plasmablasts and plasma cells by PDL241. However, as B cells are thought to have a broader role in the autoimmune process, including antigen presentation, assisting in the development of lymphoid tissue architecture within the joint, and production of inflammatory cytokines [6], further investigation of CD319 biology in the RA disease process is warranted.
The mechanism of action of PDL241 is similar to that of another anti-CD319 mAb, elotuzumab. Elotuzumab has shown potent activity in vitro and in vivo against multiple myeloma cell lines and primary multiple myeloma cells, with the activity in both settings being attributed to ADCC. Neither PDL241 nor elotuzumab mediate complement dependent cytotoxicity or direct cytotoxic activity [36]. PDL241 binds a different epitope on CD319 than elotuzumab, with the PDL241 epitope residing on the membrane distal V domain, whereas elotuzumab binds the proximal C2-Ig domain. These mAb have differential effects on CD319 function. Elotuzumab enhances the homotypic adhesion of CD319, whereas PDL241 inhibits this interaction (Collins et. al., submitted). The role of CD319 as an ADCC target may come from its polarized expression in uropods [16]. Along with the ability to associate in a homotypic manner, CD319 has also been postulated to be involved in the interaction of multiple myeloma cells with bone marrow stroma [16]. The counter-receptor for CD319 on bone marrow stromal cells has not been defined, but is likely distinct from CD319, which has expression restricted to hematopoietic cells [9]. Although the function of CD319 on plasma cells is not known, it is possible that it plays a role in the bone marrow niches for plasma cell survival. Likewise, a role for CD319 in mediating interactions of leukocytes with the stroma in the inflamed synovium has not been investigated.
PDL241 showed promising activity in the rhesus monkey CIA model. The development of clinical arthritis in the CIA model is dependent on the production of CII specific IgM [28]. Mamu-B 26-ve rhesus monkeys of Indian origin develop clinical arthritis and show a good production of CII specific IgM. Mamu-B 26+ve rhesus monkeys are resistant to the development of clinical arthritis with heavily reduced serum levels of CII specific IgM [28]. Furthermore, the production of IgM is prominently associated with early responders to induction. The activity of PDL241 on anti-collagen antibodies observed in the model was consistent with the ability of the mAb to inhibit IgM production by PBMC in vitro. Although effects were detected on other disease-related parameters, there was a minimal effect on overall clinical scores, in part due to the severe inflammatory response in this model. Due to the labor-intensive nature of this model and only limited experience with other biologic drugs that are in the public domain [26], it was not possible to benchmark currently approved therapeutics in the current study.
The in vivo study was hampered by the immunogenicity of PDL241 in this rhesus monkey model, resulting in the development of ADA leading to strong infusion reactions following the third infusion. Retrospective analysis confirmed the presence of high levels of neutralizing ADA in serum collected on day 35 before the third infusion, and moderate to high levels of IL-6 and TNF-α were detected in serum collected shortly after infusion. A 90% reduction in complement levels (as determined by the CH50 assay) was also observed in post-dose serum samples (data not shown). As the reaction was observed at later time points and not associated with the initial dose of PDL241, it is likely that the infusion reactions were mediated by immunocomplexes of ADA and PDL241. A similar response to PDL241 was observed in a multiple dose GLP toxicology study, performed in parallel to this study in cynomolgus monkeys as part of the preclinical development of PDL241. Although ADA to human immunoglobulins in NHP are relatively common and not predictive of clinical immunogenicity [37], the magnitude of the response to PDL241 has prevented further development of this antibody. It is unclear if the strong immunogenicity of PDL241 was due to highly immunogenic amino acid sequences or the biology of the PDL241-CD319 interaction. The expression of CD319 on APC [11, 38] may lead to enhanced presentation of humanized mAb leading to an enhanced ADA response. In support of this hypothesis, we have found that PDL241 was substantially more immunogenic in a huCD319 transgenic B57BL/6 mouse than in a wild-type B57BL/6 mouse (unpublished data). The association of CD319 with EAT-2 may also play a role in the enhanced immunogenicity of PDL241 [39].
Literature on the expression of CD319 on naïve B cells is not consistent, with studies showing no expression [9, 11, 33] and expression [30–32] on B cells prior to their activation and/or differentiation. PDL241 did not bind naïve B cells and did not deplete B cells in culture. There was no difference in the binding of PDL241 to the long or the short forms of CD319 when expressed on 293 cells (data not shown). The mAb used in IHC studies showed binding mainly to plasma cells in tissues with limited staining of other cell subsets (the current study, and [9]). As the sensitivity of IHC is lower than flow cytometry, the more limited expression pattern of CD319 in tissue was more likely a consequence of sensitivity rather than a reflection of different biology in tissues versus blood. The expression of CD319 on other leukocytes provides the potential for other mechanisms of action aside from the depletion of plasmablasts and plasma cells. CD319 expression has been reported on subsets of T cells, notably a subset of CD8+ T cells and activated CD4+ T cells, the majority of NK and NK T cells, DC and activated monocytes [9, 11]. Binding of PDL241 was consistent with the published expression profile of CD319. The ability of PDL241 to inhibit T cell function was not examined in this study; however, preliminary data support an inhibitory activity on T cell proliferation via depletion of CD319-expressing T cells. The expression of CD319 on pDC and mDC [38] may provide further therapeutic opportunities for anti-CD319 mAb. Although development of PDL241 was halted due to the immunogenicity concerns described above, our data highlight the potential of CD319 as a therapeutic target in a range of autoimmune diseases where CD319-expressing cells have a role in the pathology.
The expression of CD319 in RA synovium led to the investigation of the potential of CD319 as a target in RA. PDL241, a novel humanized mAb to CD319, demonstrated activity in vitro against plasmablasts/plasma cells and in a NHP model of RA. Our data highlight the therapeutic potential of targeting CD319, which may be especially relevant in anti-CD20 therapy non-responsive disease associated with the presence of plasmablasts.
Anti-drug antibodies
Biomedical Research Research Centre
Complete Freund's adjuvant
- chCII:
Chicken type II collagen
Collagen-induced arthritis
Dendritic cells
Distal interphalangeal
Enzyme-linked immunosorbent assay
Fluorescence-activated cell sorting
Formalin fixed paraffin embedded
- hu-SCID:
Human-severe combined immunodeficiency
- Ig:
Immunoreceptor tyrosine-based switch motifs
- mAb:
Monoclonal antibody
Non-human primate
Natural killer
NOD scid gamma chain knockout
Optimal cutting temperature
Peripheral blood mononuclear cells
Phosphate-buffered saline
Proximal interphalangeal
Rheumatoid arthritis
Rheumatoid factor
SLAM-associated protein
Synovial fibroblasts
Small joint swelling score
Soft-tissue swelling
Tumor necrosis factor.
The authors gratefully acknowledge David Powers, Susan Rhodes and Marjorie James for molecular biology, Rick Powers for protein purification; the members of the Preclinical and Development Sciences group of AbbVie for conducting the PDL241 PK/PD study; and Alan Wahl and Fiona Harding of AbbVie for critical evaluation of the manuscript. We also would like to thank the animal caretakers of the BPRC who have provided optimal technical assistance and care to the rhesus monkeys with CIA.
Authors' Affiliations
- McInnes IB, Schett G: The pathogenesis of rheumatoid arthritis. N Engl J Med. 2011, 365: 2205-2219. 10.1056/NEJMra1004965.View ArticlePubMedGoogle Scholar
- Vander Cruyssen B, Peene I, Cantaert T, Hoffman IE, De Rycke L, Veys EM, De Keyser F: Anti-citrullinated protein/peptide antibodies (ACPA) in rheumatoid arthritis: specificity and relation with rheumatoid factor. Autoimmun Rev. 2005, 4: 468-474. 10.1016/j.autrev.2005.04.018.View ArticlePubMedGoogle Scholar
- Genovese MC, Becker JC, Schiff M, Luggen M, Sherrer Y, Kremer J, Birbara C, Box J, Natarajan K, Nuamah I, Li T, Aranda R, Hagerty DT, Dougados M: Abatacept for rheumatoid arthritis refractory to tumor necrosis factor alpha inhibition. N Engl J Med. 2005, 353: 1114-1123. 10.1056/NEJMoa050524.View ArticlePubMedGoogle Scholar
- Edwards JC, Szczepanski L, Szechinski J, Filipowicz-Sosnowska A, Emery P, Close DR, Stevens RM, Shaw T: Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis. N Engl J Med. 2004, 350: 2572-2581. 10.1056/NEJMoa032534.View ArticlePubMedGoogle Scholar
- Martin F, Chan AC: Pathogenic roles of B cells in human autoimmunity; insights from the clinic. Immunity. 2004, 20: 517-527. 10.1016/S1074-7613(04)00112-8.View ArticlePubMedGoogle Scholar
- Browning JL: B cells move to centre stage: novel opportunities for autoimmune disease treatment. Nat Rev Drug Discov. 2006, 5: 564-576. 10.1038/nrd2085.View ArticlePubMedGoogle Scholar
- Owczarczyk K, Lal P, Abbas AR, Wolslegel K, Holweg CT, Dummer W, Kelman A, Brunetta P, Lewin-Koh N, Sorani M, Leong D, Fielder P, Yocum D, Ho C, Ortmann W, Townsend MJ, Behrens TW: A plasmablast biomarker for nonresponse to antibody therapy to CD20 in rheumatoid arthritis. Sci Transl Med. 2011, 3: 101ra192-View ArticleGoogle Scholar
- Jourdan M, Caraux A, Caron G, Robert N, Fiol G, Reme T, Bollore K, Vendrell JP, Le Gallou S, Mourcin F, De Vos J, Kassambara A, Duperray C, Hose D, Fest T, Tarte K, Klein B: Characterization of a transitional preplasmablast population in the process of human B cell to plasma cell differentiation. J Immunol. 2011, 187: 3931-3941. 10.4049/jimmunol.1101230.View ArticlePubMedGoogle Scholar
- Hsi ED, Steinle R, Balasa B, Szmania S, Draksharapu A, Shum BP, Huseni M, Powers D, Nanisetti A, Zhang Y, Rice AG, van Abbema A, Wong M, Liu G, Zhan F, Dillon M, Chen S, Rhodes S, Fuh F, Tsurushita N, Kumar S, Vexler V, Shaughnessy JD, Barlogie B, van Rhee F, Hussein M, Afar DE, Williams MB: CS1, a potential new therapeutic antibody target for the treatment of multiple myeloma. Clin Cancer Res. 2008, 14: 2775-2784. 10.1158/1078-0432.CCR-07-4246.PubMed CentralView ArticlePubMedGoogle Scholar
- Cannons JL, Tangye SG, Schwartzberg PL: SLAM family receptors and SAP adaptors in immunity. Annu Rev Immunol. 2011, 29: 665-705. 10.1146/annurev-immunol-030409-101302.View ArticlePubMedGoogle Scholar
- Bouchon A, Cella M, Grierson HL, Cohen JI, Colonna M: Cutting edge: activation of NK cell-mediated cytotoxicity by a SAP-independent receptor of the CD2 family. J Immunol. 2001, 167: 5517-5521.View ArticlePubMedGoogle Scholar
- Tassi I, Colonna M: The cytotoxicity receptor CRACC (CS-1) recruits EAT-2 and activates the PI3K and phospholipase Cgamma signaling pathways in human NK cells. J Immunol. 2005, 175: 7996-8002.View ArticlePubMedGoogle Scholar
- Kumaresan PR, Lai WC, Chuang SS, Bennett M, Mathew PA: CS1, a novel member of the CD2 family, is homophilic and regulates NK cell function. Mol Immunol. 2002, 39: 1-8. 10.1016/S0161-5890(02)00094-9.View ArticlePubMedGoogle Scholar
- Cruz-Munoz ME, Dong Z, Shi X, Zhang S, Veillette A: Influence of CRACC, a SLAM family receptor coupled to the adaptor EAT-2, on natural killer cell function. Nat Immunol. 2009, 10: 297-305. 10.1038/ni.1693.View ArticlePubMedGoogle Scholar
- Lee JK, Boles KS, Mathew PA: Molecular and functional characterization of a CS1 (CRACC) splice variant expressed in human NK cells that does not contain immunoreceptor tyrosine-based switch motifs. Eur J Immunol. 2004, 34: 2791-2799. 10.1002/eji.200424917.View ArticlePubMedGoogle Scholar
- Tai YT, Dillon M, Song W, Leiba M, Li XF, Burger P, Lee AI, Podar K, Hideshima T, Rice AG, van Abbema A, Jesaitis L, Caras I, Law D, Weller E, Xie W, Richardson P, Munshi NC, Mathiot C, Avet-Loiseau H, Afar DE, Anderson KC: Anti-CS1 humanized monoclonal antibody HuLuc63 inhibits myeloma cell adhesion and induces antibody-dependent cellular cytotoxicity in the bone marrow milieu. Blood. 2008, 112: 1329-1337. 10.1182/blood-2007-08-107292.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim JR, Mathew SO, Patel RK, Pertusi RM, Mathew PA: Altered expression of signalling lymphocyte activation molecule (SLAM) family receptors CS1 (CD319) and 2B4 (CD244) in patients with systemic lupus erythematosus. Clin Exp Immunol. 2010, 160: 348-358. 10.1111/j.1365-2249.2010.04116.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Queen C, Schneider WP, Selick HE, Payne PW, Landolfi NF, Duncan JF, Avdalovic NM, Levitt M, Junghans RP, Waldmann TA: A humanized antibody that binds to the interleukin 2 receptor. Proc Natl Acad Sci USA. 1989, 86: 10029-10033. 10.1073/pnas.86.24.10029.PubMed CentralView ArticlePubMedGoogle Scholar
- Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N: An engineered human IgG1 antibody with longer serum half-life. J Immunol. 2006, 176: 346-356.View ArticlePubMedGoogle Scholar
- Cole MS, Anasetti C, Tso JY: Human IgG2 variants of chimeric anti-CD3 are nonmitogenic to T cells. J Immunol. 1997, 159: 3613-3621.PubMedGoogle Scholar
- Drobyski WR, Gottlieb M, Carrigan D, Ostberg L, Grebenau M, Schran H, Magid P, Ehrlich P, Nadler PI, Ash RC: Phase I study of safety and pharmacokinetics of a human anticytomegalovirus monoclonal antibody in allogeneic bone marrow transplant recipients. Transplantation. 1991, 51: 1190-1196. 10.1097/00007890-199106000-00009.View ArticlePubMedGoogle Scholar
- Bakker NP, Van Erck MG, Otting N, Lardy NM, Noort RC, 't Hart BA, Jonker M, Bontrop RE: Resistance to collagen-induced arthritis in a nonhuman primate species maps to the major histocompatibility complex class I region. J Exp Med. 1992, 175: 933-937. 10.1084/jem.175.4.933.View ArticlePubMedGoogle Scholar
- Otting N, Heijmans CM, Noort RC, de Groot NG, Doxiadis GG, van Rood JJ, Watkins DI, Bontrop RE: Unparalleled complexity of the MHC class I region in rhesus macaques. Proc Natl Acad Sci USA. 2005, 102: 1626-1631. 10.1073/pnas.0409084102.PubMed CentralView ArticlePubMedGoogle Scholar
- Hart BA, Bank RA, De Roos JA, Brok H, Jonker M, Theuns HM, Hakimi J, Te Koppele JM: Collagen-induced arthritis in rhesus monkeys: evaluation of markers for inflammation and joint degradation. Br J Rheumatol. 1998, 37: 314-323. 10.1093/rheumatology/37.3.314.View ArticlePubMedGoogle Scholar
- Vierboom MP, Jonker M, Bontrop RE, 't Hart B: Modeling human arthritic diseases in nonhuman primates. Arthritis Res Ther. 2005, 7: 145-154. 10.1186/ar1773.PubMed CentralView ArticlePubMedGoogle Scholar
- Vierboom MP, Jonker M, Tak PP, 't Hart BA: Preclinical models of arthritic disease in non-human primates. Drug Discov Today. 2007, 12: 327-335. 10.1016/j.drudis.2007.02.012.View ArticlePubMedGoogle Scholar
- Black D, Duncan A, Robins SP: Quantitative analysis of the pyridinium crosslinks of collagen in urine using ion-paired reversed-phase high-performance liquid chromatography. Anal Biochem. 1988, 169: 197-203. 10.1016/0003-2697(88)90274-6.View ArticlePubMedGoogle Scholar
- Bakker NP, van Erck MG, Botman CA, Jonker M, 't Hart BA: Collagen-induced arthritis in an outbred group of rhesus monkeys comprising responder and nonresponder animals. Relationship between the course of arthritis and collagen-specific immunity. Arthritis Rheum. 1991, 34: 616-624. 10.1002/art.1780340514.View ArticlePubMedGoogle Scholar
- Pettit AR, Ji H, von Stechow D, Muller R, Goldring SR, Choi Y, Benoist C, Gravallese EM: TRANCE/RANKL knockout mice are protected from bone erosion in a serum transfer model of arthritis. Am J Pathol. 2001, 159: 1689-1699. 10.1016/S0002-9440(10)63016-7.PubMed CentralView ArticlePubMedGoogle Scholar
- De Salort J, Sintes J, Llinas L, Matesanz-Isabel J, Engel P: Expression of SLAM (CD150) cell-surface receptors on human B-cell subsets: from pro-B to plasma cells. Immunol Lett. 2011, 134: 129-136. 10.1016/j.imlet.2010.09.021.View ArticlePubMedGoogle Scholar
- Lee JK, Mathew SO, Vaidya SV, Kumaresan PR, Mathew PA: CS1 (CRACC, CD319) induces proliferation and autocrine cytokine expression on human B lymphocytes. J Immunol. 2007, 179: 4672-4678.View ArticlePubMedGoogle Scholar
- Llinas L, Lazaro A, de Salort J, Matesanz-Isabel J, Sintes J, Engel P: Expression profiles of novel cell surface molecules on B-cell subsets and plasma cells as analyzed by flow cytometry. Immunol Lett. 2011, 134: 113-121. 10.1016/j.imlet.2010.10.009.View ArticlePubMedGoogle Scholar
- Murphy JJ, Hobby P, Vilarino-Varela J, Bishop B, Iordanidou P, Sutton BJ, Norton JD: A novel immunoglobulin superfamily receptor (19A) related to CD2 is expressed on activated lymphocytes and promotes homotypic B-cell adhesion. Biochem J. 2002, 361: 431-436. 10.1042/0264-6021:3610431.PubMed CentralView ArticlePubMedGoogle Scholar
- Dechanet J, Merville P, Durand I, Banchereau J, Miossec P: The ability of synoviocytes to support terminal differentiation of activated B cells may explain plasma cell accumulation in rheumatoid synovium. J Clin Invest. 1995, 95: 456-463. 10.1172/JCI117685.PubMed CentralView ArticlePubMedGoogle Scholar
- Guyot P, Taylor P, Christensen R, Pericleous L, Poncet C, Lebmeier M, Drost P, Bergman G: Abatacept with methotrexate versus other biologic agents in treatment of patients with active rheumatoid arthritis despite methotrexate: a network meta-analysis. Arthritis Res Ther. 2011, 13: R204-10.1186/ar3537.PubMed CentralView ArticlePubMedGoogle Scholar
- Collins SM, Bakan CE, Swartzel GD, Hofmeister CC, Efebera YA, Kwon H, Starling GC, Ciarlariello D, Bhaskar S, Briercheck EL, Hughes T, Yu J, Rice A, Benson DM: Elotuzumab directly enhances NK cell cytotoxicity against myeloma via CS1 ligation: evidence for augmented NK cell function complementing ADCC. Cancer Immunol Immunother. 2013, 62: 1841-1849. 10.1007/s00262-013-1493-8.PubMed CentralView ArticlePubMedGoogle Scholar
- Brennan FR, Morton LD, Spindeldreher S, Kiessling A, Allenspach R, Hey A, Muller PY, Frings W, Sims J: Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies. MAbs. 2010, 2: 233-255. 10.4161/mabs.2.3.11782.PubMed CentralView ArticlePubMedGoogle Scholar
- Ding Y, Ju X, Azlan M, Hart DN, Clark GJ: Screening of the HLDA9 panel on peripheral blood dendritic cell populations. Immunol Lett. 2011, 134: 161-166. 10.1016/j.imlet.2010.10.010.View ArticlePubMedGoogle Scholar
- Aldhamen YA, Appledorn DM, Seregin SS, Liu CJ, Schuldt NJ, Godbehere S, Amalfitano A: Expression of the SLAM family of receptors adapter EAT-2 as a novel strategy for enhancing beneficial immune responses to vaccine antigens. J Immunol. 2011, 186: 722-732. 10.4049/jimmunol.1002105.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Products & ServicesSee also: Categories | Featured Products | More Information
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Digi-Key Electronics
3M Thermal Interface Materials at Digi-Key
Thermal interface tapes, pads, and epoxies by 3M provide high thermal conductivity by focusing on the two areas that matter most: bulk conductivity and interface conductivity. They address a crucial problem with today's low-power electronic devices - the need to dissipate the sizable amounts of heat being generated. The thermally conductive adhesive transfer tapes provide efficient thermal transfer for a wide range of applications including bonding heat sinks, heat spreaders, IC packages, power... (read more)
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Light degradation prediction of high-power light-emitting diode lighting modules
The 1 W high-power LED is mounted on an aluminum heat sink by 3M®8805 thermally conductive adhesive transfer tape at a thermal conductivity of 0.6 W/m .
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mount with 3M® 9890 thermally conductive adhesive transfer tape .
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Every American officer, regardless of branch of service, take the same oath upon their appointment to the military. They swear to "support and defend the Constitution of the United States against all enemies, foreign and domestic."
The language used is not dissimilar to that of the presidential oath of office as it appears in Article II of the Constitution: "I do solemnly swear (or affirm) that I will faithfully execute the office of President of the United States, and will to the best of my ability, preserve, protect, and defend the Constitution of the United States."
The American oaths of office are different from those of so many nations around the world, because we do not swear allegiance to a person, a religious sect, or even to the nation's citizens. America's leaders swear to uphold the Constitution, the document in which the individual freedoms that make America unique among all nations in history are enshrined and guaranteed.
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It should come as no surprise, then, that when long-service American military officers see the principles of the Constitutional repeatedly violated, disregarded, and even held in contempt, they take offense.
Retired officers, of course, have more freedom to express their views without concern for the impact such honesty might have on their careers. For example, Air Force Major General Bentley Rayburn and four-star Army General Jack Keane have both spoken out against Obama's treatment of Islamic terrorists, including his trade of several Guantanamo Bay detainees for accused deserter Bowe Bergdahl. Retired Lieutenant General William G. "Jerry" Boykin has gone so far as to call for Obama's impeachment, as has General Patrick Henry Brady.
Retired U.S. Army Major General Paul E. Vallely has publicly argued that America's current leadership has so damaged individual American liberty that it can be restored only by citizen protests to force the resignations of President Obama and other political leaders. The general has also recently been working to uncover the truth about the Benghazi attacks of 2012, which he says involved an illegal arms deal by Hillary Clinton's State Department. General Vallely and retired Air Force Brigadier General Charles Jones have formed a "citizen's council" to challenge the government more directly.
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More recently–and more remarkably–active duty officers have been giving vent to their frustrations and concerns about the current administration. Active-duty 4-star Marine General and US Southern Command Commander John Kelly has commented on the grave threat to America's existence that the current border crisis represents. Even more remarkable, the Commandant of the Marine Corp himself, General James Amos, recently made comments highly critical of the Obama administration's leadership and policy in Iraq.
That's quite a list, and it's far from complete. Nor does it include the numerous other senior officers who retired at the rank of Colonel or Lieutenant Colonel who have also spoken out against this president, his administration, and his policies.
When a politician or even a journalist makes negative comments about the leadership and practices of a sitting president, it is wise to take those with a grain of salt, asking what the motive of the speaker might be.
In the case of these retired and active-duty senior military officers, however, their motive appears to be clear–the fulfillment of the oath they swore to support and defend the Constitution of the United States of America.
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Please share this article on Facebook and Twitter to honor the men and women of America's armed services who bravely choose to speak the truth about President Barack Obama.


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Johns Hopkins Biomedical Engineering primary faculty
Jordan J. Green, PhD
Associate ProfessorAssociate Professor of Biomedical Engineering, Ophthalmology, Oncology, Neurosurgery, and Materials Science & Engineering at the Johns Hopkins University School of Medicine
Office: Smith 5017
Lab: Biomaterials and Drug Delivery Laboratory
PhD, Biological Engineering, MIT, 2007
BS, Chemical Engineering, Biomedical Engineering, Carnegie Mellon University, 2003
Dr. Jordan J. Green is an Associate Professor of Biomedical Engineering, Ophthalmology, Oncology, Neurosurgery, and Materials Science & Engineering at the Johns Hopkins University School of Medicine. He is also an executive committee member of the Institute for NanoBioTechnology and a co-founder of the Translational Tissue Engineering Center at JHU.
Dr. Green serves as the Chair of the Drug Delivery Special Interest Group of the Society For Biomaterials. He is also the CEO and co-founder of the Baltimore biotech startup company, AsclepiX Therapeutics.
His work has resulted in the publication of over 60 papers and he has received numerous awards including the 2015 American Institute of Chemical Engineers NSEF Young Investigator award, the Maryland Outstanding Young Engineer Award, the Carnegie Mellon University Recent Alumni Award, the Biomedical Engineering Society Rita Schaffer Award, and was named by Popular Science as one of the 2014 Brilliant Ten.
Dr. Green's main research interests are applying chemical engineering principles to develop biomaterials and nanobiotechnology to meet challenges in human health.
Research Interests
Dr. Green's Biomaterials and Drug Delivery Laboratory is acutely interested in biomaterials, drug delivery, gene therapy, nanobiotechnology, and cell engineering. Research findings — and the technologies developed — are applied in the fields of ophthalmology, oncology, and regenerative medicine.
The lab works within the chemistry/biology/engineering interface to answer fundamental scientific questions, and to create innovative technologies and therapeutics that can directly benefit human health.
Current projects include:
- Development of safe and effective biodegradable nanoparticles for DNA and siRNA delivery to treat cancer
- Polymeric microparticle-based biological treatments for age-related macular degeneration
- Design of biomimetic artificial antigen presenting cells for immunobioengineering
- Enabling technologies for tissue engineering and regenerative medicine
Publications Search
From PubMed | Google Scholar Profile
Selected Publications
Randall A Meyer, Joel C Sunshine, Karlo Perica, Alyssa K Kosmides, Kent Aje, Jonathan P Schneck, Jordan J Green. Biodegradable Nanoellipsoidal Artificial Antigen Presenting Cells for Antigen Specific T-Cell Activation. Small 2015, 11 (13), 1519 –1525.
Antonella Mangraviti, Stephany Yi Tzeng, Kristen Lynn Kozielski, Yuan Wang, Yike Jin, David Gullotti, Mariangela Pedone, Nitsa Buaron, Ann Liu, David R Wilson, Sarah K Hansen, Fausto J Rodriguez, Guo-Dong Gao, Francesco DiMeco, Henry Brem, Alessandro Olivi, Betty Tyler, Jordan J Green. Polymeric Nanoparticles for Nonviral Gene Therapy Extend Brain Tumor Survival in Vivo. ACS nano 2015, 9 (2), 1236–1249.
CJ Bishop, B Abubaker-Sharif, T Guiriba, SY Tzeng, JJ Green. Gene delivery polymer structure-function relationships elucidated via principal component analysis. Chemical Communications 2015.
Hugo Guerrero-Cázares, Stephany Y Tzeng, Noah P Young, Ameer O Abutaleb, Alfredo Quiñones-Hinojosa, Jordan J Green. Biodegradable polymeric nanoparticles show high efficacy and specificity at DNA delivery to human glioblastoma in vitro and in vivo. ACS nano 2015 8 (5), 5141 –5153.
Green, J.J. 2011 Rita Schaffer Lecture: Nanoparticles for Intracellular Nucleic Acid Delivery. Annals of Biomedical Engineering 2012, 40(7):1408–18.
Bhise, N.S., Shmueli, R.B., Gonzalez, J., Green, J.J. A novel assay for quantifying the number of plasmids encapsulated by polymer nanoparticles. Small 2012, 8(3):367–73.
Sunshine, J.C., Sunshine, S., Bhutto, I., Handa, J., Green, J.J. Poly(ß-amino ester)-Nanoparticle Mediated Transfection Of Retinal Pigment Epithelial Cells in vitro and in vivo. PLoS ONE 2012, 7(5): e37543.
Tzeng, S.Y., Hung B.P., Grayson, W.L., and Green, J.J. Cystamine-terminated poly(beta-amino ester)s for siRNA delivery to human mesenchymal stem cells and enhancement of osteogenic differentiation. Biomaterials 2012, 33(32): 8142–51.
Sunshine, J.C., Peng, D.Y., Green, J.J. Uptake and transfection with polymeric nanoparticles are dependent on polymer end-group structure, but largely independent of nanoparticle physical and chemical properties. Molecular Pharmaceutics 2012, 9(11): 3375–83.
Tzeng, S.Y. and Green, J.J. Subtle changes to polymer structure and degradation mechanism enable highly effective nanoparticles for siRNA and DNA delivery to human brain cancer. Advanced Healthcare Materials 2013, 2(3):468–80.


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== Search results for ssl packet sniffer ==
Searched file: ssl packet sniffer
*Filename: ssl packet sniffer.zip
*Year: 2015
*Total Downloads: 153451
*Uploaded: 02/26/2015 04:45:07
*Type: WinZip
*Supported OS: All
== Searches related to ssl packet sniffer: ==
02/26/2015 04:45:07
Simple ARP/ND spoofing and password sniffing for Windows (and some really basic SSL stripping too). Nighthawk is an experimental implementation of ARP/ND spoofing ...Packet Sniffing with Wireshark and Tcpdump v1.1 . 1 Setting up the packet sniffer applications . In determining how to set up a network sniffer, the topography and ...Network Sniffer is a tool that can help you locate network problems by allowing you to capture and view the packet level data on your network. The Colasoft Capsa ...Meterpreter has the capability of packet sniffing the remote host without ever touching the hard disk. This is especially useful if we want to monitor what type of ...11/14/2011Â hubs will broadcast all the packets to all the computers. Switches are a bit smarter and will try send each packet to the respective computer.A packet sniffer, sometimes referred to as a network monitor or network analyzer, can be used legitimately by a network or system administrator to monitor and ...Secure Socket Layer (SSL) SSL provides communication security between two hosts. It provides integrity, authentication and confidentiality. It is used most commonly ...2/23/2012Â [NOTICE]: Due to spam in comments i have set it to approval only. I will approve all comments that are not spam. Even hate and troll comments will be ...2. How can I defend myself against packet sniffers? 2.1 How can I stop people from sniffing my data? While you can configure your local network to make sniffing hard ...Free Network Analyzer is a software network packet sniffer and protocol analyzer for Windows platform. Using this free network monitoring software you may intercept ...
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Where is the information stored when the satisfaction survey is submitted?
Answer The entry for the overall experience is stored in the column Rating and that column is found in the workitem table. The responses for the rest of the questions are saved to the Comment field.
For information on how to customize the satisfaction survey, please refer to:
How do I customize the Satisfaction Survey in Helpdesk 6.0 SP5? http://www.symantec.com/business/support/index?page=content&id=HOWTO9489
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http://www.MMAWeekly.com/ -- Johny Hendricks has been rocketing towards a title shot against UFC welterweight champion Georges St-Pierre, dispatching of three of the top 170-pound fighters in the wor watch video >>
Johny Hendricks versus Nick Diaz Full Fight Breakdown by Paulie G watch video >>
Johny Hendricks talks GSP fight, Carlos Condit, Nick Diaz & more watch video >>
UFC middleweight champion Michael Bisping recounts how a fight with Georges St-Pierre nearly came to be, but then, just as quickly, fell away. After the GSP fight blew up, Bisping got stuck in fight watch video >>
Georges St-Pierre and Nick Diaz get in a heated discussion before their fight at UFC 158. Subscribe to keep up with everything MMA; http://tinyurl.com/MMAdigest watch video >>
http://fightnetwork.com/ - Johny Hendricks stopped by the Fight Network studio ahead of UFC 200 to give his thoughts on his UFC 167 bout vs. Georges St-Pierre and teasing a possible rematch with a re watch video >>
That time Nick Diaz Chased Georges St. Pierre Around Hotel lobby looking for a street fight watch video >>
http://www.smackhisface.com That time Nick Diaz Chased Georges St. Pierre Around Hotel lobby looking for a street fight watch video >>
Johny Hendricks says Nick Diaz, GSP, Ben Askren & Robbie Lawler can all shove it! Paulie G Agrees! watch video >>
In preparation for his welterweight title fight against Georges St-Pierre, Johny Hendricks tackled a brutal obstacle course, inspired by Sparta, circa 400 B.C. watch video >>
In this boxing and mma video, Nick Diaz talks about what he thought of Manny Pacquiao's fight, wanting someone to buy out his UFC contract, and what he thought of Hendricks fight with Lawler. For mo watch video >>
Nick Diaz joins Showdown Joe to discuss why he attended UFC 171. watch video >>
http://fightnetwork.com - Fight Network's John Pollock, John Ramdeen and Robin Black give us a complete breakdown following UFC on FOX 9: Johnson vs. Benavidez 2 from Sacramento, CA., plus we hear fr watch video >>
MMA H.E.A.T. brings you the UFC 167: Georges St-Pierre vs Johny Hendricks post fight press conference LIVE from the MGM Grand Garden Arena in Las Vegas, Nevada. Be sure to visit http://www.MMAHEAT.c watch video >>


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Rezidence Lundborg
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Rezidence Lundborg Hotel Reviews
Based on reviews from across the web.
Rezidence Lundborg Hotel Details
Check-in: 2:00 PM
Check-out: 11:00 AM
Property Location With a stay at Rezidence Lundborg, you'll be centrally located in Prague, steps from Lesser Town Bridge Tower and Lennon Wall. This hotel is within close proximity of Franza Kafka Museum and Charles Bridge. Rooms Make yourself at home in one of the 13 individually decorated guestrooms, featuring kitchens. Your Select Comfort bed comes with down comforters, and all rooms are furnished with queen sofa beds. Computers are provided along with complimentary wireless Internet access. Private bathrooms with shower/tub combinations feature jetted bathtubs and hair dryers. Rec, Spa, Premium Amenities Pamper yourself with onsite massages or take in the view from a garden. This hotel also features complimentary wireless Internet access, gift shops/newsstands, and wedding services. Dining Take advantage of the hotel's room service (during limited hours). Quench your thirst with your favorite drink at a bar/lounge. Business, Other Amenities Featured amenities include a 24-hour business center, a computer station, and express check-in. For a surcharge, guests may use a roundtrip airport shuttle (available on request) and a train station pick-up service.


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More Videos...
88 Accord at the north west 3geez meet 09
Stock except for the short ram.
3GEEZ tribute
a tribute to the guys at 3GEEZ.com. and to the 86-89 accord. this is my first video ever made so don't hate. we may have different opinions of what looks good, but we respect the work everyone has put into their car to make it one of a kind. lol, dont hate on the music. i couldn't think of any good songs that would go with this video so i just threw in some honda songs.
3geez video
dis is slide show of Honda accord made from 86 to 89 (3geez.com), it is just made for fun and these r not my pics and song is from wangan midnight maximun tune and i do not claim any of these things
1989 Honda Accord at the drag strip
Ran an 18.7. I thought I ran a 15 at first, but I looked at the wrong numbers. I know this is slow, but its a 4 cylinder with a carburetor geared for economy.


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A'kin Pure Alchemy Cellular Radiance Serum is a potently active serum high in bio-available anti-oxidant extracts. Ideal for all skin types including sensitive and fragile it energises brightens and revitalises your skin.
Thank you for visiting Akin Pure Alchemy Cellular Radiance Serum (23ml) on happybeauty.co.uk
We bring the products from many websites to one website for you to search, save and compare.


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Kino! 2013: New Films from Germany
April 18–
24, 2013
Continuing a long tradition of celebrating emerging visions from German filmmakers, we are pleased to present MoMA's 35th annual series of new films from Germany. Selected from major international festivals, including this year's Berlin International Film Festival, the series features popular narrative, documentary, and avant-garde works that demonstrate Germany's vital role as an incubator of cinematic talent.
Organized by Rajendra Roy, The Celeste Bartos Chief Curator of Film, The Museum of Modern Art, with Nicole Kaufmann, Project Coordinator, German Films Service + Marketing (Munich) and its New York representative, Oliver Mahrdt. KINO! is presented in association with the Goethe-Institut New York and the German Consulate General New York.
Licensing of MoMA images and videos is handled by Art Resource (North America) and Scala Archives (all other geographic locations). All requests should be addressed directly to those agencies, which supply high-resolution digital image files provided to them directly by the Museum.
Requests for permission to reprint text from MoMA publications should be addressed to email@example.com.


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By Rocsi Diaz | 02/29/2012 - 14:09
The NBA All Star Game was this past weekend in Orlando, Florida and Pitbull lit up the stage!
Rocsi's Inside Track: My Favorite Latin Pop Songs From Back-In-The-Day
Read more
By Sugey Palomares | 02/28/2012 - 16:06
Don Omar is leading the race for the most Latin Billboard Music Award's nominations.
Don Omar Is Moving to Hollywood
Read more
By Sugey Palomares | 02/27/2012 - 15:13
Madison Square Garden was filled with love struck Romeo Santos fans for his final sold-out performance in New York City on Friday night. The energy was electrifying, as the heartthrob bachatero got on-stage to perform his first song of the night, "Soy Feliz." Thankfully, the romantic track was simply the prelude to hours of bachata hits including Aventura classics like "Por un Segundo" and "Obsesión."
Read more
By Sugey Palomares | 02/27/2012 - 13:18
Leave it to Mr. 305 to get the party started!
Pitbull set the stage on fire for the 2012 NBA All-Star Game at the Amway Theater in Orlando, Florida. The Cuban rapper performed an electrifying medley of all of his top hits including, "International Love" and "Give Me Everything" with help from his all-star collaborators Ne-Yo, Chris Brown, and Nayer.
Read more
By Sugey Palomares | 02/23/2012 - 11:20
Jump aboard!
Read more
By Sugey Palomares | 02/22/2012 - 11:29
Demi Lovato is getting real and uncensored about the struggles that come with recovery for MTV's new documentary Demi Lovato: Stay Strong. In 2010, the 19-year-old singer and actress entered a rehabilitation center in Chicago where she spent a few months battling an eating disorder and deep emotional issues rooted from her childhood.
Read more
By Sugey Palomares | 02/22/2012 - 10:57
Romeo Santos has tapped into the ultimate formula for success in his debut album "Formula, Vol. 1," which features addictive bachata hooks and lyrics that make you swoon. Aventura's former front man's latest single "Mi Santa" has hit number one on Billboard's airplay charts. The hit also features famous Spanish flamenco guitarist Tomatito, who laces the track with a touch of eloquence.
Read more
By Rocsi Diaz | 02/21/2012 - 15:20
While traveling this weekend, I decided to browse my iPod playlists and landed on my favorite Latin pop section. To my amazement I heard songs that I haven't heard in forever, and loved every minute of my old school playlist.
One of the songs on my list is Enrique Iglesias', "Miente" off of his album "Vivir." I remember as a little girl kissing this album cover and singing this song to the top of my lungs! I really fell for his raspy voice and then of course the lyrics to the song.
Read more
By Rocsi Diaz | 02/14/2012 - 13:31
If it wasn't for the energetic and vibrant performance by Bruno Mars, and the quick presentation appearances from Marc Anthony and NY Giant Victor Cruz, Latinos were pretty much unseen at the 2012 Grammys.
Read more
By Sugey Palomares | 02/10/2012 - 17:28
The Grammy Awards may draw attention to fashionable looks and crazy celebrity speeches, but its true essence has always been about the music. Mainstream audiences may have taken sometime to appreciate all of the Latin musical genres, but just a small taste has had them hooked! We are honoring our talented culture by showcasing the top Latino Grammy performances of all time. Watch with pride below and stay tuned for Bruno Mars to rock the stage this Sunday!
Read more
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Tap Tap Words
By: Arpita S
Welcome to the walkthrough for the iOS game Tap Tap Words, if you are looking for ways to make it to the next level, and to improve gaming experience as a master player, you've come to the right place. Here you will find Tap Tap Words tips, tricks and guides, as well as the opportunity to share your own discoveries to help others move on and beat the game.
If you can't find the answer to your question you are welcome to post it in the comments section below. This way others who know the answer can help you by sharing their own tips and advices.
Tap Tap Words game app is developed by Arpita and available on iTunes and Google Play. For more details on the app, check out our review.
- App name: Tap Tap Words
- Seller: Arpita
- Category: Puzzle
- Updated: Apr 9, 2015
Check out these tutorial for all levels of Tap Tap Words. We'll continue to update this list with the latest answers as new levels are released.
Tap Tap Words Answers
- Basic Game Guide Part One - tap two closedsyllable words
Hit like if this helped you!
Until the Walkthrough is completed, feel free to ask for help in the comments section.
Looking for a trick all your friends? Check out the Tap Tap Words cheats list to find collectibles guides, easter eggs and secrets.


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Latest Stories
RPG Roundtable #7 - Part 20 Comments
July 10, 2007
Silverfall Review0 Comments
April 4, 2007
Help us solve the toughest (or most puzzling) game puzzles and challenges.
Our attempt to answer issues which most commonly occur on IGN's wikis.
About This Game
- Summary
- Specifications
- Features
- Game Editions
Since the dawn of time, the inhabitants of Nelwë have relied on mighty elemental wizards to keep order. But the invention of steam power has overturned this ancient social order. Disillusioned by the introduction of new technology, certain sections of the population have turned instead to the power of nature. Taking advantage of the conflicts tearing apart the factions of nature and science, the forces of darkness lay siege to the magic domains in an attempt to seize power.
From the peaks of Silverfall the Fair to the mist-enshrouded city of Cloudworks, Players must fight these dark forces, choosing to rely on either science or magic to help them. Whichever they choose will determine the pathy they follow through-out the game.
- Supported Functions
- Number Of Players: 1-8
- Online
- Online - VS Number Of Players: 8
- Online - Co-Op Number Of Players: 8
- 150+ creatures and bosses
- Create your own character from a choice of four races and release your inner beast by transforming into a fearsome, flesh rending were-beast
- Build your team of two from a choice of eight companions. Each of them has their own story, background, abilities and can endow you with quests. The way you treat them will alter their loyalties – they can become your friend for life or even a lover
- Manage your character's evolution by choosing from 130+ skills and spells. Create precisely the character you want to play and adapt it to your style of play
- Two multiplayer modes – the first allows the player to connect to any party, compose their team and play in co-op or PVP (up to eight players); the second sees the player compose a team at the beginning and play through the entire story together.
- Dazzling light, shadow and particle effects
- Real-time streaming of universe minimises loading between regions
- Ragdoll physics and particle effects lend greater reality to spell effects
SilverfallPublisher: Monte CristoRelease Date: March 9, 2007MSRP: 34.99 GBP
Silverfall: Earth AwakeningRelease Date: August 15, 2008
SilverfallPublisher: AtariRelease Date: March 20, 2007MSRP: 39.99 USD
Silverfall: Earth AwakeningPublisher: Monte Cristo


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This feature is not supported on the Wolfram Cloud.
This feature is not supported on the Wolfram Cloud.
gets OpenCLMemory into the CPU and the Wolfram System.
- The OpenCLLink application must be loaded using Needs["OpenCLLink`"].
- OpenCLMemory is returned by OpenCLMemoryLoad or OpenCLMemoryAllocate.
基本范例 (3)基本范例 (3)
First, load the OpenCLLink application:
This registers Range[10] with the OpenCLLink memory manager returning a handle to the memory:
Images can be loaded as OpenCLMemory:
When OpenCLMemory is retrieved, the original image is returned:
Graphics objects can be loaded as OpenCLMemory:
The Graphics object is rasterized when loaded:
The head of the retrieved data is an Image:


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Ben whishaw
Gerelateerde onderwerpen verkennen
"I'm your new Quartermaster." - Ben Whishaw
Eli Mullan [Ben Whishaw] Elam Mullan's son. Has his eyes on being Ceres' next Minister of Foreign Affairs and is convinced Sinead is after the position too. She isn't. He considers himself her rival. It's one-sided.
The most amazing thing is when you find yourself watching someone in the café or something doing something weird. It's amazing what people do, isn't it, when you just look at them, when you take the time to look.Ben Whishaw
Ben Whishaw [photo, but composition reminds me of artist-whose-name-eludes-me]
Ben Whishaw
Ben Whishaw - better known to most as the new Q in Bond films.
Still of Ben Whishaw in "The Hour"-one very talented actor.
Ben Whishaw News, Ben Whishaw photographed by Syndi Huang. Interview...
Ben Whishaw in the original National Theater production of "His Dark Materials" in 2004
Ben Whishaw in portrays the character of Robert Frobisher in the film "Cloud Atlas"......


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Well now I really need your help so all of you throqw in your two cents worth.
This spring I am building a water garden of fairly decent size. After working
for several hours to come up with the best spot for the pond, my wife made
another of her famous and irrevocable decisions - The pond goes where half of
my hosta garden was last year..and for that matter still is.. The problem is
that I have not blown the leaves off my hostas yet and they have not even
begun to grow so it's going to be difficult to locate all of them..
So,, the questions are:
1. Is it too early to uncover them"
2. How do you locate them if there is no growth. Some of them are obvios
since the scapes from last year are still standing.
3. Can I dig them and put them in gallon pots untill I can get out and
prepare a new bed for them?
If you can help me I'd be much obliged.
Hosta La Viesta
To sign-off this list, send email to email@example.com with the
message text UNSUBSCRIBE HOSTA-OPEN


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Distances are calculated in a straight line and may not reflect actual travel distance.
Let us help calculate the repayments on this property.Use our mortgage calculator
A beautiful and lovingly restored two bedroom conversion flat located on the prestigious Cator Estate.
The current owners have undertaken an extensive refurbishment programme resulting in a wonderful blend of period charm and contemporary features.
Originally built in 1867 by local trio Rowlands-Stansby-Green this residence was converted into flats in the 1940s, and this particular property occupies the top floor. The grandeur of the building is evident not only from the exterior but as you enter the main front door into the impressive communal hallway. Accommodation briefly comprises; two double bedrooms, open plan kitchen/reception room, modern shower room and built in storage areas. There has been considerable thought put into the stylish finishes, not least the shower room complete with his and hers showers and exquisite bathroom furniture and the smart kitchen with solid composite work-surfaces, induction hob and oak flooring. Other nice touches include exposed brick walls in both bedrooms and lovely high ceilings. Outside, there is off street parking to the front and a well maintained communal garden to the rear.
Manor Way is found on the exclusive Cator Estate, arguably the finest address in Blackheath. The tree lined roads of the estate give a lovely rural feel yet Blackheath Station can deliver you to London Bridge in sixteen minutes, Charing Cross in twenty minutes and London Victoria in twenty-four minutes. Only one stop along the line takes you to Lewisham Station where it is possible to pick up the DLR service directly to Canary Wharf and Bank. Local amenities in Blackheath Village include an array of independent cafés and coffee shops alongside more familiar stores. You will also find a wonderful choice of bars, pubs and restaurants to suit all tastes. Beyond the village the wonderful expanse of the heath is a real gem of South East London providing over two hundred acres of idyllic recreation area.
Grand communal entrance
Two bedrooms
Open plan reception room
Shower room
Storage room
Communal garden
Recently refurbished
Share of freehold
Contact branch for relevant Energy Performance Certificate


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Autumn is mixing her color palette and unpacking her brushes to paint a gorgeous, colorful season for this autumn palette 50th anniversary card! Inspired by the colors of autumn, we customized this 50th anniversary party invitation with colorful leaves and a rough brown background. The ribbon graphic can be custom-colored for you, and we can also personalize this with a wedding or recent photo of the couple (in original color or vintage sepia). See more ideas & personalize this design for your own celebration here (simply request the leafy, photo-less design in the notes field): Fine Fall Anniversary Invitation.
For a beautifully matched set, pair with our fine fall thank you cards.


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Rates in SGD (taxes & fees included), shown as:
Nightly Average
S$32 Avg. price/night
Sri Krishna Palace Business Hotel
No. 50/2, Nethaji Road, Hosur 635109, India
Find Hotel Rates
Recent Searches
Wego searches multiple travel sites so you can find and compare the best rates.
Sri Krishna Palace Business Hotel Hotel Reviews
Based on reviews from across the web.
Sri Krishna Palace Business Hotel Hotel Details
Check-in: 1200
Check-out: 1200
Sri Krishna Palace Business Hotel is one of the popular budget hotels in Hosur. It enjoys close proximity to the bus-stand. Guests can relax and enjoy in any of the economy, premium and executive rooms for a grand stay. A multi-cuisine restaurant which serves delicious meals to its diners is available in this hotel. An array of modern facilities like small meeting rooms offering audio visual equipment and high speed internet access to surf their important mails are also offered within this premise.
No. 50/2, Nethaji Road Hosur 635109, India


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With all of these IGN Editor lists of favorite games being published lately, instead of arguing/agreeing with them, I felt that it would be more productive for me to create my own list and see what my fellow gamers think about it.
10. Digimon World (PS1)
I remember buying this game when the original Digimon series was in its' prime. And although the idea of raising a Digimon (having them eat, exercise, battle, think a better looking Tamagachi) may seem hokey now, back in 2000 it was the coolest thing in the world.
9. Dragonball Z Budokai 3 (PS2)
While I enjoy all of the games in this series, the fluidity of this game really put the other ones to shame. Not to mention the awesome cel-style graphics, the customizable characters, the amount of characters (Bardock?! Yes Please!), free roaming maps, and just overall infinite replay value (preferably when playing 1v1 against friends.)
8. Rogue Squadron (N64)
That box art just says it all for me. From the opening level of flying around Tattooine, I was hooked. While Rogue Leader may have made several control/visual improvements over this first entry, I am still partial to the first in the series, and not just because there were codes that let me fly the Naboo starfighter (before Phantom Menace was even released) and the car from Men In Black. The missions were challenging but not impossible, the music was fantastic as always, voice acting was top notch; it is overall just an excellent game.
7. Baten Kaitos (Gamecube)
A truly underrated gem. While I was never really a fan of the card battle system, I was able to overlook that with Baten Kaitos because the game was able to pull me in on the level of Final Fantasy games. It wasn't just because of the art style (which was gorgeous) but character development, story progression, and overall it felt like a very unique game, if you haven't played it and still have a Gamecube or a BC Wii, give this game, and its prequel/sequel Baten Kaitos Origins a go.
6. Mass Effect 3 (PS3, XBOX 360, PC)
There are few games where I feel emotionally connected to so many characters at once, and the Mass Effect trilogy happens to rank among the top games in that category, especially Mass Effect 3. While there are many critics who will bash this game because of its controversial ending, it cannot be denied that it has one of, if not the best and most engaging stories in video game history. I played through the series several times so I could experience all of the different outcomes, and I am not ashamed to admit that I almost shed a tear when some of my crew members died (I'm looking at you Mordin and Legion). Suffice it to say that this entire trilogy is simply amazing, and they are all worth playing, at the very least, 3 times each.
5. Kingdom Hearts (PS2)
Like many fans of this series, I was at first very hesitant to try this game out. I loved Square and I loved Disney, but was it possible for my two loves to come together and be like lamb and tuna fish? (name that reference) Thankfully the answer was a resounding YES. Although the game was plagued by some buggy camera controls (which the HD remix has thankfully fixed) the rest of the game was a blast to play. KH was able to seamlessly and cleverly weave the characters from the Final Fantasy franchise with characters and worlds from Disney, with at least one exception IMO (Little Mermaid). The combat is fluid and fast paced, voice acting is excellent, music is memorable, and an unforgettable story.
4. Final Fantasy X (PS2)
What can be said about FFX that hasn't been said before? Amazing story, music, characters, level designs, voice acting (OK, maybe no one ever said that before, but we can overlook that for now) FFX does for me the same thing the Mass Effect trilogy did for me, it got me to become emotionally invested; so by the time you find out Auron had been dead all along, or after you have beaten Yu Yevon and Yuna goes to hug you and falls through you, you feel the hurt and pain the characters in the game are feeling too. I have already beaten this game several times, and I am looking forward to beating it AND it's awesome sequel again when the HD collection is released next month.
3. Star Wars: Knights of the Old Republic (XBOX, PC)
In my opinion, the greatest RPG in existence. What I love about this game the most is that it caters to more than just one type of player. Some people automatically assume that since it's a Star Wars game that you automatically are going to end up a Jedi/Sith using lightsabers and force powers; while you definitely have that option, you are by no means limited to it. On my first play through of KOTOR, I chose the soldier class and specialized in using primarily long range weapons like sniper and assault rifles; other players may prefer to level up their hacking and computer skills in order to turn electronics against their opponents. However you choose to play this game it is amazing. Than of course there are the obvious things, such as: amazing story with a lot of alternate routes to take, great characters, awesome music, great voice acting, amazing level designs, etc.
1&2. Legend of Zelda: Ocarina of Time & Legend of Zelda: Majoras Mask (N64)
Like many Zelda fans, I had a hard time choosing between these two classic and superb titles. On one hand we have OoT, widely considered the greatest game of all time (for good reasons). With its stunning level designs, amazing story and an unrivaled musical score, it is a force to be reckoned with. On the other hand, we have Majoras Mask; considered by some to be the black sheep of the Zelda family because of dark/depressing themes, lack of dungeons and the use of the 3 day cycle. What it lacks in the dungeon department, it more than makes up for it in the improved character development, a deeper and less predictable story, unique mask change system, and a lengthy side quest tracker that will keep you occupied and entertained for quite awhile. And did I mention it also introduced the world to Tingle? Kooloo-Limpah! So needless to say, I am a diehard fan of BOTH of these amazing games and have decided to rank them both 1&2; depending on my mood, one can be better than the other and vice-versa.
So that is my list! I hope that everyone enjoyed reading through this. If not, I am sorry I wasted your time, and I hope you find a way to regain the last 5 minutes of your life.
Thought this was hilarious


text:
Wilson adds huge spin potential to the incredible feel and precision for which the Six One franchise is known. Boasting Spin Effect Technology, the Six.One 95S comes loaded with an open 18x16 string pattern. The result is a stringbed which grips the ball very effectively before snapping it out of the stringbed with extra spin. From the baseline our TW playtesters noted a comfortably crisp response with enough pop to effectively drive the ball. Additionally, we felt very plugged into the ball's trajectory and depth, giving us the confidence to swing bigger when applying pace and spin. At net this racket is quite speedy while also delivering decent stability for when the pace goes up. There's also plenty of feel for dropping the ball short. All in all the Six.One 95S offers a wonderful middle ground for the player who wants some extra spin but does not want to trade away the exceptional control that comes with an 18 main pattern.
|Head Size:
95 in² / 613 cm²
|Length: 27 in / 68,5 cm
|Unstrung Weight: 10.9 oz / 309 g
|Unstrung Balance: 31,5 cm / 9 pts HL
|Beam Width: 22mm / 22mm / 22mm
|Composition: Graphite Basalt Matrix
|Power Level: Low-Medium
|Stroke Style: Medium-Full
|Swing Speed: Medium-Fast
|Racquet Colours:
White / Red
|Grip Type: Wilson Sublime Grip
|String Pattern:
18 Mains / 16 Crosses
Mains skip: 8T 10T 8H 10H
One Piece
No Shared Holes
|String Tension: 51-61 pounds / 23-27 kg
Wilson Tour Molded 2.0 Red 15 Pack Bag
2 rackets plus Wilson Tour Molded 2.0 Red 15 Pack Bag is 200,70 €.
Savings: 15,00 €
ProKennex Ki 5 (300g) Racket
Fast, precise and very spin friendly. This light and comfortable player's racket is ideal for intermediate and advanced players. Headsize; 645cm². Standard Length. String Pattern: 16x20
Yonex VCORE Si 98 (285g) Racket
Offering a phenomenal combination of speed and precision, this racket is ideal for the player who wants great control and spin without the burden of weight: Headsize: 630cm². Weight: 285g