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The structural characteristics of autoreactive-T cell receptor (TCR) engagement of major histocompatability (MHC) class II-restricted self-antigens is established, but how autoimmune-TCRs interact with self-MHC class I has been unclear. We examined how CD8+ T cells kill human islet β-cells, in Type-1 diabetes, via autoreactive-TCR (1E6) recognition of an HLA-A*0201-restricted glucose-sensitive preproinsulin peptide. Rigid 'lock-and-key' binding underpinned the 1E6-HLA-A*0201-peptide interaction, whereby 1E6 docked similarly to most MHCI-restricted TCRs. However, this interaction was extraordinarily weak, due to limited contacts with MHCI. TCR binding was highly peptide-centric, dominated by two CDR3-loop-encoded residues, acting as an 'aromatic-cap', over the peptide MHCI (pMHCI). Thus, highly focused peptide-centric interactions associated with suboptimal TCR-pMHCI binding affinities might lead to thymic escape and potential CD8+ T cell-mediated autoreactivity.
The major histocompatibility complex (MHC) on human chromosome 6 is the most important genetic locus for human autoimmune diseases, indicating that T cell recognition of self antigens initiates and drives pathology1, 2. Most, but evidently not all, self-reactive T cells are eliminated in the thymus by negative selection3. Hence, there is a pressing need to better understand the molecular interactions through which disease-relevant T cell receptors (TCRs) bind to self-derived peptide-MHC (pMHC) ligands and escape thymic negative selection to populate the periphery. Structural studies of numerous non-autoimmune TCR-pMHC complexes have demonstrated that this can be a variable and versatile interaction4-14 in which a prototypic diagonal TCR-pMHC docking mode is utilized with the variable (V)α and Vβ domains of the TCR positioned over the α2- and α1-helices of the MHC molecule, respectively4, 15. In contrast, the current structural database for human autoantigen-reactive TCRs in complex with their self-ligands is highly limited. Three complexes exhibit atypical binding properties when compared to pathogen-specific TCR-pMHC complexes13, 16, 17, such as a predominant focus on the N-terminal region of the peptide or excessive tilting 13, 16, leading to the notion that autoreactive TCRs have an abnormal docking mode. However, only four structures in total have been described to date and all are MHCII-restricted and specific for myelin basic protein (MBP)13, 14, 16, 17, an autoantigen targeted in multiple sclerosis. At present, no structural or molecular data exist for other human autoimmune diseases. More importantly, there has been no exploration of the molecular interaction between an autoreactive TCR and pMHCI displayed on the surface of target cells that are destroyed as the central component of autoimmune disease pathology.
To address this knowledge gap, we turned to type 1 diabetes (T1D), an organ-specific autoimmune disease focused on the islets of Langerhans, in which β-cells are destroyed, leading to lifelong insulin dependence. Recent genome wide association studies have highlighted a key role for MHCI genes in determining disease susceptibility (HLA A*02, HLA A*11, HLA A*24 and HLA B*39)1 with a relative risk comparable to, or greater than, more classically associated genes such as INS and PTPN22. As the major role of MHCI is to present peptide antigens to CD8+ cytotoxic T lymphocytes, there are sound reasons for proposing that CD8+ T cells specific for β-cell-derived peptides presented by MHCI molecules play a pivotal role in β-cell destruction. Indeed, CD8+ T cells dominate the characteristic islet mononuclear cell infiltrate observed post mortem in patients studied close to diagnosis18 and are required to mediate efficient adoptive transfer of disease in animal models19. As proof-of-concept, we recently generated a CD8+ T cell clone (1E6) specific for the major β-cell autoantigen, preproinsulin (PPI) using a blood sample obtained from a patient studied 3 months after the onset of T1D, diagnosed using criteria of the American Diabetes Association and including acute onset of symptoms, glycosuria, random plasma glucose of >11.1mmol/l and positivity for autoantibodies to glutamic acid decarboxylase-65. The 1E6 clone mediates β-cell-specific killing via recognition of a highly distinctive HLA A*0201-presented signal peptide epitope (PPI15-24) that exhibits glucose-dependent presentation on the surface of human β-cells20. The contact between the TCR of this clone and pMHCI on the β-cell surface is thus representative of a critical, disease-determining molecular interaction in a common human autoimmune disease.
Here, we present the structures of the TCR expressed by the CD8+ β-cell cytotoxic T cell clone 1E6, its cognate HLA A*0201-ALWGPDPAAA antigen (A2-ALW), and the 1E6-A2-ALW complex. This first structure of an auto-reactive TCR-pMHCI complex furthers our understanding of how autoreactive T cells may escape into the periphery by revealing a novel mechanism for autoreactive TCR recognition of an MHCI-restricted self-antigen.
To consolidate the role of 1E6 T cells in T1D, we extended our previous studies20 on 1E6-mediated killing, using human islet cells purified from a further three HLA A*0201+ organ donors. Previously, we reported robust killing when islet cells were pre-treated with cytokines as a means to increase HLA A*0201 surface expression20 thus recapitulating the well-described islet hyper-expression of MHCI that is seen in human T1D21 However, here we have shown that untreated islet cells with resting levels of MHCI expression, and without the addition of exogenous PPI15-24 peptide, are also highly sensitive to killing by 1E6 T cells (Fig. 1). These data demonstrate that 1E6 T cells are capable of engaging with naturally occurring levels of pMHCI ligand (Supplementary Fig. 1) and can mediate disease-related effector functions as a consequence. Moreover, CD8+ T cells with specificity for this key β-cell target are enriched in the circulation of a majority of patients with T1D as shown by interferon-γ (IFN-γ) ELISPOT studies20 and PPI15-24-loaded HLA A*0201 (A2-ALW)-multimer staining of peripheral blood lymphocytes22, and show a preferential effector memory phenotype (data not shown). Thus, β-cell-reactive CD8+ T cells specific for A2-ALW are likely to mediate important effector functions in T1D patients20.
Next, we probed the specificity of 1E6 T cells using a comprehensive peptide mutagenesis scan. The index (ALWGPDPAAA) sequence was used as a blueprint, and each residue was systematically mutated along the backbone with all remaining 19 amino acids. The production of tumor necrosis factor (TNF) was used as the activation readout. Using this approach, we found that the 1E6 TCR was tolerant to changes in peptide residues Ala1, Leu2, Ala8, Ala9, Ala10; in some cases, amino acid substitutions at these residues generated larger responses compared with the index peptide (e.g. Ala1 to Arg1, and Leu2 to Gln2) (Fig. 2). Anchor residue modifications can directly alter TCR binding affinity and T cell sensitivity23 thus the enhanced responses observed could reflect, at least in part, indirect modifications to the conformation of the peptide that aid TCR binding. In contrast, modifications to the central Gly-Pro-Asp-Pro motif were not well tolerated. Modifications to this region of the peptide reduced activation by at least half in all instances, whereas changes to other regions of the peptide were substantially less critical, and in some cases increased activation compared to the wildtype sequence. This is consistent with previous structural data showing that TCRs generally focus on the central residues of the peptide, which typically bulge out of the MHC groove4, 15. Overall, the functional mutagenesis scan demonstrated the critical importance of the central Gly-Pro-Asp motif in peptide recognition by the 1E6 CD8+ T cell clone.
We next determined the affinity of the 1E6-A2-ALW interaction using surface plasmon resonance (SPR). Typically, the affinity of a pathogen-specific TCR-pMHCI interaction lies in the region of 1-10 μM24. In contrast, the binding affinity of the 1E6-A2-ALW interaction was very low (KD ≈ 278μM ± 7.6 μM at 25°C), with kinetics that were too rapid to measure (Table 1, Supplementary Fig. 2). In order to confirm that this weak interaction was not temperature-dependent, we also measured the binding affinity at 5°C (KD ≈ 274.5μM) and 37°C (KD ≈ 335μM), the latter reflecting physiological conditions (Table 1). Thus, similar to some other self-TCR-pMHCII interactions17, 24, the affinity of this MHCI-restricted autoreactive TCR was very low and within the overall spectrum of TCR-pMHCI interactions for which biophysical data are available. Indeed, to the best of our knowledge, this is the lowest TCR-pMHC affinity recorded for any natural human agonist ligand.
To investigate the structural basis of a CD8+ T cell response to an HLA A*0201-restricted, preproinsulin-derived epitope, we solved the structure of the 1E6 TCR in complex with A2-ALW in two different crystal forms at 2.6 and 2.7Å resolution (Supplementary Table 1). The ternary complexes were virtually identical in the two different crystal forms, and thus the analysis was restricted to the complex solved at higher resolution. The electron density at the interface was unambiguous, permitting structural analysis to be undertaken (Supplementary Fig. 3). Further, we solved the structure of the binary A2-ALW complex and the uncomplexed 1E6 TCR to 1.7 and 2.6 Å, respectively (Supplementary Table 1), enabling assessment of the degree of conformational change that took place upon complex formation. Indeed, while plasticity of either the TCR and/or the peptide is a hallmark of the TCR-pMHC interaction8, 12, 25, 26, neither the 1E6 TCR nor the A2-ALW complex moved appreciably during ligation. This indicated that the 1E6 autoreactive TCR engaged the pMHCI in a rigid 'lock and key' manner (Supplementary Fig. 4, 5).
Contrary to reports that some autoimmune TCRs bind to pMHC with atypical docking modes13, 16, 17, the 1E6 TCR bound in a conventional, centrally-located orientation, with the TCR α-chain positioned over the α2-helix and the TCR β-chain positioned over the α1-helix of the MHCI (Fig. 3a). Indeed, the 58.4° TCR docking angle (calculated as in4) for the 1E6-A2-ALW complex was within the range observed for other human pathogen-specific TCR-pMHC complexes (32°-80°), with the 1E6 TCR being located over the solvent exposed Gly-Pro-Asp bulge of the ALW peptide (Fig. 3a and Fig.3b). The surface complementarity (SC) index across the interface was 0.61, which also falls within the normal range for TCR-pMHCI complexes (0.60-0.72)11. Further, the total buried surface area (BSA) of the 1E6-A2-ALW complex was 1640 Å2, which falls at the lower end of the range observed for human TCR-pMHCI complexes (1470-2450 Å2). Thus, the 1E6-A2-ALW complex exhibits overall characteristics typical of most previously determined TCR-pMHCI structures.
Although in most TCR-pMHC structures solved to date the number of TCR contacts with peptide is less than the number of contacts with the restricting MHC molecule4, the 1E6-A2-ALW interaction was unusually peptide-centric (see below) with a limited MHC contact footprint compared to other anti-pathogen TCR-pMHCI complex structures. For example, the crystal structure of the AS01 TCR in complex with an HLA-A*0201 restricted, Epstein Barr virus derived, antigen (A2-CLG)11, demonstrated far broader TCR interactions with both the MHC surface and the peptide (Fig. 3c and Fig. 3d). On average, human TCRs make ~4 H-bonds and ~66 Van der Waals (vdW) contacts with the MHC surface4. A TCR bound to a super-bulged 13-mer peptide made the fewest contacts with MHC seen to date4, with only 2 H-bonds, 1 salt-bridge and 36 vdW contacts with the MHC (calculated independently using ≤4Å cut-off for vdW and ≤3.4Å cut-off for H-bonds and salt bridges)27. In comparison, the 1E6 TCR formed only 2 H-bonds and 24 vdW contacts with MHC (Table 2). At the interface, only the CDR3α loop interacted with the MHC surface, whereas both the CDR2β and CDR3β loops made MHC contacts (Fig. 4a). The CDR3α loop utilized; Asp94 to make 1 salt bridge and 2 vdW contacts with MHC residue Lys66, Ser95α to make 1 H-bond and 1 vdW with Arg65 and a solitary vdW with Lys66, and Ser96α to make 3 vdW with Arg65 (Table 2; Fig. 4b and Fig. 4c). Trp97β from the CDR3β loop made a network of 2 vdW contacts each with MHC residues Ala150 and Val152. In addition, there was 1 vdW contact between Ala101β and MHC residue Gln155, and 3 vdW contacts between Lys102β and MHC residue His151 (Table 2; Fig. 4b and Fig. 4c). The TCR CDR2β loop contacted the MHC through a total of 9 vdW interactions, comprising; 1 vdW between Asn50β and Gln72, 1 vdW between Asn51β and Val76, 3 vdW between Val53β and Gln72, 2 vdW between Ile55β and Arg65, and 2 vdW between Asp56β and Arg65. The prominent role of the CDR3 loops, especially the CDR3α loop, in contacting the MHC contrasts with the current view that the germline encoded CDR1 and CDR2 loops govern interactions with the MHC28, 29.
Previous TCR-pMHC structural analyses have unearthed the existence of three conserved MHC contact points (positions 65, 69 and 155; the "restriction triad")27, although the dependency of these positions in mediating TCR recognition is not absolute4. Of the nine MHC residues that were contacted by the TCR, Arg65 made a sizeable network of interactions with both the TCR α and β chains. However, although Arg65 was a chief MHC residue contacted by the 1E6 TCR, only 1 (>3.4 Å) vdW contact was formed with the MHC 'gatekeeper' residue Gln155, and no contacts were made with the other restriction triad residue, Arg69. This suggests that, for this autoreactive TCR-pMHCI complex, contacting the conserved "triad" was not obligatory for productive engagement30.
In contrast to typical TCR recognition of MHC molecules bound to 8-10mer peptides4, the 1E6 TCR formed a large number of peptide-mediated contacts compared to the MHC interactions. Specifically, 6/8 (75%) of the H-bonds and salt bridges, and 53/77 (69%) of the vdW contacts, were between the TCR and the peptide (Table 2). The TCR α-chain used only its CDR3α loop to contact the peptide, forming a total of 2 H-bonds and 32 vdW contacts, while the TCR β-chain bound the peptide via its CDR1β and CDR3β loops, forming a total of 4 H-bonds and 21 vdW contacts. The central positioning of the 1E6 TCR over the ALWGPDPAAA peptide enabled contacts with the central 5 amino acids of the peptide (GPDPA) (Fig. 5a and Fig. 5b). The TCR CDR3α and CDR3β loops formed a tight binding pocket around the GPDPA motif and accounted for the majority of the peptide contacts, whereas the CDR1β loop played a lesser role during peptide binding (Fig. 5b). While the 1E6 TCR was positioned in a canonical diagonal fashion over A2-ALW (Fig. 3a), the CDR3 loops of the 1E6 TCR were twisted into a perpendicular orientation over the peptide, enabling a greater number of TCR contacts to be formed with Pro5 (Fig. 3b). Although the 1E6 TCR interacted with 50% of the residues in the peptide, most of the interactions were focused in a contact zone that included just 2 residues; Pro5 and Asp6. Indeed, Pro5 and Asp6 accounted for 4/6 of the H-bonds and 34/53 of the vdW contacts between the TCR and the peptide (Table 2; Fig. 5c and Fig. 5d). Pro5 formed 1 H-bond with TCR residue Asp94α and an important network of 23 vdW interactions with Arg92α, Asp94α, Ser95α and Tyr97α within the TCR CDR3α loop (Fig. 5c). Asp6 formed 1 H-bond and 5 vdW contacts with Tyr97α in the TCR CDR3α loop, as well 1 H-bond and 2 vdW contacts with Tyr30β (CDR1β), and 1 H-bond and 4 vdW contacts with Trp97β (CDR3β) (Fig. 5d). The structural importance of peptide residues Pro5 and Asp6 is consistent with the mutagenesis data (Fig. 2). Thus, the 1E6 TCR is highly focused upon a minimal peptide motif dominated by peptide residues Pro5 and Asp6.
The dominant role of peptide residues Pro5 and Asp6 during TCR binding was mirrored by the dominant role of two contact residues in the 1E6 TCR in which Tyr97α and Trp97β from the CDR3α and CDR3β loops, respectively, dominated the interface (Table 2) and sat directly over the central Gly-Pro-Asp motif of the peptide (Fig. 5b-5d). Every pMHC contact made between the 1E6 TCR α-chain, and A2-ALW occurred via a germline-encoded residue (Table 2). Indeed, the 11-residue long 1E6 CDR3α loop was heavily germline-encoded with only 3 N-nucleotide insertions encoding for Gly93. The two CDR3α contact residues, Asp94 and Tyr97, were encoded by the TRAV12-3 gene and the TRAJ12 junctional region gene, respectively. The longer than average 14-residue CDR3β loop enabled the formation of a two-turn α-helix, a secondary structure not previously seen in TCR CDR loops4. This unusual α-helix presented the Trp97 side chain in the correct orientation to make H-bonds with Asp6, stack its aromatic ring against Pro7 and Ala8, and make contacts with residues on the MHC α2-helix. The fact that Trp97 was the only residue in the CDR3β loop to contact the peptide underscores the importance of this residue in the recognition of A2-ALW (Table 2; Fig. 5d). The CDR3β loop was constructed from the TRBV12-4, TRBD2-2 and TRBJ2-4 genes, with six N-nucleotide insertions encoding Trp97, Lys99 and Leu100. Trp is rarely found in CDR3 loops as it is not encoded in the junctional region of any of the 47 TRAV genes or 54 TRBV genes. Trp is found in just one of 57 TRAJ junctional regions (TRAJ34) and does not occur in any of the 13 TRBJ genes. Furthermore, Trp is also one of just two amino acids encoded by only one of the 64 genetic codons. Thus, the manufacture of CDR3 loops including Trp is statistically less probable during V(D)J recombination. Overall, when considering TCR germline coding regions and N-nucleotide encoding, Trp is the rarest amino acid in CDR3 loops. Further, prominently surface exposed Trp residues are often a signature for mediating protein-protein interactions31. Thus, Trp97β alongside Tyr97α is ideally suited to form a receptor-binding interface; indeed, this combination of residues formed an 'aromatic cap' that dominated TCR contacts with the A2-ALW complex. These highly focused interactions between just two TCR residues and two peptide residues governed the antigen specificity of the 1E6 TCR.
Despite mounting evidence that MHCI-restricted CD8+ T cells play a pivotal role in the development of T1D, the structural and biophysical basis for such an interaction with target β-cells has not been studied. To investigate the molecular basis for the recognition of an HLA A*0201-restricted preproinsulin epitope (A2-ALW) by a CD8+ T cell clone, we solved the structures of the autoreactive 1E6 TCR and A2-ALW in isolation and in complex. Overall, the 1E6 TCR bound to A2-ALW in a canonical diagonal fashion, with the CDR3 loops positioned over the central bulge of the peptide. Nevertheless, despite this standard docking orientation, the 1E6 TCR engaged A2-ALW with an extremely low affinity, attributable to a lower than average BSA and limited MHC contact footprint. Further, several other features of the complex are noteworthy. In contrast to most TCR-pMHC structures determined to date, this autoreactive TCR-pMHCI interaction was characterized by a rigid 'lock and key' conformation that is more typical of innate receptors or natural killer T cell TCR-CD1d-Antigen interactions32, 33. Moreover, the interaction was dominated by the CDR3 loops, consistent with a previously proposed system in which non-germline-encoded CDR3 loops drive the energetic landscape of recognition34, and yet conflicting with current theories regarding the role of the CDR1 and CDR2 loops as determinants of MHC-restriction28, 29, 35, 36. Our findings, that T cells specific for this antigen are highly enriched in T1D patients20, 22, and that 1E6 T cells kill resting β-cells in vitro, suggest that ultra-low affinity binding and highly limited MHC footprint are still sufficient to have an important impact on the destructive processes that lead to insulin deficiency and T1D.
Unusually, the 1E6-A2-ALW interaction was predominantly focused through just two aromatic TCR residues, with Tyr97α and Trp97β accounting for 64% of the contacts. Residue-focused binding was mirrored by the peptide, in which just 2/5 TCR contact residues (Pro5 and Asp6) comprised 63% of the contacts. Thus, the interaction between the 1E6 TCR and the ALW peptide was exquisitely focused and governed by a binding mechanism dominated by just two TCR and two peptide residues. Aromatic residues, and surface exposed Trp residues in particular, commonly participate in protein-protein interactions31, yet are relatively rare in TCR CDR3 loops. Nonetheless, Tyr97α and Trp97β appeared to be the driving force in this otherwise weak TCR-pMHCI interaction, acting as an aromatic cap that perches on a central region of the peptide.
There is considerable interest as to how T cells bearing TCRs that interact with autoantigens escape negative selection in the thymus and populate the periphery. Comparison of 1E6-A2-ALW and four other self-reactive human TCR-pMHC complexes with a typical HLA A*0201 pathogen-reactive complex highlights some of the differences in TCR binding to autoimmune and microbial antigens observed to date. These differences suggest several possible mechanisms to explain how self-reactive T cells might escape negative selection. First, a DR4-MBP-specific TCR was shown to bind cognate antigen with high affinity and normal topology14. This is the strongest TCR affinity reported to date for any MHCII-restricted antigen, and lies in the range reported for pathogen-specific MHCI-restricted TCRs24. However, the DR4-MBP epitope was highly unstable, leading to the hypothesis that T cells responding to low antigen densities in the thymus could receive a weak signal for positive selection, but then activate in response to higher antigen densities in the periphery. Second, self-reactive TCR-pMHC complexes, such as Ob.1A12-DR2a-MBP, 3A6-DR2-MBP and hy.1B11-DQ-MBP, were found to exhibit unusual conformations compared to more conventional pathogen-specific TCR-pMHC interactions13, 16, 17. Such unusual TCR-pMHC binding orientations may allow T cells bearing these TCRs to bypass negative selection signals. Our own findings suggest a third route of thymic escape. The 1E6 TCR bound A2-ALW conventionally, but with a very low affinity that was highly peptide-centric. The low affinity of 1E6 TCR may be below the threshold required to induce negative selection but is sufficient to induce killing of β-cells20. It is tempting to speculate that, for this TCR at least, the presence of cognate pMHCI in the thymus is highly determining with regard to selection. It is noteworthy that the INS gene encoding PPI is transcribed and translated in the thymus and that possession of variable numbers of tandem repeats of a consensus sequence located 5′ of the coding region is the strongest genetic risk for T1D outside the MHC37. Homozygosity for alleles containing few repeats (AA at rs689) confers a >2-fold relative risk of disease and is associated with 2-3 fold-reduced thymic transcription level compared with homozygous protection (TT at rs689)38. It is proposed that reduced thymic INS expression impairs central tolerance to PPI and this paradigm is supported by the demonstration that non-obese diabetic mice with targeted deletion of INS2 (syntenic to human INS) display enhanced T cell reactivity to insulin and more rapid and penetrant disease39. The T1D patient from whom 1E6 was obtained has AA at rs689. Notably, all three of the above mechanisms represent atypical recognition parameters that may help self-reactive T cells to bypass thymic deletion.
The observation that clone 1E6 has the lowest TCR affinity for pMHC described to date, yet remains able to kill resting human HLA-A*0201 β-cells expressing low levels of HLA class I, is a further potentially important observation in relation to disease pathogenesis, since it suggests that such clones have the potential to engage non-conditioned targets. Most disease models of T1D pathogenesis and initiation elaborated to date argue that islet inflammation comes first, and that the resulting cytokine-induced islet hyper-expression of HLA class I facilitates CD8+ T cell killing. Our data raise the possibility that CD8+ T cell killing could be an early event. Pre-existing low affinity CD8+ T cells could also be relevant in the setting of islet transplantation, in which donor β-cells express resting levels of HLA class I and therefore constitute targets for 1E6-like CD8+ T cells. Similarly, the future use of gene-modified β-cell replacement therapy may need to include designs that limit HLA class I expression to avoid disease recurrence. Finally, our observations raise the important consideration as to whether 1E6-like autoreactive T cells with ultra-low affinity TCRs are originally primed by self- or non-self ligands. We consider that our findings make the latter more likely, especially when viewed in the context of numerous studies implying a link between viruses, islet autoimmunity and T1D. Extensive searching of databases of viral proteins against ALWGPDPAAA, or modifications tolerated by 1E6 TCR, followed by examination of stimulation of 1E6 in vitro have not to date revealed any potential virus-derived ligands (data not shown). However, the number of complete sequences of viruses that might be considered of highest relevance (eg the human enteroviruses), and are in the public domain, are currently limited and pathogen cross-reactivity will remain an important question to be that needs to be addressed. We have recently shown that the 1E6 clone recognizes over a million distinct decamer peptides in the context HLA A*020140. A large number of peptides acted as substantially better agonists than the preproinsulin-derived peptide ALWGPDPAAA with the most potent peptide identified, RQFGPDFPTI, differing from this 'index' sequence at 7 of 10 amino acids. These findings serve to highlight the enormous potential of TCR degeneracy to be a causative factor in autoimmune disease.
In summary, we present the first structure of a human TCR in complex with an autoreactive MHCI-restricted antigen. Collectively, our observations suggest that CD8+ T cells expressing the 1E6 TCR could evade negative selection due to a 'light-touch' MHC contact footprint and weak TCR binding. In the periphery, β-cells operating in the presence of elevated glucose levels, combined with the pro-inflammatory milieu that leads to hyper-expression of MHCI, might drive high levels of surface A2-ALW expression and render these cells susceptible to antigen-experienced, 1E6-like CD8+ T cells20, 41. Such T cells with low avidity for self likely require priming through contact with an infectious agent. It is possible that the unusual features of 1E6 TCR binding, focusing on just two residues in the peptide, as seen here, might increase the chances of pathogen-derived peptide cross-recognition. Indeed, it has been speculated that such 'hotspot mimicry' limited to a small number of key features within the TCR-binding footprint could contribute to the initiation of multiple sclerosis42. Collectively, these data suggest a mechanism by which 1E6-like CD8+ T cells avoid thymic culling to populate the periphery and cause disease.
Human islet cells were isolated as described previously43. Pancreata were retrieved with the consent of donors' relatives and permission from the Ethical Review Committee of King's College Hospital. Islet-enriched cell fractions were cultured in CMRL1066 containing 5.6 mM glucose, supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 μg/ml) and L-glutamine (2 mM) (Invitrogen) for 2 weeks. Cells were allowed to become monolayer cultures and were cultured with 16 mM glucose in either the presence or absence of IL-1β (50 IU/ml; Strathmann Biotec), TNF (2,500 IU /ml), IFN-γ (500 IU/ml; Miltenyi Biotec) and IFN-α (1,000IU/ml; Roche Laboratories) for 16-24 hr to upregulate HLA class I expression. Cytotoxicity was analyzed using a non-radioactive Europium TDA (EuTDA) cytotoxicity assay with DELFIA® Technology (Perkin Elmer) according to the manufacturer's instructions. Briefly, human islet cells (106) were incubated for 20 min at 37°C in 2 ml of CMRL1066 (complete) with 3 μl of fluorescence-enhancing ligand. After washing with PBS (3 times), 50 μl of labeled islets cells (5 × 103) were seeded in triplicate into V-shaped 96-well plates followed by 50 μl of effector cells (CD8+ T cell clone) at varying ratios in X-Vivo 15/5% AB serum containing IL-7 (10 ng/ml), IL-15 (0.1 ng/ml) and 2.5% Cellkines (Helvetica Healthcare). The cultures were incubated for 4 hr at 37°C, then 20 μl of supernatant was transferred into a clear, flat-bottom microtitration plate followed by the addition of 200 μl/well of enhancement solution. After 15 min at room temperature with shaking, the europium signal was measured using a FLUOstar OMEGA™ Time-Resolved Fluorescence reader (BMG Labtech). To determine maximum lysis, islet cells alone (5 × 103 in 100 μl) were treated with 20 μl lysis buffer. Spontaneous release was measured from wells containing only labeled islet cells in 100 μl medium. Specific cytotoxicity was calculated using the formula: % specific release = (experimental-spontaneous release) × 100/(maximum-spontaneous release).
For the recognition screen, 2 × 103 1E6 CD8+ T cells were cultured in triplicate for 16 hr with individual peptides representing every positional variant (n=190) of the wildtype PPI15–24 epitope sequence (ALWGPDPAAA) at a concentration of 1 μg/ml. Supernatant was harvested and analyzed for TNF production by ELISA according to the manufacturer's instructions (Life Technologies, Paisley, UK).
CD8+ T cell clones specific for A2-ALW were generated as described previously20. The 1E6 TCR, HLA A*0201 α-chain and β2m sequences were generated by PCR mutagenesis (Stratagene) and PCR cloning. For the 1E6 TCR, a disulphide linked construct was used to produce the soluble domains (variable and constant) for both the α and β chains44. The soluble HLA A*0201 α-chain (α1, α2 and α3 domains), tagged with a biotinylation sequence, and β2m were also cloned and used to make the HLA A*0201 protein. The TCR α and β chains, HLA A*0201 α-chain and β2m sequences were inserted into separate pGMT7 expression plasmids under the control of the T7 promoter.
Competent Rosetta DE3 E. coli cells were used to produce the 1E6 TCR α and β chains, and the HLA A*0201 α-chain and β2m, in the form of inclusion bodies (IBs) using 0.5 mM IPTG to induce expression as described previously44. TCR and pMHCI proteins were refolded as previously described24. Refolded proteins were purified initially by ion exchange using a Poros50HQ™ column and finally gel filtered into BIAcore buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% (v/v) Surfactant P20) or crystallization buffer (10 mM TRIS pH 8.1, 10mM NaCl) using a Superdex200HR™ column. Protein quality was analyzed by Coomassie-stained SDS-PAGE.
Biotinylated pMHC was prepared as described previously45.
Binding analysis was performed using a BIAcore T100™ equipped with a CM5 sensor chip as reported previously45. Briefly, between 200 and 400 response units (RUs) of biotinylated pMHC was immobilized to streptavidin, which was chemically linked to the chip surface. Equilibrium analysis was performed using ten serial dilutions, prepared in triplicate, for each sample and injected over the relevant sensor chips at 25°C. The 1E6 TCR was injected over the chip surface using kinetic injections at a flow rate of 45 μl/min. To assess temperature dependency, this method was repeated at 5°C and 37°C. Results were analyzed using BIAevaluation 3.1™, Microsoft Excel™ and Origin 6.1™. The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = (P1x)/(P2 + x)).
1E6-A2-ALW, 1E6 TCR and A2-ALW crystals were grown at 18°C by vapour diffusion via the sitting drop technique. For 1E6-A2-ALW, optimal crystals were obtained with 0.2 M sodium citrate, 0.1 M Bis Tris propane pH 6.5, 20% w/v polyethylene glycol (PEG) 3350. For A2-ALW, optimal crystals were obtained with 0.2 M potassium/sodium tartrate, 0.1 M Bis Tris propane, pH 6.5, 20% w/v PEG 3350. For 1E6 TCR, optimal crystals were obtained with 0.2 M sodium iodide, 20% w/v PEG 3350. All crystals were soaked in 30% ethylene glycol before cryo-cooling. All crystallization screens and optimization experiments were completed using an Art-Robbins Phoenix dispensing robot (Alpha Biotech Ltd, UK). Data were collected at 100 K at the Diamond Light Source (DLS), Oxfordshire, UK. All datasets were collected at a wavelength of 0.976Å using an ADSC Q315 CCD detector. Reflection intensities were estimated with the XIA2 package46 and the data were scaled, reduced and analyzed with SCALA and the CCP4 package47. The structure was solved with molecular replacement using AMORE48. The model sequence was adjusted with COOT49 and the model refined with REFMAC5. Graphical representations were prepared with PYMOL50. Data reduction and refinement statistics are shown in Supplementary Table 1.
We thank the staff at Diamond Light Source for providing facilities and support. This work was funded by the BBSRC (grant BB/H001085/1), the Wellcome Trust (WT086716 to AKS to WT095767 to DKC), the JDRF (7-2005-877 and 1-2007-1803 to MP; 17-2009-806 to AKS, MP, DAP and AS), EU FP7 (241447 NAIMIT) and NIHR cBRC at Guy's & St Thomas' NHS Foundation Trust and King's College London. Fellowship support was received from RCUK (PJR), NHMRC (JR), WORD (JJM), MRC (DAP) and Wellcome Trust (LW). We are grateful to M Zhao for help preparing the human islets and John Todd for INS genotyping.
METHODS Methods and any associated references are available in the online version of the paper at http://www.nature.com/natureimmunology/.
Accession codes: Protein data bank: 1E6-A2-ALW complex (P1 space group), 3UTS; 1E6-A2-ALW complex (P21 space group), 3UTT; 1E6 TCR, 3UTP; A2-ALW, 3UTQ.
Note: Supplementary information is available on the Nature Immunology website.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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All hotels in Folkestone, England, United Kingdom
1 room, 2 adults
22-26 The Leas, Folkestone, England, CT20 2DY, United Kingdom, 800-491-6126
The Southcliff, Kent (county)
Lowest price$30
Best Price Guarantee
Folkestone Victorian hotel with bar/lounge
- Free WiFi in lobby
Main amenities
- 70 guestrooms
- Breakfast available
- Bar/lounge
- 2 meeting rooms
- 24-hour front desk
- Daily housekeeping
- Computer station
- Conference space
- Wedding services
- Luggage storage
- Lobby TV
What's around
- Heart of Folkestone
- Leas Cliff Hall (0.1 mi / 0.2 km)
- Folkestone Harbour (0.6 mi / 0.9 km)
- Leas Lift (0.7 mi / 1.1 km)
- Folkestone Beach (0.7 mi / 1.2 km)
- Lower Leas Coastal Park (1.2 mi / 1.9 km)
Very very outdated, shabby and dirty, the breakfast room is appalling
Tired old seafront hotel. Staff reasonably good but décor pretty run down. No secure…
TripAdvisor Traveller Rating: 3
From 374 reviews
Enter dates to check prices
Hotel Info: 866-539-8117from $30
- Twin Room, 2 Single Beds
- Standard Double Room
- Double Room
- Double Room Single Use
- Twin Room, Sea View
- Standard Twin Room
- Single Room (Value)
- Standard Double Room, Balcony, Sea View
- Double Room, Sea View
Recommended hotels
At a glance
Key facts
Hotel size
- This hotel has 70 rooms
- This hotel is arranged over 4 floors
- Check-in time starts at 3 PM
- Check-out time is 10:30 AM
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
- Minimum check-in age is 18
Travelling with others
- No cribs (infant beds)
- Pets not allowed
- Free WiFi in public areas
- No parking
Nearby Two Star Kent (county) hotels
In the hotel
Food and drink
- English breakfast (surcharge)
- Bar/lounge
Things to do
- Use of nearby fitness center (discount)
- Golfing nearby
- Scuba diving nearby
Working away
- Number of meeting rooms - 2
- Conference space
- Computer station
- 24-hour front desk
- Luggage storage
- Wedding services
- Number of buildings/towers - 1
- Year Built
- Elevator/lift
- Designated smoking areas (fines apply)
- Television in lobby
In the room
Home comforts
- Coffee/tea maker
- Iron/ironing board (on request)
Freshen up
- Private bathroom
- Shower/tub combination
- Free toiletries
Be entertained
- 19-inch LCD TV
- Digital TV channels
Stay connected
- Phone
- Daily housekeeping
Top Kent (county) hotels
Recently reviewed hotels in Kent (county)
- Dunkerley's Restaurant and HotelFrom
"Nice hotel in a perfect location. Our room was large for a European standard with a nice, large shower. We didn't eat in the restaurant other than the included breakfast which was…"Jun 21, 2015
- Best Western Plus Dover Marina Hotel & SpaFrom
"Great stay overall. Parking should be better-signed. Room layout cramped. Spectacular views of the English Channel, Dover Castle, the White Cliffs of Dover and the pier."Jun 20, 2015
- The George in RyeFrom
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Special features
DiningThe Office - Onsite bar.
The Southcliff, Kent (county)'s small print
Also known as
- Southcliff Folkestone
- Southcliff Hotel
- Southcliff Hotel Folkestone
- The Southcliff Hotel
Only registered guests are allowed in the guestrooms.
Optional extras
Self parking costs GBP 2 per day
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.

subset: commoncrawl


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Ferrari Donates 1st F12 Berlinetta for Sandy Relief
Nov. 23 (Bloomberg) -- On today's "Weird Wall Street," Bloomberg News discusses bizarre stories in business. They speak on Bloomberg Television's "Street Smart." (Source: Bloomberg)
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Latest Titles With Rupert Everett
- Job Types
- Movies or TV
- Genres
- Keywords
- IMDb Rating
- Instant Watch Options
- In Theaters
- On TV
- Release Year
1. The Happy Prince
Comedy, Drama, History | Announced
The Happy Prince is the untold story of the last days in the tragic times of Oscar Wilde. A person who observes his own failure with ironic distance and regards the car crash of his life with detachment and humor.
Writer: Rupert Everett | Director: Rupert Everett | Stars: Colin Firth, Hugh Dancy, Emily Watson, Tom Wilkinson
2. The B Team (II)
Comedy | Announced
An action comedy reality movie about CIA spies going undercover on the most watched TV series in the world, to save humankind.
Writer: Michael Brody | Director: Chris Cottam | Stars: Rupert Everett, Alexandra Paul, John Cleese, Kelly Packard
3. Miss Peregrine's Home for Peculiar Children (2016)
Adventure, Fantasy | Filming
Teenager Jacob follows clues that take him to a mysterious island, where he discovers the crumbling ruins of Miss Peregrine's School for Peculiar Children. As Jacob explores the abandoned bedrooms and hallways, he discovers that its former occupants were far more than peculiar; they possessed incredible powers. And they may still be alive.
Writers: Jane Goldman, Ransom Riggs | Director: Tim Burton | Stars: Eva Green, Samuel L. Jackson, Kim Dickens, Asa Butterfield
4. A Royal Night Out (2015)
97 min | Drama, Romance, Thriller
On V.E. Day in 1945, as peace extends across Europe, Princesses Elizabeth and Margaret are allowed out to join the celebrations. It is a night full of excitement, danger and the first flutters of romance.
Writers: Trevor De Silva, Kevin Hood | Director: Julian Jarrold | Stars: Jack Reynor, Sarah Gadon, Emily Watson, Rupert Everett
Votes: 505
5. Altamira (2015)
Drama | Post-production
Writers: Olivia Hetreed, José Luis López-Linares | Director: Hugh Hudson | Stars: Antonio Banderas, Rupert Everett, Golshifteh Farahani, Nicholas Farrell
The Graham Norton Show (2007– )
Rupert Everett/Miranda Hart/Greg Davies/Electro Velvet
43 min | Comedy, Talk-Show
Writer: Rob Colley | Director: Steve Smith | Stars: Graham Norton, Rupert Everett, Miranda Hart, Greg Davies
Votes: 7
7. The Kumars (2014– )
60 min | Comedy
Stars: Sanjeev Bhaskar, Vincent Ebrahim, Meera Syal, Harvey Virdi
Votes: 63
8. Rosenn (2014)
Writer: Yvan Le Moine | Director: Yvan Le Moine | Stars: Hande Kodja, Rupert Everett, Stanislas Merhar, Jacques Boudet
Votes: 12
9. Love for Sale with Rupert Everett (2014 TV Mini-Series)
Star: Rupert Everett
Votes: 14
The Kumars (2014– )
Episode #1.2
Writers: Sanjeev Bhaskar, Madeleine Brettingham, Fergus Craig, George Jeffrie, Nish Kumar, Tom Leopold, Maya Sondhi, Bert Tyler-Moore | Director: Sasha Ransome | Stars: Sanjeev Bhaskar, Vincent Ebrahim, Meera Syal, Harvey Virdi
ITV News London (1993– )
Episode dated 11 December 2014
Stars: Rupert Everett, Charlene White
This Week (2003– )
Episode dated 6 March 2014
News, Talk-Show
Stars: Andrew Neil, Michael Portillo, Diane Abbott, Rupert Everett
13. Justin and the Knights of Valour (2013)
PG | 90 min | Animation, Adventure, Family
A young boy becomes a man as he embarks on a quest to become a knight.
Writers: Matthew Jacobs, Manuel Sicilia | Director: Manuel Sicilia | Stars: Antonio Banderas, James Cosmo, Michael Culkin, Charles Dance
Votes: 3,855
14. The Return (VII) (2013)
25 min | Short
Writer: Karl Lagerfeld | Director: Karl Lagerfeld | Stars: Geraldine Chaplin, Rupert Everett, Amanda Harlech, Arielle Dombasle
Votes: 10
Loose Women (1999– )
Episode #17.153
Stars: Carol Vorderman, Lisa Maxwell, Janet Street-Porter, Sherrie Hewson
Loose Women (1999– )
Episode #17.75
Stars: Carol Vorderman, Denise Welch, Carol McGiffin, Jane McDonald
17. Parade's End (2012 TV Mini-Series)
287 min | Action, Drama, Romance | Active
Revolves around a love triangle between a conservative English aristocrat, his mean socialite wife and a young suffragette.
Stars: Benedict Cumberbatch, Rebecca Hall, Roger Allam, Adelaide Clemens
Votes: 6,410
18. The Other Wife (2012 TV Mini-Series)
180 min | Romance
Two part drama about a wife who finds out her husband has another wife and child after he is killed in a plane crash.
Stars: Rupert Everett, Natalia Wörner, John Hannah, Matt Barber
Votes: 55
Rosamunde Pilcher (1993– )
Die andere Frau, Teil 1
Drama, Romance
Director: Giles Foster | Stars: Matt Barber, Sarah Childs, Hilary Connell, Rupert Everett
Rosamunde Pilcher (1993– )
Die andere Frau, Teil 2
Drama, Romance
Lord Carey-Lewis Loveday's testament leaves his Cornish estate, Nancherrow, to his penniless daughter Loveday, who loves it even more then he did. DEviting her life to it soon wrecks her ... See full summary »
Director: Giles Foster | Stars: Matt Barber, Hilary Connell, Rupert Everett, Miguel Herz-Kestranek
Parade's End (2012– )
Episode #1.1
Action, Drama, Romance
In 1908 civil servant Christopher Tietjens marries the pregnant Sylvia Satterthwaite though the father is probably her married lover Gerald Drake,as Christopher's brother Mark points out. ... See full summary »
Writers: Tom Stoppard, Ford Madox Ford | Director: Susanna White | Stars: Lucinda Raikes, Rebecca Hall, Benedict Cumberbatch, Stephen Graham
Votes: 217
The Other Wife (2012– )
Episode #1.1
Writer: Rosamunde Pilcher | Stars: Rupert Everett, Natalia Wörner, John Hannah, Matt Barber
Parade's End (2012– )
Episode #1.2
Action, Drama, Romance
Sylvia returns to Christopher,largely for the financial security and attends his mother's funeral,where her showy appearance shocks the mourners. Realising she is reviled she goes to a ... See full summary »
Writers: Tom Stoppard, Ford Madox Ford | Director: Susanna White | Stars: Rebecca Hall, Benedict Cumberbatch, Janet McTeer, Ned Dennehy
Votes: 177
The Other Wife (2012– )
Episode #1.2
Writer: Rosamunde Pilcher | Stars: Rupert Everett, Natalia Wörner, John Hannah, Matt Barber
Parade's End (2012– )
Episode #1.3
Action, Drama, Romance
Despite receiving a white feather for cowardice Vincent becomes a very influential author and marries Edith after Duchemin kills himself. They are the subject of gossip and scandal which ... See full summary »
Writers: Tom Stoppard, Ford Madox Ford | Director: Susanna White | Stars: Benedict Cumberbatch, Rebecca Hall, Adelaide Clemens, Dominic Coleman
Votes: 159
Parade's End (2012– )
Episode #1.4
Action, Drama, Romance
Christopher is in Rouen with his godfather General Campion and the unbalanced McKechnie,his job being to kit out fresh troops for the front. His desire to see that the men are humanely ... See full summary »
Writers: Tom Stoppard, Ford Madox Ford | Director: Susanna White | Stars: Anna Skellern, Rebecca Hall, Ronald Pickup, Leslie de Gruyter
Votes: 146
Parade's End (2012– )
Episode #1.5
Action, Drama, Romance
1917;- Christopher,McKechnie and Perowne have all been sent to fight in the trenches,surrounded by carnage and madness. When Bill,the commanding officer,suffers a breakdown from stress ... See full summary »
Writers: Tom Stoppard, Ford Madox Ford | Director: Susanna White | Stars: Roger Allam, Elliot Levey, Benedict Cumberbatch, Patrick Kennedy
Votes: 153
This Morning (1988– )
Episode dated 19 September 2012
Director: Ashley S. Gorman | Stars: Phillip Schofield, Holly Willoughby, Rupert Everett, Paul Merton
29. Black Mirror (2011– )
60 min | Drama, Sci-Fi, Thriller
A television anthology series that shows the dark side of life and technology.
Creator: Charlie Brooker | Stars: Daniel Kaluuya, Toby Kebbell, Rory Kinnear, Hayley Atwell
Votes: 33,522
30. Hysteria (2011)
R | 100 min | Comedy, Romance
The truth of how Mortimer Granville devised the invention of the first vibrator in the name of medical science.
Writers: Stephen Dyer, Jonah Lisa Dyer, Howard Gensler | Director: Tanya Wexler | Stars: Maggie Gyllenhaal, Hugh Dancy, Jonathan Pryce, Felicity Jones
Votes: 23,084
31. Fern (2011– )
Hour-long weekday chat show featuring a mix of real people's true stories, celebrity interviews, cooking and entertainment.
Stars: Fern Britton, Michael Ball, Ben Barnes, Wayne Bartholomew
Votes: 6
32. Shrek: Once Upon a Time (2011 TV Movie)
59 min | Documentary
Stars: David Tennant, Antonio Banderas, Richard Blackwood, Bill Damaschke
Votes: 10
Fern (2011– )
Episode #1.20
Stars: Fern Britton, Wayne Bartholomew, Rupert Everett, Craig Revel Horwood
Loose Women (1999– )
Episode #15.174
Stars: Andrea McLean, Lisa Maxwell, Carol McGiffin, Denise Welch
Paul O'Grady Live (2010– )
Episode #2.1
Stars: Paul O'Grady, Jon Briggs, Charlotte Church, John Cleese
Black Mirror (2011– )
Fifteen Million Merits
62 min | Drama, Sci-Fi, Thriller
In a bleak,automated future Britain, Bing is one of millions who pedal exercise bikes to create energy as a living. Their currency is merits, tokens with which to buy food from vending ... See full summary »
Writers: Charlie Brooker, Konnie Huq | Director: Euros Lyn | Stars: Daniel Kaluuya, Jessica Brown Findlay, Rupert Everett, Julia Davis
Votes: 3,976
37. Wild Target (2010)
PG-13 | 98 min | Action, Comedy, Crime
A hitman tries to retire but a beautiful thief may change his plans.
Writers: Lucinda Coxon, Pierre Salvadori | Director: Jonathan Lynn | Stars: Bill Nighy, Emily Blunt, Rupert Grint, Rupert Everett
Votes: 27,518
38. The People Speak UK (2010)
Documentary, History
The People Speak is a celebration of the forgotten and famous-dissenters, rebels, and visionaries-from Britain's past and present. Through dramatic and musical performances based on primary... See full summary »
Creators: Anthony Arnove, Colin Firth | Directors: Anthony Arnove, Colin Firth, Stuart McDonald | Stars: Nonso Anozie, Saffron Burrows, John Castle, Anna Chancellor
Votes: 15
39. Paul O'Grady Live (2010– )
60 min | Talk-Show
Stars: Paul O'Grady, Jon Briggs, Gary Barlow, Cilla Black
Votes: 13
Chris Moyles Quiz Night (2009– )
Alan Carr, Mischa Barton and Rupert Everett
Stars: Mischa Barton, Alan Carr, Peter Dickson, Rupert Everett
Loose Women (1999– )
Episode #14.200
Stars: Kate Thornton, Lisa Maxwell, Carol McGiffin, Lynda Bellingham
Who Do You Think You Are? (2004– )
Rupert Everett
Director: Michael Waldman | Stars: Rupert Everett, Mark Strong
Votes: 8
43. St Trinian's 2: The Legend of Fritton's Gold (2009)
106 min | Adventure, Comedy
The girls of St. Trinians are on the hunt for buried treasure after discovering headmistress Miss Fritton is related to a famous pirate.
Writers: Piers Ashworth, Jamie Minoprio, Nick Moorcroft, Jonathan M. Stern | Directors: Oliver Parker, Barnaby Thompson | Stars: Rupert Everett, Colin Firth, David Tennant, Talulah Riley
Votes: 5,646
44. Madonna: Celebration - The Video Collection (2009 Video)
211 min | Music
Directors: Fabien Baron, Steve Barron, Harold Becker, Luc Besson, Chris Cunningham, Jonas Euvremer, David Fincher, James Foley, Michael Haussman, Alek Keshishian, Jamie King, Mary Lambert, Melodie McDaniel, Jean-Baptiste Mondino, Tom Munro, Arthur Pierson, Brett Ratner, Johan Renck, Eugene Riecansky, Nathan Rissman, Guy Ritchie, Herb Ritts, Matthew Rolston, Mark Romanek, Peter Rosenthal, Earle Sebastian, Susan Seidelman, Philipp Stölzl, Traktor, Bobby Woods, Jonas Åkerlund | Stars: Madonna, Danny Aiello, Sacha Baron Cohen, Erika Belle
Votes: 128
45. Alan Carr: Chatty Man (2009– )
60 min | Talk-Show
Interview talk show hosted by comedian Alan Carr
Stars: Alan Carr, David Walliams, Mollie King, The Saturdays
Votes: 454
46. Chris Moyles Quiz Night (2009– )
Stars: Chris Moyles, Peter Dickson, Peter Crouch, Ozzy Osbourne
Votes: 99
47. The Scandalous Adventures of Lord Byron (2009– )
48 min | Documentary, History
Star: Rupert Everett
Votes: 12
48. Hi Society: The Wonderful World of Nicky Haslam (2009 TV Movie)
60 min | Documentary
Writer: Hannah Rothschild | Director: Hannah Rothschild | Stars: David Bailey, Graydon Carter, Rupert Everett, Christopher Gibbs
The Scandalous Adventures of Lord Byron (2009– )
Episode #1.1
Documentary, History
Star: Rupert Everett
Alan Carr: Chatty Man (2009– )
Episode #1.7
Writer: Stewart Williams | Stars: Alan Carr, Kevin Bishop, The Enemy, Rupert Everett


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Edit hours
East Village
Their ice cream with caramel &sea salt is so good. Get a loyalty card and get 1 ice cream free when you've had 10. Get 50% off if u buy the t-shirt they wear in the shop - have to wear it to qualify
Really amazing love the big pimp and bea Arthur
The salty pimp. Get it in a dish for a drip-free experience.
Stop looking at the wall. You want Salty Pimp believe me.
Great soft-serve. Try the salty pimp
Try the Salty Pimp or American Globs.
Good cones but a lil pricy
Amazing ice cream!
Dulce leche
Don't believe the hype. Just like a McDonald's cone + lots of stuff on top.
There's a reason that all of the reviews mention the Salty Pimp. Get it and be glad you did.
Get ANYTHING and you won't be disappointed. Well, except in the fact that you don't have room to get EVERYTHING.
A bit of a queue but the ice cream was ridiculously delicious.
Long line in front of the door, if you want to taste NY ice cream you can trt
Very friendly staff
Salty pimp, the tip is the best part!
Salty pimp
Ask to merge the salty pimp and the bea Arthur.
Get one even if it's cold out.
77 degrees after the big chill and this place was packed. My ice cream was worth the wait tho. It's soft serve with fun flavours. Nothing too serious!
Love their techniques and combos! Worth the wait!
I had a small love affair with an American Globs on a sunny afternoon. Would recommend to any/everyone.
Quick service and the salty pimp was divine! Vanilla ice cream with salty caramel and dulce de leche and then dipped in chocolate.
Creative soft serve sundaes with a healthy serving of kitsch
Every flavor is good but American Globs is the best
Deadpool recommends the Bea Arthur 👌
So much happiness in this place. And killer ice cream.
The Bea Arthur is bæ
Free sprinkles!! Get a punch card too for free ice cream.
Good ice cream, don't mind the name.
Yellow cake soft serve is to die for
The salted Pimp rules the Street! 👍
Everything is good.
Killer ice cream.
Great Lil ice cream shop. Watch out, no seating.
Great use for the unicorn sticker in the Swarm app!
Rocky roadhouse!
Be adventurous and hack your own creations. "The limit does not exist!"
Horchata shake is good. Ginger-curry is unique .
Mexican affogayto. Salty pimp. Bea Arthur
It's tasty; don't get me wrong. But, I was expecting more than fancied up soft serve. The Salty Pimp is good, but I wish they'd double dip it so the chocolate shell would last more than 3 minutes
You can't go wrong with anything here. My favorite is vanilla ice cream in a gluten free cone with Nutella inside 😋
so beautiful place ;)
The store was adorable, the ice cream was yummy just wasn't what I was expecting at all. It's more of a ice cream cake in a cone, to many fillings not enough ice cream. #iwantedbubblegum
Big selection of freshly made ice creams. I especially love the malteds. Usually a line in the summer but well worth the wait.
Blueberry gobbler was so good :-)
Great ice cream. Try the Salty Pimp
Bea Arthur!!!
The Bea Arthur is the greatest ice-cream!


text:
Posts tagged "Chicago Hauntings"
150th Anniversary of the Emancipation Proclamation and a Haunted Portrait of Abraham Lincoln in the City of Evanston?
Meet The Blogger
Ray Johnson
Ray Johnson is a former criminal investigator in Du Page County, Illinois. He was born in Chicago and has spent his entire life in the Chicagoland area. He is a graduate of The University of Illinois at Chicago and has taught College Classes in Criminal Justice at the College of Du Page in Glen Ellyn as well as lecturing on Chicago folklore and history and teaching adult education classes on historical research techniques. He is a former Chicago Area Representative of the Association of Professional Genealogists, a member of the Jackson Park Advisory Council and a member of the Hyde Park Historical Society. He is a life-long fan of Chicago history and especially the stranger side of Chicago's history which really makes history come alive for adults and kids alike. His first published work is "Chicago's Haunt Detective", Schiffer Publishing, Atglen, PA, 2011. His website for the stranger side of Chicago is HauntDetective.com He also runs a historical research service from his other website, HistoryCop.com. His second book, "Chicago History - The Stranger Side", Schiffer Publishing, Atglen, PA, 2014 was released this February. The 1893 World's Columbian Exposition is one of Ray's favorite historic topics in Chicago history and he recently started a not for profit called "Friends of the White City" Contact Ray Email email@example.com Websites http://www.hauntdetective.com http://www.historycop.com http://www.friendsofthewhitecity.org Facebook https://www.facebook.com/HistoryCop Twitter @ChiTownHistory
History Cop's Books
Recent posts
Chicago Mayor Approves City-Wide Use of Fireworks on the 4th of July 1893 »Posted Thursday at 9:00 pm
Sundae in the Park Fundraiser for Jackson Park Advisory Council - May 17, 2015 »Posted May 8, 2015 at 10:53 am
Fox News and Female Bystander both Witness Police Shooting in Baltimore That Didn't Happen »Posted May 4, 2015 at 4:49 pm
List of Chicago Police Officers Killed and Injured at Chicago Haymarket Affair »Posted May 4, 2015 at 8:22 am
Undocumented Shoppers Antagonized by Police during Baltimore Riots! »Posted April 29, 2015 at 1:14 pm
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Latest on ChicagoNow
All Wildwood Kantharos in the Purple Violetfrom Picks & Ponderings by Paul Mazur
posted today at 6:31 pm
X's and O's idea: Running San Antonio's "Loop"from Chicago Bulls Confidential by Morten Stig Jensen
posted today at 5:12 pm
White Sox Minor League Recap 7/3/2015: Affiliates go 7-2 with explosive offense all around.from Future Sox by Daniel Shapiro
posted today at 3:29 pm
Epistalory wisdom from Thomas Jefferson to mark the 4th of Julyfrom The Quark In The Road by Aquinas wired
posted today at 1:31 pm
My favorite July 4th celebrationsfrom Candid Candace by Candace Jordan
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Read these ChicagoNow blogs
Life with a Side of BrowniesViewing life as an adventure. Acknowledging that it requires regular doses of chocolate.
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Read these ChicagoNow Bloggers
David Telismanfrom Chocolate Diapers:
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The United Nations Office for
Free tagging:
The proceedings of the Fourth Session of the Global Platform for Disaster Risk Reduction are now available. The Platform, a biennial forum with the latest goal of improving implementation of
Download the March 2013 Updates
In this issue:
UN-SPIDER at a glance
Representatives of UN agencies, funds and programmes completed an action plan this week that will accelerate the integration of
The UN system task team on the post-2015 UN development agenda has developed their second Thematic Think Piece on Disaster Risk and Resilience to support discussions on the post-2015 development agenda. The paper outlines the modus operandi of the International Strategy for
An early view of
The Fourth Session of the Global Platform "Invest Today for a Safer Tomorrow" comes at a time when there is unprecedented international momentum to reduce
International Strategy for Disaster Reduction, ISDR
Portuguese Government
Municipality of Amadora
The United Nations International Strategy for
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On The Ground: Discovery Lands European Olympic Rights Through 24Night On The Towns CAA reps top two NBA picks in Karl-Anthony Towns, D'Angelo Russell Tony's Hawks New Hawks Owner makes firm statement that he'll call the shots. Red Faced Red Sox sponsors claim club's on-field woes now affecting business. ... Sports Business Journal, 1 week ago
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3,438 Matches for Patricia Lopez
Patricia O. Lopez 33 yrs
33-year-old Patricia O Lopez lives in Austin, Texas, but has also spent time in Lockhart, Texas. She attended the Lockhart High School and she is employed by Graduate Student. Patricia enjoys listening to Pop Rock. How To Lose A Guy In Ten Days and The Passion Of Christ are some of the films Patricia loves.What is PeekScore?
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Reading Log 2015
by Oyin
1. Marriage, Divorce & Remarriage – Kenneth E. Hagin
2. The Circle Maker – Mark Batterson
3. How to get the most from your reading your Bible – Adam Houge
4. How Faith Works – Fred Price
5. Loveology – John Mark Comer
1. Marriage, Divorce & Remarriage – Kenneth E. Hagin
2. The Circle Maker – Mark Batterson
3. How to get the most from your reading your Bible – Adam Houge
4. How Faith Works – Fred Price
5. Loveology – John Mark Comer
If you are a Christian, at one point or another in your walk you have heard or read the part of the bible that says
For I know the plans I have for you, declares the Lord, plans for welfare and not for evil, to give you a future and a hope. (Jeremiah 29:11 ESV)
We read it and get excited about our lives, I mean who wouldn't!?! We just got reassured & a 100% guarantee of great things happening to us. I have read this scripture so many times and also got peace in the midst of storms. I have read it and just hoped for the sun to shine. I have read it in times of smooth sailing and just worshiped because He is a good God and gave me this promise even without me asking but something interesting happened to me today. As I read this scripture again, I got a deeper and heart jumping revelation into this promise. For once, I lifted the covers and saw what else was hidden in this promise. I guess you can say the deep things of God were revealed unto me and I'm super excited to share it with you today.
When God promised plans of peace & welfare for me, this is what else He promised. He promised me & you:
The list goes on & on! This is exciting! So now when you read this scripture, what does it tell you? It says,
For I know the plans I have for you, declares the Lord, plans for (good fortune/health/happiness/prosperity/abundance/…..) and not for evil, to give you a future and a hope.
You get the picture right? :)
So now when you renew your faith with this verse, see all the other things God has in store for you. Confess them in faith and see the hand of God move with gladness in your life.
Whatever goal or dream God has placed in your heart, you're going to see it come to pass. It might not happen when you thought it would happen, and it might not happen the way you thought it would happen, but God is going to work in such a way as to exceed your wildest expectations too. You won't have to make it happen in your strength. All you have to do is persevere— just don't give up. Surround yourself with hope and watch God bless you in ways you never thought possible.
Eye has not seen and ear has not heard and has not entered into the heart of man, [all that] God has prepared (made and keeps ready) for those who love Him [who hold Him in affectionate reverence, promptly obeying Him and gratefully recognizing the benefits He has bestowed] . 1 Corinthians 2:9
I believe that God has many pleasant surprises waiting for you, things He has prepared that are waiting for you. Keep your hopes up!
~ Excerpt from Get Your Hopes Up by Joyce Meyer.
And this is the confidence that we have in him, that, if we ask any thing according to his will, he heareth us: And if we know that he hear us, whatsoever we ask, we know that we have the petitions that we desired of him.
1 John 5:14-15
Have you ever been in prayer and suddenly had the disturbing sensation that God simply isn't listening?
It's happened to all of us. We limp along praying the same old prayers, half-hoping those prayers are heard and half-suspecting they're not.
Today I want to show you how to resolve that dilemma once and for all. But let me warn you, I'm not going to slap you on the back and assure you God will listen to any old doubtful, self-centered thing you say to Him. He won't. He's only promised to listen to prayers that are prayed according to His will. The Apostle John says when you pray like that, you'll know that you have the petitions you desire of Him.
The word petition is defined as "a formal written request addressed to a sovereign superior of a particular right or grace," and that's exactly what you need when you're praying about something serious.
How do you put together a solid petition, one that's in line with the will of God?
First, you have to roll up your sleeves and dig into the written Word. Find scriptures that apply to your situation and make those the foundation for your petition.
Then get on your knees and consult with the Holy Spirit. Let Him help you develop your petition in detail. The best way to do that is to spend time praying in other tongues. (See Romans 8:26-27.) Expect God to reveal things from His heart to yours. He wants you to know His will. So, while you're praying, listen!
Last of all, write it down. Make a formal written request by writing down every scripture you found. Also, as you're listening to the Holy Spirit for the details, write down the impressions and ideas He gives you.
Take your time. Let the Spirit develop your prayer. Let the truth He reveals to you settle in and begin to work in you. Get your petition firmly in mind, then when you're ready, present it to God.
Believe me, He'll be listening
by Kenneth Copeland.
What is the Spirit of God saying to you today? What is He telling you about your spiritual development…your family…and your finances? If you're facing trouble, what word of victory has He spoken to you?
As a born-again child of the living God, you ought to know the answers to those questions.
I've learned by experience, however, the majority of believers don't. I can tell by just listening to them talk. "Oh, my goodness," they'll say, "I'm in a crisis and I don't know what to do!"
If that's your situation, I'm going to be straight with you. You'd better go to God and find out what to do. You'd better be quiet long enough to hear what He has to say and pay attention to it. You'd better get rid of that fear and start believing God. Otherwise, the devil is going to end up getting the best of you.
If you read the Bible you'll see God has been trying to get His people to understand that for thousands of years. In fact He gave those exact instructions to an Old Testament king named Ahaz back in the days of the prophet Isaiah. Ahaz was in serious trouble at the time. He had two enemy armies coming against him and he had no idea what to do about it. So God sent His Word to Ahaz through the prophet Isaiah and gave him a whole new perspective.
Instead of agreeing with Ahaz about how powerful his enemies were, God let him know they didn't impress Him much. He referred to them as two "smoking stumps" (Isaiah 7:4, The Amplified Bible). Then He assured Ahaz their plans against him would not stand if he would simply obey the following instructions: Take heed…be quiet…fear not (verse 4)…and believe(verse 9).
Check Your Receiver
Before you head down the road looking for a prophet like Isaiah to tell you what God is saying to you, let me save you a trip. You don't need a prophet to tell you what to do. You have a better covenant than Ahaz had. You're not just a servant of God like people were in the Old Covenant. Through the blood of Jesus, you've become a full-fledged son and the Bible says, "For as many as are led by the Spirit of God, they are the sons of God" (Romans 8:14).
Jesus confirmed that fact in John 10 when He said that His "sheep hear his voice; and he calls his own sheep by name and leads them out. And when he brings out his own sheep, he goes before them; and the sheep follow him, for they know his voice" (John 10:3-4, New King James Version).
As a child of Almighty God, you have the right and the spiritual ability to hear the voice of God for yourself!
"But, Brother Copeland," you might say, "God never speaks to me."
Sure He does. You're just not listening.
Have you ever turned on your television and found something was wrong with the picture? Maybe it was fuzzy or the sound was crackling with static. Or maybe you couldn't get a picture at all. Did you grab the telephone, call NBC and tell them that something was wrong with their transmitter? Did you get some television executive on the line and say, "Hey, you guys must not be broadcasting anymore. I turned on my set and you weren't there!"
Certainly not! That didn't even occur to you. You were smart enough to figure out the problem most likely wasn't with the transmitter. The problem was with the receiver. The problem was with your own television set.
So instead of blaming the broadcaster, you started trying to find the problem on your end. You might have checked the electrical connections. You might have looked to make sure the cable was hooked up. You might have checked the settings on the television to make sure they were all adjusted correctly.
What's more, you stayed with it until you got it fixed. Why? Because you never doubted the fact that the networks were still broadcasting. You understood if you could just successfully tune in, they would be there for you.
Find His Frequency
Don't you think we ought to have at least as much faith in God as we have in the television networks? They can break down. They can fail us. But God never will. He has given us His Word. He has promised He'll lead us by His Spirit and enable us to know His voice. So if we're having difficulty with those things, we need to stop blaming Him and determine where we're missing it.
Actually, that's not hard to do. When we have trouble hearing God's voice it's almost always for one of the following four reasons.
We don't believe He is speaking.
We're not paying attention.
We're allowing some kind of interference to drown out His voice.
We just plain don't know what frequency He is on.
Let's focus right now on that last point. Exactly how do you locate the frequency God uses to speak to you?
I'll tell you right off the bat, you don't do it by using those fleshly paddles on the sides of your head—your natural ears. Some people try to hear God that way. They think if He would just speak audibly to them everything would be solved.
They're mistaken. Our natural ears weren't designed to hear the voice of God.
We were created to hear Him in our inner man or what the New Testament calls "the hidden man of the heart" (1 Peter 3:4).
As Hebrews 3:7-8 says, "Wherefore (as the Holy Ghost saith, Today if ye will hear his voice, harden not your hearts.)"
God leads His children not by outward voices or signs and wonders but by the inward witness of "the hidden man of the heart". So to find His frequency, the first thing you'll have to do is tune in—not with your head but with your heart!
Learn to Discern
How do you know the difference between your head and your heart?
Scripturally speaking, your head is your soul which is comprised of your mind, will and emotions. Your heart is your spirit which is the core of your being that has been born again and joined to the Spirit of God. Granted, discerning the difference between the voice of your soul and the voice of your spirit can be challenging and it takes spiritual wisdom to do it. But the Bible tells us clearly how to develop that wisdom: We do it by spending time in the written Word of God.
Hebrews 4:12 tells us that the Word is "quick, and powerful, and sharper than any twoedged sword, piercing even to the dividing asunder of soul and spirit, and of the joints and marrow, and is a discerner of the thoughts and intents of the heart." It contains the voice of God in its most tangible form.
God always agrees with His written Word and His Word always agrees with Him. In fact, Psalm 138:2 says He has magnified His Word even above His Name. That means God has put His Name on His written Word the way we would put our name at the bottom of a contract. He has given us His Word as a covenant and signed it in the Name of Jesus by the blood of Jesus.
Since God cannot lie, there is no way He will ever do or say anything contrary to that Word. He has absolutely joined Himself to it forever. So the first place God takes us to train us to recognize His voice is to His written Word. He uses it to tune our spiritual ears to the real so that we can easily recognize a counterfeit.
Have you ever watched an impersonator mimic a celebrity? If he is good at what he does, you'll think, I know who that guy is impersonating. It's so-and-so. He sounds just like him! But if you were to put the real personality right next to the impersonator, the differences would be glaring. If you could see them both at the same time, you'd think, That guy doesn't sound much like him at all!
That's the way it is with God's written Word. The better you know it, the more you've heard God's voice speaking to you through it, the easier it is for you to tell the difference between His voice and another voice. The easier it is for you to divide your soul from your spirit and discern the difference between the voice of your head and the voice of your heart.
When you're trained to hear God's voice in His Word, the devil won't be able to sneak deceptions in on you, either. When he tries to razzle-dazzle you with some religious-sounding voice that says, I love you, my son. But it's just not my will to heal you at this time, you won't buy it. You'll rise up and say, "That's not the voice of God. That's a lie from hell because it doesn't agree with the Word that says 'by His stripes ye were healed.'"
Let me warn you though, you won't get that kind of training just casually reading the Bible now and then. You won't get it by knowing generally what it says…or by mixing what it says with your own opinions. If you desire to truly learn to hear the voice of God in the Word, you'll have to do with it what God told Ahaz to do.
You'll have to take heed to it. You'll have to pay attention!
Zero In
Paying attention is more than just mentally assenting to a scripture and saying, "Oh yes, hallelujah. Amen." To pay attention means to lock in on what's being said and make a firm decision that from now on, you will see this matter the way God sees it instead of the way you've always looked at it.
To do that, you'll have to do the second thing God told Ahaz to do. You'll have to be quiet. You'll have to shut up all the mental jabbering you've been doing, stop thinking about your own opinions and listen—really listen—to what the Word says.
Do you realize it's possible to read great quantities of scripture and never really hear what God is saying in it? It's not only possible, people do it all the time. Here's how it happens. We'll read a verse or two and something in them will trigger another train of thought. We may start thinking about what Aunt Sally said about that scripture. Or what Grandma used to say. Or we may even let our mind wander off on something else altogether—all the while we're still reading our Bible! We end up with only a vague idea of what we read because we weren't really paying attention to the Word, we were paying attention to our own thoughts.
Reading the Word of God that way is like shooting scattershot at a flock of birds—you don't ever hit anything. To hear the voice of God through His Word, you have to take aim and zero in on every word as you read it. Instead of just breezing through the Bible reading it like you'd read a novel or a history book, read it deliberately.
Meditate on it by asking yourself, What does that mean to me? How does it change my life? Ask the Lord to reveal specifically what He is saying through that Word to you.
Read every verse with the attitude that This is God speaking to me and I am going to do what He tells me to do. Make a quality decision you're going to act on that Word as quickly as you would the word of your doctor, lawyer or a very close, trusted friend.
Determine in advance that you're not going to bend the Word to fit your lifestyle. On the contrary, you're going to bend your lifestyle to fit that Word. With that attitude, your spiritual ears will be open to hear whatever God has to say.
Fear Not!
"But, Brother Copeland, I'm afraid I'll do those things and I still won't be able to hear the voice of God."
Well, stop being afraid of that! Fear not!
Instead take a step of faith and believe what Jesus said. He said you're His sheep and you hear His voice. So stop doubting Him and calling Him a liar. Don't ever, ever, EVER again say, "I can't hear God's voice."
Start agreeing with Jesus. Start believing and agreeing with the Word. Say, "The Word says I can hear God's voice and I believe it! I do hear the voice of God!"
Then put your faith into action. Open the ears of your heart and start listening for the voice of the Spirit, especially when you're reading the Word. Pay attention not just to the activity in your brain but to the quickenings in your inner man (primarily the area just below your chin, or chest area).
If you're not sure where to locate those stirrings, just think back to a time when you had what we sometimes call a "hunch." Suddenly something just dawned on you and you knew or understood something you hadn't known before. Those kinds of dawnings come from your born-again spirit.
Purpose to become more aware of the promptings that arise from your spirit because they are the leading of God. Learn to trust them. The Holy Spirit will help you to step out on them a little at a time.
Initially He won't be giving you risky, world-shaking kinds of leadings. When you're first learning to identify God's voice, it will be mostly yes and no answers, not to go sell everything you own and invest it in some off-the-wall business venture. He'll begin by showing you simple truths from the Word that you can act on. He'll reveal from the Word, for example, how you can obey more fully the law of love. He'll show you ways to bless the people around you.
The more you trust His voice and follow His leadings, the more clearly you'll find you can hear from Him. Before long, hearing from God won't be an occasional event but an everyday part of life. And when someone asks you, "What is the Spirit of God telling you today?" you won't hesitate a moment.
You'll know exactly what to say.
– Kenneth Copeland
Here I am before You, falling in love and seeking Your truth
Knowing that Your perfect grace has brought me to this place
Because of You I freely live, my life to You, oh God, I give
So I stand before You, God
I lift my voice cause You set me free
So I shout out Your name, from the rooftops I proclaim
That I am Yours, I am Yours
All the good You've done for me, I lift up my hands for all to see
You're the only one who brings me to my knees
To share this love across the earth, the beauty of Your holy worth
So I kneel before You, God
I lift my hands cause You set me free
So I shout out Your name, from the rooftops I proclaim
That I am Yours, I am Yours
All that I am, I place into Your loving hands
And I am Yours, I am Yours
Here I am, I stand, with arms wide open
To the One, the Son, the Everlasting God
What do you do when you're lost for words?
They all fall short, so I just stand in awe
Stand in awe of you
Melodies come and go
You remain, so I stand in awe
Stand in awe of you
You reign forever and hold me together
And come whatever, you love me
You reign forever and hold me together
And come whatever, I love you
What do you do when God forgives
And gives his life so you can live in awe?
We live in awe of you
Melodies come and go
You remain, so I stand in awe
Stand in awe of you
You reign forever and hold me together
And come whatever, you love me
You reign forever and hold me together
And come whatever, I love you
You reign forever and hold me together
And come whatever, you love me
You reign forever and hold me together
And come whatever, I love you
Holy, holy, we lift up holy hands
All for your praise, your honor and glory
Holy, holy, we lift up holy hands
All for your praise, your honor and glory
Holy, holy, we lift up holy hands
All for your praise, your honor and glory
Holy, holy, we lift up holy hands
All for your praise, your honor and glory
You reign forever and hold me together
And come whatever, you love me
You reign forever and hold me together
And come whatever, I love you
You reign forever and hold me together
And come whatever, you love me
You reign forever and hold me together
And come whatever, I love you
I'll sing forever, I'll sing forever
I'll sing forever, I love you
~ Jesus Culture [Kim Walker Smith]


text:
Product Information
Product Information
Dragon's Crown - PS Vita
Brandish your blade, dust off your grimoire, and rally your online adventuring party. Dragon?s Crown thrusts you into a fantastical medieval world packed with dungeons to explore, vile monstrosities to smite, and nearly endless online adventure. Your mission is to dive into the dank underworld, uncovering treasure and strengthening your mettle as you unlock the secret behind the ancient dragon threatening the world. --Embark on quests that take you across varying dungeon worlds, where you'll battle tremendous dragons and other powerful bosses in exhilarating combat. Hunt for hidden treasure, items and weapons that will propel you on your quest. Decide to team up with a legion of friends or take the road alone ? whatever you choose, prepare for nonstop action and intense, role-playing immersion on your hunt for the Dragon's Crown.--Descend into a labyrinth underground world on a quest to unlock the secrets behind a mighty dragon that threatens the land-Hunt for hidden treasure, items and weapons that will maximize your power and aid you on your mission-Embark on a variety of different, daring quests throughout endless dungeon worlds in addicting, long-lasting gameplay-Battle deadly dragons and other fearsome foes in massive boss battles-Customize and raise your Character to experience the world in exciting new ways-Join forces with your friends and conquer monsters together
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I sincerely long for love in my life. I primarily look for a long-term relationship with people living close to me, but I keep my doors open. I would consider seeing someone "located anywhere".
I think that making a good general presentation is hard. If you like to get to know me better I would love to write to you.
I am not an athlete, but I do work out regularly. I generally lift free weights. Come winter I will be out skiing.


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Though I am not sure that I understand the exact effect that the heat from the C02 Laser is having on the melanocytes, it seems that Melanotan might be the answer. I've searched Melanotan discussion forums and have seen personal results. Is the "vaporizing" of tyrosine causing the hypopigmentation? Can Melanotan ameliorate or cure hypopigmentation from CO2 Laser Resurfacing? Thanks.
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BJP vice president Mukhtar Abbas Naqvi and other senior leaders have nevertheless upped the ante against the present government, saying it had gone to the "ventilator" and would collapse soon under its own weight.
Sharpening the party's attack on the UPA government, Naqvi said, "The government is on the ventilator. But if the government manages to run after somehow getting support from here and there then it will not be in the interest of the country."
"The Congress led UPA government is in an ICU. It is a lame duck government surviving on the support of two contradictory parties," BJP leader Rajiv Pratap Rudy said. The BJP leader also said that government allies were now trying to find excuses to abandon the "sinking ship" of Congress. "The allies one after the other are trying to find excuses and break away. None of them wants to carry the burden of Congress to the next general elections," he said.
All top leaders however have reiterated that the party is not going to move a no confidence motion against the incumbent government.


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2201 West College Parkway
Carson City, NV 89703
Carson City, NV 89703
Tel: (775)445-3000
3,976 students
Public institution
Public institution
Western Nevada College is a public, open admissions college dedicated to providing affordable, quality educational opportunities for those residing in our seven-county service area. Our students come from a variety of social, ethnic, and economic backgrounds and vary in their reasons for attending our college; some are the first in their family to attend college while others hold post-graduate degrees. Western Nevada College welcomes all who desire the opportunity to learn. The strength of our college emanates from its diversity, and we affirm the right of all to pursue and disseminate knowledge free of discrimination and prejudice. All members of the college community � faculty, support staff, and administrators � are committed to the values of free and unfettered inquiry; tolerance of, and respect for, difference; the nurturing of human potential; good citizenship; and civility. These values are reflected in all of the college's policies and practices.
School Highlights:
Western Nevada College serves 3,976 students (32% of students are full-time).
The college's student:teacher ratio of 40:1 is higher than the community college average of 22:1.
Minority enrollment is 30% of the student body (majority Hispanic), which is less than the state average of 54%.
School Overview
The teacher population of 100 teachers has declined by 33% over five years.
|Western Nevada College
|Community College Avg.
|Carnegie Classification
|Associate of Arts Colleges
|Associate of Arts Colleges
|Institution Level
|Four or more years
|At least 2 but less than 4 years
|Institution Control
|Public institution
|Total Faculty
|100 staff
|59 staff
|Total Enrollment
3,976 students
|1,282 students
|Student : Teacher Ratio
|# Full-Time Students
1,275 students
|810 students
|# Part-Time Students
|2,701 students
|1,017 students
|% American Indian/Alaskan
|% Asian
|% Hawaiian
|% Hispanic
|% Black
|% White
|% Two or more races
|% Unknown races
|Diversity ScoreThe chance that two students selected at random would be members of a different ethnic group. Scored from 0 to 1, a diversity score closer to 1 indicates a more diverse student body.
|In-State Tuition Fees
|Out-State Tuition Fees
|% Students Receiving Some Financial Aid
School Notes:
Western Nevada College is a comprehensive community college which serves more than 6,000 students each semester within an 18,000-square-mile service area. One of four community colleges within the Nevada System of Higher Education, it is accredited by the Northwest Association of Schools and of Colleges and Universities. WNC reaches out to its urban and rural communities with campuses in Carson City, Fallon and Minden/Gardnerville as well as instructional centers in Dayton, Fernley, Hawthorne, Lake Tahoe, Lovelock, Smith Valley, and Yerington. Distance education courses, including interactive video and Internet classes, allow even those in remote locations ready access to higher education. The college offers some 50 academic degrees and certificates in areas as diverse as engineering and construction technology, nursing, musical theatre, golf facilities management, geographic information systems, criminal justice and graphic design. Students may also complete up to two years of many baccalaureate degree programs, such as teacher preparation, for transfer to a university. At present, WNC continues to expand, providing new facilities, services and opportunities. Construction is nearing completion on the 35,000-square-foot Joe Dini Jr. Library and Student Center. The college's first free-standing library building will have student study rooms, a video presentation room for satellite downlinks, and a hands-on computer teaching room. One floor will be dedicated entirely to student activities. The Jack C. Davis Observatory, which is being built entirely with private donations, will also be completed this spring. The 2,800-square-foot observatory will house three telescopes, including two 16-inch and one 10-inch reflecting telescope. WNCC's main campus, located in the Silver State's capital, offers students an interesting blend of environments: the comfortable pace of a small city combined with the political bustle of state government. The Carson City campus is also close to some of the best scenery and outdoor recreation in the West. The campus rests on the eastern slope of the Sierra Nevada mountains, and the spectacular beauty of Lake Tahoe is only 12 miles away.
Nearby Schools:
- College Location Mi. Students
South Lake Tahoe Lake Tahoe Community College
One College Drive
South Lake Tahoe, CA 96150
(530)541-4660 20.4 Mi | 2458 students
Sparks Career College of Northern Nevada
1421 Pullman Dr.
Sparks, NV 89434
(775)856-2266 24.8 Mi | 556 students
Reno Truckee Meadows Community College
7000 Dandini Blvd
Reno, NV 89512
(775)673-7000 26.5 Mi | 11683 students
Folsom Folsom Lake College
10 College Parkway
Folsom, CA 95630
(916)608-6572 80.4 Mi | 8233 students
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5000 Rocklin Road
Rocklin, CA 95677
(916)624-3333 81.2 Mi | 18564 students
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Ophthalmologist (eyes)
6 years of experience
Accepting new patients
6 years of experience
Accepting new patients
352 E Butler Ave
Doylestown, PA 18901 Phone number & directions
Doylestown, PA 18901 Phone number & directions
License & Identifications
A medical license is required for a doctor to practice medicine in a particular state. Requirements vary by state but most require, at a minimum, post-graduate training in the doctor's specific specialty. An NPI number is a national identifier unique to an individual health care provider and is managed by the Center for Medicare & Medicaid Services (CMS).
Learn more about Dr. Emily Decarlo:
View the full profile to get information on:
- Qualifications
- What's important to you
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Patient Reviews
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Specialties & Research
Dr. Emily Decarlo, MD is board certified in Ophthalmology.
Education & Awards
Dr. Decarlo graduated from University of Pennsylvania , Philadelphia.
Dr. Decarlo has 3 hospital affiliations:
- Warminster Hospital
- Abington Memorial Hospital
- Wills Eye Hospital
Home > Find an Ophthalmologist > PA > Doylestown Ophthalmologists > Dr. Emily Decarlo, MD
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- Additional Information
- Dr. Emily A Decarlo MD is a female Ophthalmologist, has 6 years of experience and practices in Ophthalmology.
- Where does Dr. Emily A. Decarlo practice?
- Dr. Emily Decarlo has 2 office locations.
Dr. Emily Decarlo practices Ophthalmology near Doylestown, PA.
Additional practice locations include: Southampton, PA.
See Doylestown Ophthalmologists.