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The IL receptor common γ (γc) chain is required for the formation of high affinity cytokine receptor complexes for IL-2, IL-4, IL-7, IL-9, and IL-15, and for signals regulating cell survival, growth, and differentiation. Our current understanding of how γc chain associates with multiple ligands and receptor subunits is drawn largely from its structural homology to the human growth hormone (hGH) receptor and known structure of the hGH/hGH receptor complex. These receptors share distinct features in their extracellular portions and are believed to function by a mechanism of ligand-induced association of receptor subunits. Here, we report the first directed mutational analysis of the human γc chain by alanine scanning conducted across seven regions likely to contain residues required for intermolecular contact. Functionally distinct, neutralizing anti-γc mAbs were employed to define critical residues. One particular mAb, CP.B8, unique in its ability to inhibit IL-2-, IL-4-, IL-7-, and IL-15-induced proliferation and high affinity cytokine binding of normal T cells as an intact mAb and as a Fab fragment, localized critical residues to four noncontinuous stretches, namely residues in loops AB and EF of domain 1, in the interdomain segment, and in loop FG of domain 2. Notably, these residues form a contiguous patch on the γc chain surface in a three-dimensional structural model. These results provide functional evidence for the location of contact points on γc chain required for its association with multiple ligands.
X-linked SCID (XSCID)3 is a disease characterized by profoundly diminished cellular and humoral immunity (1, 2). In XSCID patients, mature T cells and NK cells are greatly reduced or absent due to impaired development of these cell types. Peripheral B cells are found at normal or relatively high numbers although specific Ab production is defective. XSCID is caused by mutation in the gene encoding the IL receptor common gamma chain, referred to hereafter as the γc chain (3, 4).
The γc chain is a subunit of the IL-2R (5) and of other cytokine receptors, including those for IL-4, IL-7, IL-9, and IL-15 (6, 7). The IL-2R is composed of two (IL-2Rβ/γc) or three (IL-2Rα/IL-2Rβ/γc) chains, whereas the IL-4, IL-7, and IL-9 receptors each have a unique α-chain that associates with γc. The IL-15R also utilizes three chains, a unique α-chain, IL-2Rβ, and γc (8, 9). Partnering of γc with other subunits increases receptor affinity for cytokine. In addition, it is absolutely required for many ligand-induced signals (10, 11, 12). This critical role in cytokine signaling is mediated by association of the γc cytoplasmic portion with Janus kinase 3 (Jak3) (13, 14). In addition to regulating T and NK cell development, the γc chain may regulate the activation, differentiation, and death of peripheral T cells (15, 16, 17), as well as that of a variety of other mature cell types, including monocytes, macrophages, granulocytes, and certain intestinal epithelial cells (18, 19). Thus, it is important to define both structurally and functionally the role of the γc chain in the association and activation of the γc chain-dependent cytokine receptors.
The γc chain and many receptor subunits that associate with the γc chain belong to a large family of structurally related receptors that mediate cell growth and differentiation, known as the Class I cytokine receptor family or hemopoietin receptors (20, 21, 22). These receptors are type I transmembrane glycoproteins that have a distinct structure, consisting of about 200 amino acids that form two fibronectin (FN) type III domains in their extracellular portion. Their hallmark is the presence of canonical motifs, a set of four conserved cysteines in the membrane distal domain and a WSXWS motif in the membrane proximal domain. In addition, each domain is comprised of seven β strands whose sequences are conserved between members of the family, while loop sequences connecting the β strands vary between family members and putatively contain residues that mediate distinct intermolecular contacts. These receptors are believed to function by a mechanism of ligand-induced association of receptor subunits, the best characterized example of which is provided by the homodimeric receptor for hGH (23). By contrast, the IL-2Rα- and IL-15Rα-chains are structurally distinct from this superfamily, though related to each other. Soluble ligands for these receptors are also structurally related to each other and belong to the four-helix bundle family of cytokines (24).
Hypothetical molecular models have been constructed of the γc chain complexed with IL-2 and the IL-2Rβ-chain, and of the γc chain complexed with IL-4 and the IL-4Rα-chain (25, 26). These models are based on a structural paradigm provided by the crystal structure of hGH bound to its receptor. In this case, two identical receptor molecules are known to complex with a single molecule of hGH (27). This structure, together with independent mutational analysis of the hGH receptor, have defined the hormone/receptor and interreceptor chain contacts and their relative contribution to complex formation (28). However, the extracellular residues on the γc chain, which are critical for interacting with various cytokines and receptor subunits, are still poorly defined. DNA sequence analysis of naturally occurring γc chain mutations have made only limited contributions to delineating this structure/function relationship since many XSCID patients harbor nonsense mutations, frame-shift mutations, or splicing defects (as reviewed in 6 . While at least some point mutations have been identified that abrogate or diminish high affinity IL-2 binding (4, 29, 30, 31, 32) and many other point mutants have been identified (33), cell surface expression and/or function of these mutants is not well characterized.
Herein we report the first mutational analysis of the human γc chain, providing functional evidence for the location of critical sites required for γc chain activity. This structure/function analysis was accomplished through the use of distinct γc chain-specific mAbs as probes and a panel of alanine substitution mutants of the γc chain. Alanine substitutions were targeted to interconnecting loop residues in each of the two fibronectin type III domains. Mutations that selectively impaired the binding of a unique neutralizing mAb, CP.B8, were of particular interest, given the ability of the Ab to inhibit high affinity cytokine binding and cytokine-dependent cell proliferation as a Fab fragment. Residues critical for CP.B8 binding were localized in the AB and EF loops of the N-terminal domain, the interdomain (ID) segment, and the FG loop of the membrane proximal domain. These results provide experimental evidence for the identity of amino acid residues comprising the cytokine binding interface on the γc chain, or in close proximity to those comprising this interface, which are required for effective recruitment of the γc chain and formation of a signal-transducing complex. Common residues required for the interaction of the γc chain with IL-2, IL-4, IL-7, and IL-15 are indicated. In addition, the potential utility of the distinct anti-γc chain mAbs as tools for mutational diagnosis and for defining the nature of defects caused by XSCID genotypes is discussed.
Materials and Methods
Primary human cells
Human PBMC were isolated from healthy donors by Ficoll-Paque density gradient centrifugation (Pharmacia Biotech, Piscataway, NJ). PBMC were cultured in a 37°C, 5% CO2 humidified incubator at 106 cells/ml in RPMI 1640 medium containing 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml PHA (Difco Laboratories, Detroit, MI) for 3 days to polyclonally activate T lymphocytes. Where indicated, PBMC were T cell enriched before PHA activation by depleting monocytes, B cells, and NK cells with magnetic beads coated with anti-CD14 and anti-CD19 mAbs (Dynal, Lake Success, NY) and with an anti-CD56 mAb (Perseptive Diagnostics, Cambridge, MA).
Cell lines
Stable transfectants expressing the human γc chain on their surface (L929/γc) were generated by electroporation of the murine L929 fibroblast cell line (American Type Culture Collection (ATCC), Manassas, VA) with a full length human γc chain cDNA in the pMDR901 expression vector, and a plasmid containing the neomycin resistance gene. L929 cells were maintained in Eagle's MEM supplemented with 10% FBS, 1 mM nonessential amino acids, and 4 mM l-glutamine, while L929/γc cells were grown in the same medium supplemented with 0.27 μg/ml of geneticin (Life Technologies, Grand Island, NY).
DNA constructs
The full-length human γc chain cDNA was cloned from a Jurkat T cell cDNA library constructed in the pCDM8 vector (34), using an oligonucleotide probe matching nucleotides 592–620 of the published sequence (5). The isolated clone, p4A1, contained a NotI insert encoding the γc cDNA with a single nucleotide difference from that of Takeshita et al. (5); a T in the coding region of clone p4A1 replaced a C at nucleotide 673 of the published sequence. Thus the protein encoded by p4A1 contains a methionine instead of a threonine in the extracellular portion at position 220, located in predicted β strand F of domain 2, numbering from the signal sequence (5). However, this encoded form of the γc chain was functionally competent as measured by its ability to mediate high affinity IL-4 binding when cotransfected into COS-7 cells with the IL-4Rα-chain, resulting in a dissociation constant (KD) of 200 ± 100 pM,4 an affinity approximately threefold higher than that mediated by the IL-4Rα-chain alone, and in agreement with Russell et al. (35). In addition, the encoded γc chain mediated IL-2 binding when cotransfected with the IL-2Rβ-chain, with a KD of approximately 1 nM, also in agreement with published results (7). Similar cytokine binding data were obtained with a myc/γc cDNA expression construct (D. Olson et al., manuscript in preparation) engineered to encode the full-length γc chain with a nine-residue myc peptide tag (EQKLISEEDL) fused to the 5′ end of the mature protein, thereby allowing detection of cell surface-expressed γc chain and mutants thereof with the anti-myc mAb 9E10. This expression construct was made as follows. A blunt end/BsaHI fragment encoding the myc tag, and the first six residues of the mature γc chain protein (the terminal L of the myc peptide being the initial γc chain residue) was formed by annealing complementary oligos. This fragment was then fused downstream of a NotI/blunt end fragment encoding the VCAM-1 gene signal sequence and upstream of a 1361-bp BsaHI/NotI fragment encoding the γc cDNA from residue seven of the mature protein to the 3′ end. The resulting NotI fragment was inserted into the NotI site of the pCDM8 expression vector (34) for transient expression studies and into the NotI site of the pMDR901 vector for stable expression. A γc-Ig fusion protein was constructed by PCR amplification of the γc chain extracellular sequence from template plasmid p4A1, using the following 5′ primer containing a NotI cloning site, 5′-AACTGCAGCGGCCGCCATGGTGAAGCCATCATTACC-3′, and the following 3′ primer that contains a SalI cloning site, 5′-GACTTTGTCGACATTCTCTTTTGAAGTATTGC-3′. The resulting 789-bp PCR fragment was cut with NotI and SalI and ligated 5′ of a 693-bp SalI/NotI fragment encoding the ten amino acids of the human IgG1 hinge region and full sequence of the human IgG1 CH2 and CH3 regions isolated from pSAB144 (36). This NotI fragment was then cloned into a NotI cleaved expression vector pSAB132. The resulting γc-Ig fusion construct, pLB001, encodes the amino terminal 254 amino acids of the mature human γc chain fused to ten amino acids of the hinge region of human IgG1 and the CH2 and CH3 constant domains of IgG1.
γc chain mutagenesis
Alanine substitutions were targeted to interconnecting loop residues based on sequence homology between the γc chain and the hGH receptor (25, 26). For a given interconnecting loop, double and triple point mutations were made, thereby changing two or three contiguous amino acid residues to alanine. Successive mutations were designed so as to scan through the loop sequences. Mutations were made in the myc/γc cDNA sequence carried in a sequencing vector, pLB013, with the USE mutagenesis kit according to manufacturer's specifications (Pharmacia Biotech). The mutations were confirmed by the presence of a novel internal restriction site and by DNA sequencing. DNA fragments containing the mutant sequence were subcloned into the myc/γc cDNA expression vector, replacing the corresponding wild-type sequence, and were confirmed by presence of the novel restriction site.
Generation of anti-γc chain mAbs
mAbs specific for the human γc chain were generated by immunizing female RBF mice with a γc-Ig fusion protein transiently expressed after electroporation of COS-7 cells with pLB001. The γc-Ig, affinity purified from a 2- to 3-day culture supernatant, was applied to protein A-Sepharose 4B Fast Flow resin (Pharmacia Biotech), washed with 25 mM Na2HPO4 (pH 5.0) and 100 mM NaCl to remove any bound bovine IgG derived from the culture medium, and eluted with 25 mM NaH2PO4 pH 2.8 with immediate postelution neutralization. Mice were immunized with γc-Ig bound to protein A-Sepharose resin, and fusion of spleen cells to the FL653 myeloma (Fisher Scientific, Pittsburgh, PA) was conducted according to standard procedures. Clones were screened for binding to the immunogen by solid phase ELISA and by differential immunofluorescent staining of L929 cells vs L929/γc cells. All of the selected mAbs were of the mouse IgG1 subclass and were affinity purified with protein A-Sepharose 4B resin, with elution at pH 5.0 with immediate neutralization.
Immunofluorescent staining
Murine anti-γc mAbs, anti-myc mAb 9E10, and MOPC 21 control IgG1 were added at specified concentrations to 3 × 105 cells suspended in PBS containing 1% BSA and 0.02% NaN3. In the case of L929 lines or COS-7 transfectants, adherent cell monolayers were recovered from culture by washing with PBS and treatment with 5 mM EDTA, 1% BSA for 10 min at 37°C. Cells were incubated with primary Ab for 30 min at 4°C and washed three times in the same buffer. Bound mAb was detected by incubation with phycoerythrin-conjugated goat anti-mouse IgG H+L (Jackson ImmunoResearch Labs, West Grove, PA). Cells were washed three times and fixed in 2% paraformaldehyde. Fluorescence was measured with a FACScan (Becton Dickinson, San Jose, CA). Cross-competition of biotin-conjugated mAbs was conducted by incubation of 5 × 105 PHA-activated PBMC with 10 μg/ml of unconjugated mAb for 45 min at 4°C, and then 2 μg/ml of biotinylated mAb was added. The mixture was incubated for an additional 30 min at 4°C and washed three times; then phycoerythrin-conjugated streptavidin (Jackson ImmunoResearch) was added to detect binding of biotinylated mAb. After 30 min at 4°C, cells were washed three times, and fluorescence was measured.
Analysis of γc mutants
Plasmid DNA prepared from CsCl gradients for each of the mutant constructs, along with the parental myc/γc cDNA (positive) and pCDM8 vector (negative) controls, were electroporated into COS-7 cells. After 3 days, cells were recovered and analyzed by immunofluorescent staining with 10 μg/ml of anti-γc mAbs, anti-myc mAb 9E10, and MOPC 21 control IgG1. Using mean fluorescence intensity (MFI) values as a measure of binding, specific anti-γc mAb binding was obtained by subtracting background binding obtained with MOPC 21 IgG1. Similar background values were obtained for binding of anti-γc mAbs to mock transfected cells. MAb binding to mutant γc molecules is presented as a percentage of the positive control (% binding) and normalized for expression level based on mAb 9E10 staining. A value of 100% indicates that mAb binding to a mutant γc chain molecule is unaffected by the mutation.
Cell proliferation assays
The MLR of freshly isolated human PBMC was assessed by culturing them with irradiated (3000 rad) allogeneic PBMC in RPMI 1640 medium supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured in flat-bottom 96-well plates, with responder cells at 2 × 105 cells/well, and responder/stimulator cell ratios of 1:1 and 2:1. Cells were cultured in the presence and absence of mAb, with triplicate wells for each culture condition, and assessments employed at least four independent donor/stimulator pairs. Cultures were maintained for a total of 4 to 5 days and pulsed with 2 μCi of [3H]thymidine during the last 16 h. Proliferation was measured by [3H]thymidine incorporation using an LKB plate reader (Pharmacia LKB, Gaithersburg, MD). Data are expressed as % inhibition of proliferation in cultures treated with mAb or control IgG relative to untreated cultures.
Cytokine-dependent proliferation assays were conducted using human T cell-enriched PBMC after 3 days of stimulation with PHA, followed by an overnight incubation at 106 cells/ml in medium without PHA. Recovered cells were then cultured in 96-well round-bottom plates at 5 × 104 cells/ well in the presence or absence of anti-γc chain mAbs or control IgG at specified concentrations. After 30 to 45 min at 37°C, recombinant human cytokines, IL-2, IL-4, IL-7, or IL-15, were added at specified concentrations ranging from 1.1–3.3 ng/ml, generally supporting 50 to 80% of the maximal growth response. Cells were cultured with cytokine for 40 to 44 h, and growth was measured by [3
IL-7-dependent growth of freshly isolated PBMC (37, 38) also was assessed using T cell-enriched PBMC cultured at 5 × 104 cells per round-bottom well with 1.1 ng/ml of IL-7 for 5 days, with growth measured by [3H]thymidine incorporation during the final 8 to 16 h of culture and data expressed as the mean cpm of triplicate wells.
Cytokine binding assays.
PHA-activated PBMC were recovered after 3 days of culture, washed with PBS, and resuspended in PBS, 1% FCS. Cells were added to Falcon 2052 tubes (Becton Dickinson Labware, Lincoln Park, NJ) with 1.5 × 106 cells per tube and incubated with and without mAbs in a total volume of 150 μl for 1 h at 4°C on a rotating platform. mAbs employed were anti-γc chain mAbs, anti-IL-2Rβ-chain mAb Mik-β2, MOPC 21 and UPC 10 control IgG, and Fab fragments of CP.B8 and MOPC 21. 125I-labeled IL-2 (New England Nuclear, Boston, MA) was then added in 50 μl to achieve a final concentration of 10 pM radiolabeled IL-2, designed as such to assess the effect of mAbs on high affinity IL-2 binding (7). The cells were incubated for 30 additional min while shaking at ambient temperature and then washed twice with 2 ml of PBS, 1% FBS; the cell pellet was counted in a Wallac (WALLAC, Gaithersburg, MD) 1470 gamma counter. Each condition was assessed in duplicate tubes in a given experiment; the cpm values were averaged and corrected for nonspecfic IL-2 binding by deducting the amount of cpm bound from a sample that contained 10 pM radiolabeled IL-2 and a 100-fold molar excess of nonradioactive IL-2 according to standard methods. Data are expressed as percent inhibition of IL-2 binding relative to that obtained in the absence of mAb.
Statistical analysis
For the MLR cultures and cytokine binding assays, comparisons over several groups were made using a one-way ANOVA, followed by Dunnett's test for multiple comparisons to a control group (two-tailed). P values < 0.05 were taken to be statistically significant. For the analysis of mAb binding to γc chain mutants, two group comparisons were made using an unpaired two-tailed Student t test, comparing each percent binding value to 100%.
Functional inhibition by distinct mAbs specific for the human cytokine receptor γc chain
mAbs specific for the human γc chain bind to the surface of L929/γc transfectants (Fig. 1⇓B) and show no detectable binding to the parent L929 cell line (Fig. 1⇓A) relative to an isotype-matched IgG control. Profiles for three such mAbs are shown and are representative of the results obtained with a broader panel of anti-γc chain mAbs. These mAbs also detect the naturally expressed molecule, as shown by immunofluorescent staining of human PHA-activated T lymphocytes (Fig. 1⇓C). The specificity of these mAbs was also confirmed by their ability to immunoprecipitate the full-length γc chain from the surface of the HUT 78 cell line (data not shown).
The ability of individual mAbs to cross-block binding of other mAbs was determined to establish the relationship between their epitopes. The results are summarized in Table I⇓. Binding of each biotin-conjugated mAb was inhibited specifically by its respective unconjugated form with only 0 to 11% maximal inhibition by the unconjugated IgG1 control. The pattern of cross-competition between different mAbs revealed five distinct epitope groups. One epitope group is comprised of mAbs AF.F4, AK.F12, and CQ.C11, given nearly complete cross-competition between members of this group but not with other mAbs, except BI.B12. Similarly, mAbs AE.F8, CJ.F4, and AH.H8 are related to each other but are distinct from other mAbs, except BI.B12. By contrast, mAb CP.B8 exhibits a unique specificity, only partially cross-blocking with other mAbs, AK.F12 and CQ.C11 in particular. Likewise, mAb AE.C9 constitutes a unique specificity. The effect of mAb BI.B12 indicates some overlap or spatial proximity between the BI.B12 epitope and epitopes recognized by two other epitope groups, represented by AF.F4 and AE.F8, respectively.
Functional activity of the γc chain-specific mAbs was assessed by measuring their effect on proliferation of freshly isolated human PBMC cultured with allogeneic targets, a response known to be highly IL-2 dependent. Representative MLR data for several mAbs are shown in Figure 2⇓, indicating that different γc chain-specific mAbs were able to inhibit significantly the proliferative response. On average, partial but significant inhibition was observed with mAbs AF.F4 and CQ.C11, i.e., 30% inhibition was achieved by 1 μg/ml of either mAb. A plateau or bell-shaped inhibition curve was observed as mAb dose increased further. Similar patterns of inhibition were observed with other mAbs in the panel. By contrast, inhibition by mAb CP.B8 tended to increase with increasing mAb dose, an average of 60% inhibition observed at the highest dose tested (100 μg/ml). Greater inhibition was observed when different noncross-blocking mAbs were used in combination; for example, 78% blocking was achieved by the CP.B8/AF.F4 pair with 50 μg/ml of each mAb.
These findings demonstrate that the Ag-induced expansion of mature lymphocytes from normal individuals is γc-chain dependent, with significant functional inhibition with different mAb specificities.
Anti-γc chain mAbs display broad cytokine inhibitory activity: unique mechanism of action of mAb CP.B8
To determine whether or not these γc chain-specific mAbs would inhibit the functional responses to different cytokines, we assessed the growth of PHA-activated T cell blasts in response to exogenous IL-2, IL-4, IL-7, and IL-15. Individual cytokines were added at doses sufficient to induce at least 50% of maximum stimulation, and the growth response of cells cultured in the presence or absence of anti-γc chain mAbs was measured. In the absence of added cytokine, only low levels of proliferation occurred. Representative results show that mAb CP.B8 and other mAbs inhibited the IL-2-, IL-15-, and IL-4-induced responses (Fig. 3⇓, A, B, and D, respectively), although inhibition was relatively greater in the IL-15 and IL-4 cultures. As observed previously for MLR cultures, CP.B8-mediated inhibition increased with increasing mAb dose. By contrast, other mAbs, including AF.F4, AK.F12, CQ.C11, AE.F8, BI.B12, and AE.C9, achieved maximum inhibition between 1 to 10 μg/ml, with a plateau or decreased inhibition thereafter (Fig. 3⇓). Similar results were obtained for IL-7-induced cellular responses (Fig. 3⇓C); however, the inhibitory capability of mAb CP.B8 was not consistently apparent when assessing the IL-7-dependent response of PHA-blasts. MAb CP.B8 did significantly inhibit the IL-7-induced growth of freshly isolated PBMC (Fig. 3⇓C, inset). Thus, anti-γc chain mAbs belonging to different epitope groups exhibited broad neutralizing activity against four specific cytokines whose receptors employ the γc chain.
The requirements for mAb-mediated inhibition were dissected by employing Fab fragments of mAbs representative of the different epitope groups. The effects of intact mAbs and their Fab fragments on cytokine-induced growth of PHA-blasts were compared directly (Fig. 4⇓, A and B). While the intact mAbs AF.F4, CQ.C11, BI.B12, and AE.C9 reproducibly inhibited IL-2-dependent cell proliferation, their Fab fragments failed to inhibit the IL-2-induced growth response even at high doses. Nonetheless, these Fab fragments were active based on the ability of each Fab preparation to inhibit completely the binding of its intact biotinylated mAb to PHA-blasts, at doses of 0.4 to 1.25 μg/ml Fab and 1 μg/ml intact biotinylated mAb. In side by side assessments, complete inhibition of binding of each biotinylated mAb was achieved by 0.4 μg/ml of its unconjugated, intact mAb (data not shown). By contrast, the ability of mAb CP.B8 to inhibit IL-2-induced responses was clearly retained by its Fab fragment (Fig. 4⇓C).
The ability of CP.B8 to block cytokine responses as an intact mAb or as a Fab fragment indicated that it did not require the size, cross-linking, or Fc-mediated functions of an intact mAb. Rather, the data suggest that CP.B8 mediates its effect by blocking directly a critical site on the γc chain required for the recruitment of γc chain into a productive signaling complex. Therefore, the ability of CP.B8 to interfere with high affinity IL-2 binding was examined using 125I-labeled IL-2 at a concentration of 10 pM, the KD for high affinity IL-2 binding. Our results show that the binding of radiolabeled IL-2 to PHA-blasts was inhibited in a dose-dependent manner by mAb CP.B8 and by its Fab fragment (Fig. 5⇓A), with no inhibition by the IgG1 control mAb MOPC 21 or its Fab fragment. Inhibition achieved by mAb CP.B8 was comparable to that exhibited by the anti-IL-2Rβ-chain mAb Mik-β2 (Fig. 5⇓A). By contrast, mAb CQ.C11 exhibited a significant but low level of inhibition whereas no significant inhibition was observed with other mAbs (Fig. 5⇓B). Thus, these data indicate that mAb CP.B8 can interfere with the formation of a stable high affinity cytokine/receptor complex, apparently by blocking directly a critical site on γc chain, whereas inhibition by other mAbs requires additional steric effects and/or other mechanism(s).
mAb binding to human γc chain alanine substitution mutants
The unique specificity and functional activity of mAb CP.B8 provided a valuable tool to map regions of the γc chain critical for its function. A panel of γc chain mutants was constructed, and mAb CP.B8 binding to these mutants was assessed and compared with the binding to the wild-type molecule. Binding of other mAbs selected from three distinct epitope groups was also assessed. This potentially offered a means to discern structural changes that caused a generalized loss of mAb binding activity, such as gross conformational changes.
Alanine scanning mutagenesis was conducted through seven putative interconnecting loops based on molecular models identifying these sequences, and indicating their potential contribution to the γc interface with cytokine or other receptor subunits (25, 26). Their location is shown in the linear γc sequence (Fig. 6⇓, see bolded residues) and in a three-dimensional molecular model (Fig. 7⇓A). Alanine substitutions were made to reveal the contribution of interactions made by side chains while minimizing conformational effects (39). In general, loops were scanned by substituting two or three residues at a time, with an overlap of one residue in consecutive mutants, as listed in Table II⇓. Mutations that caused a significant reduction in mAb binding are boxed as shown.
We found that mAb CP.B8 binding was completely abrogated or reduced significantly by mutations in five of the seven loops, namely loops AB and EF of domain 1, the interdomain segment, and loops BC and FG of domain 2. The reductions in mAb CP.B8 binding were not due to gross changes in γc folding since the binding of other mAbs, such as BI.B12 and CJ.F4, was generally retained. Binding of other mAbs was also retained in many instances. For example, substitutions in residues F43N44V45, N128L129V130, V130I131P132, and F205N206P207, which reduced mAb CP.B8 binding to 9%, 0%, 21%, and 28%, respectively, caused little, if any, significant reduction in the binding of AF.F4, AK.F12, CQ.C11, and AE.F8. Thus, mutations that preferentially impair mAb CP.B8 binding were identified in three noncontiguous stretches by alanine substitutions at F43N44V45, N128L129V130I131P132, and F205N206P207. In the latter two cases, mAb CP.B8 binding was retained by sequential mutants P132W133 and P207L208, indicating that changes in the N128L129V130I131 and F205N206 portions are critical.
In some instances, reduced binding of mAb CP.B8 and a subset of other mAbs was observed. For example, mutants L126Q127N128, H159C160L161, and L161E162H163 significantly reduced binding of mAb CP.B8 and other mAbs, including AE.F8, by >50%. Since there was no significant cross-blocking between mAbs CP.B8 and AE.F8 (Table I⇑), these data suggest that loss of binding is due to an indirect effect of the mutation, possibly consequent to a change in folding that affected multiple but not all epitope groups. In addition, there are mutations that reduce binding of mAb CP.B8 and one or more members of the AF.F4 epitope group, i.e., AF.F4, AK.F12, and/or CQ.C11. Notable examples are mutant V45E46Y47 and mutations in residues K98E99I100H101L102 in the EF loop of domain 1. Given that partial cross-blocking was observed between mAb CP.B8 and members of this group (Table I⇑), these mutations likely localize residues that are important to their respective epitopes. However, it is unlikely that identical residues are shared by the CP.B8 epitope and other mAb epitopes. Rather, considering that three residues are substituted at once in any given mutant, these mutations most likely localize one or more residues important for mAb CP.B8 binding and one or more adjacent residues important for the binding of other mAbs. As such, these mutated stretches may contain critical points of contact for ligands of the γc chain.
The results of this mapping analysis are summarized schematically in Figure 6⇑, with bars indicating sequences at which alanine substitution markedly reduced mAb binding. Notably, the residues that impair mAb CP.B8 binding and that are distant in the linear sequence are in close proximity in the context of the three-dimensional structural model (Fig. 7⇑B), namely residues in loops AB and EF of domain 1, in the interdomain segment, and in loop FG of domain 2. Consistent with this, mAb CP.B8 bound to the intact γc chain but did not bind detectably to either domain 1 or domain 2 when expressed alone on the surface of COS-7 transfectants whereas other mAbs did bind to these isolated γc chain domains (A. Jakubowski, unpublished observation), indicating that residues contributed by both domains 1 and 2 form the epitope for mAb CP.B8.
In summary, mutational analysis of the γc chain and epitope mapping for a unique neutralizing mAb have identified sites on the γc chain required for mAb CP.B8 binding. The availability of multiple distinct mAb specificities has allowed us to distinguish residues that contribute uniquely to the conformational epitope of this Ab. Moreover, these data provide important functional evidence for the localization of γc residues required for functional responses to multiple cytokines.
Targeted mutational analysis of the human γc chain and use of a panel of distinct neutralizing anti-γc chain mAbs have identified residues required for mAb binding to this molecule. The ability of these mAbs to block γc chain function was established by their ability to inhibit cytokine-dependent T cell proliferation in vitro, including T cell responses to IL-2, -4, -7, and -15. Partial, but significant, inhibition was achieved by many of the anti-γc chain mAbs of four different epitope groups. In general, a maximum of 30 to 50% inhibition was observed at 1 to 10 μg/ml mAb, with a plateau or decreased inhibition at higher doses. We speculate that the decreased inhibition at higher doses reflects a shift from bivalent to monvalent binding, consistent with the lack of inhibitory activity exhibited by Fab fragments of these mAbs. While their mechanism of action is still unclear, most mAbs appear to inhibit γc chain function with little, if any, effect on cytokine binding. By contrast, one particular mAb, CP.B8, and its Fab fragment inhibited cytokine-dependent proliferation as well as high affinity IL-2 binding, the degree of inhibition increasing with mAb dose. Similarly, mAb CP.B8 and its Fab fragment inhibit γc chain-dependent IL-4 binding using 80-pM radiolabeled IL-4 (D. Olson, unpublished observations). The unique capability of CP.B8 to inhibit γc chain function as an intact mAb or as a Fab fragment, strongly supports the notion that CP.B8 acts by blocking critical sites on the γc chain. As such, mAb CP.B8 was particularly valuable for mapping contact points on the γc chain, providing evidence for their role as points of intermolecular contact for γc association with cytokines or other receptor subunits.
Critical residues for mAb CP.B8 binding to the γc chain have been localized to four noncontiguous stretches of the γc chain sequence. These residues are in loops AB and EF of domain 1, the interdomain segment, and loop FG of domain 2. While it is difficult to completely rule out possible indirect effects of mutations, changes in these sequences selectively reduced mAb CP.B8 binding with little, if any, effect on binding of other noncross-blocking mAbs. By contrast, changes in loop BC of domain 2 impaired binding of mAb CP.B8 as well as the binding of the noncross-blocking mAb AE.F8. We propose that the residues critical for mAb CP.B8 binding are identical, or are in close proximity, to those required for binding of the γc chain to cytokines or other receptor chains. This conclusion is supported further by the effects of γc chain mutation on IL-2 binding (D. Olson et al., manuscript in preparation). Interestingly, the discontinuous γc chain residues implicated by our studies map to a contiguous patch formed at the interface between domains 1 and 2 when placed in a three-dimensional structural model based on the hGH receptor paradigm. To ensure that the residues forming this contiguous patch were exposed on the surface of the γc chain and would therefore be accessible for binding to mAb CP.B8, calculations of the surface solvent accessibility were conducted using the program CHARMM (40). Of the 16 residues highlighted in Figure 7⇑B as being important for mAb CP.B8 binding, only 2, V45 and V96, were buried, with all the other residues having surface accessibilities of between 7.4 and 111.3 Å2, with a mean value of 51.2 Å2. Therefore, while V45 and V96 may not make contact with mAb CP.B8, all other residues are likely to be sufficiently exposed to interact directly with the Ab. This finding is strikingly similar to that defined for the hGH receptor, wherein the 11 most important receptor residues constituting the functional epitope for binding to hGH map to a contiguous patch, comprised of residues from four interconnecting loops, namely loops AB and EF of domain 1, the ID segment, and loop BC of domain 2 (28). As such, the contiguous patch on the γc chain identified by our studies most likely constitutes a common surface for the interaction of the γc chain with its cytokines.
Thus, many of the important γc chain residues revealed by our studies are closely aligned with hGH receptor residues known to mediate contact with hGH. Notably, Y103 of the γc chain is aligned with W104 of hGH receptor, one of two tryptophan residues (W104 in loop EF of domain 1 and W169 in loop BC of domain 2) of the hGH receptor contributing more than 75% of the binding free energy for hGH (28). The importance of the γc chain Y103 residue is supported by our finding that substitution of L102Y103Q104 reduced mAb CP.B8 binding to 13%. In addition, the binding of mAbs AF.F4 and AK.F12, which partially cross-block with mAb CP.B8, was reduced by 50% or greater. Other areas of alignment include F43N44V45E46Y47 of the γc chain and R43E44 of the hGH receptor, both situated in the AB loop of domain 1, and V130I131 of the γc chain and D126E127 of the hGH receptor, both situated in the ID segment. By contrast, there is no γc chain residue homologous to W169 of the hGH receptor, and mutation in the BC loop of γc chain domain 2 diminished mAb CP.B8 binding but also impaired AE.F8 binding, suggesting that these residues are not direct contact sites on the γc chain. Alternatively, residues in the FG loop of domain 2 of the γc chain (F205N206) also were identified as critical. While there are no contributing residues in the homologous loop in the hGH receptor, the FG loop of the IL receptor common β chain (βc), another cytokine receptor family member, has been shown to contain a Y residue critical for binding of IL-3, IL-5, and granulocyte-macrophage (GM)-CSF (41, 42). The βc chain also requires residues in the BC loop of domain 2, since substitution of Y, H, and I residues therein impaired the binding of GM-CSF and IL-5, but not of IL-3 (43).
While the ability of mAb CP.B8 to inhibit IL-2-, IL-4-, IL-7-, and IL-15-induced responses indicates a common surface for the interaction of the γc chain with multiple cytokines, it is possible that the γc chain residues required for binding to different cytokines are overlapping but not identical. Consistent with this, He et al. (44) reported mAbs specific for the murine γc chain that preferentially inhibited IL-4 vs IL-7 responses. Our studies suggest that residues required for binding IL-2 and IL-7 may be overlapping but distinct from those required for binding to IL-4 and IL-15, since we have noted that mAb CP.B8 inhibits IL-4 and IL-15 responses to a relatively greater degree than IL-2 and IL-7 responses of PHA-blasts. Alternatively, the differential activity of mAb CP.B8 could reflect quantitative differences at a variety of other levels, including the density of cytokine-specific receptors and/or the frequency of responsive cells, since IL-7-induced responses of freshly isolated PBMC were more readily inhibited than those of PHA-blasts. In addition, a given cytokine response may be preferentially inhibited due to differences in the degree to which the γc chain preassociates with other specific receptor chains in the absence of cytokine (45) and/or due to differences in the affinity of the γc chain for complexes of cytokine and cytokine-specific receptor chain.4
Our mutational analysis also explored the identity of contact residues that potentially mediate interaction of the γc chain with other cytokine receptor subunits. Given evidence that the γc chain cannot bind ligand independent of other cytokine-specific receptor chains (7), it is postulated that interchain contacts may be established, thereby stabilizing complex formation. In the hGH/hGH receptor complex, interreceptor contacts occur at the base of the membrane proximal domains, largely through residue Y200 (27). Since the γc chain is analogous to the second hGH receptor molecule, it was mutated in homologous regions of domain 2. Alanine scanning through loop residues L143S144E145S146Q147 resulted in no significant decrease in mAb binding. Furthermore, preliminary data with alanine substitutions at residues S190V191D192Q194K195R196 in the EF loop of domain 2 of the γc chain also indicated little, if any, effect on mAb binding (A. Jakubowski, unpublished observation). While we find no evidence for the existence of interreceptor chain contacts, we cannot rule them out since our mAbs did not map to this candidate region. Further studies are needed to resolve this issue.
The targeted mutagenesis of the human γc chain reported herein provides evidence delineating the structure/function relationship for this molecule. The analysis of naturally occurring γc chain mutants from XSCID patients has also made contributions to defining this relationship (6). Of particular relevance for our discussion are XSCID mutations that cause single amino acid changes in the extracellular region of the γc chain, excluding those that alter canonical cytokine receptor motifs or residues within β strands. Several mutations have been reported to abrogate or reduce high affinity IL-2 binding based on the activity of EBV-transformed B cell lines or of cells transfected with mutant sequences derived from the XSCID patients. Interestingly, these include mutation E46K in loop AB of domain 1 (31), A134V in loop EF of domain 1 (30), and R204C in loop FG of domain 2 (29). However, expression of these XSCID γc chain mutants at the level of the cell surface was confirmed only in the case of the A134V mutant, using the nonblocking anti-human γc chain mAb TUGh4 (18). Several other key residues have also been suggested based on XSCID mutation I131N in loop EF of domain 1 and L161S in loop BC of domain 2, as reviewed by Leonard et al. (6) and, more recently, by Puck et al. (33), who have executed an extensive survey of XSCID patient sequences, reporting 18 different point mutations in the extracellular region of the γc chain, all of which are expressed at the mRNA level. However, these analyses continue to be limited by the availability of mAbs specific for the human γc chain (18, 46), none of which are reported to have blocking activity. Our panel of anti-γc chain mAbs will therefore be useful for evaluating the expression of XSCID γc chain mutants, thereby contributing to diagnosis of mutations and to defining the functional basis for these genetic defects. Taken together, our approach and the previous XSCID mutant analyses provide strong support for the critical role of the AB and EF loops of domain 1 and the FG loop of domain 2 in mediating the interaction of γc chain with cytokine. In addition, our data further address the role of the BC loop in domain 2, providing evidence that changes in this loop indirectly affect the cytokine binding site on the γc chain.
In summary, we have provided functional evidence for the critical role of four noncontiguous stretches of the human γc chain sequence that together comprise a common interface for the interaction of the γc chain with other molecules, most likely its multiple cytokines. This was achieved through the use of a panel of novel anti-γc chain mAbs representing five different epitope groups, including the unique blocking mAb CP.B8. The identification of these key sequences provides practical guidance for the development of small molecules that bind to or mimic this receptor interface, enabling inhibition of γc chain recruitment and thereby the regulation of cytokine receptor function in pathologic settings.
We thank Mark Krivopal, Daisuke Tsujimoto, and Michael Cohen for excellent technical contributions, Dr. Adrian Whitty for helpful discussions during the course of this work, and Dr. Herman Van Vlijmen for determining the surface solvent accessibility of the γc chain.
↵1 Equal contributions were made by these authors.
↵2 Address correspondence and reprint requests to Dr. Linda C. Burkly, Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142. E-mail address: Linda_Burkly{at}biogen.com
↵3 Abbreviations used in this paper: XSCID, X-linked SCID; γc, common γ chain; hGH, human growth hormone; ID, interdomain; MLR, mixed lymphocyte reaction.
↵4 A. Whitty, N. Raskin, D. L. Olson, C. W. Borysenko, C. D. Benjamin, and L. C. Burkly. Interaction affinity between cytokine receptor components on the cell surface. Submitted for publication.
- Received March 23, 1998.
- Accepted June 1, 1998.
- Copyright © 1998 by The American Association of Immunologists


text:
Antanas Mockus when he was mayor of Bogota, dressed as Supercitizen
The entire South American continent is firmly left leaning. One of the few exceptions recently has been Colombia. Well, come this Sunday*, a very eccentric man, a Green no less, may become president. This will be interesting. Tensions between Colombia and Venezuela will lessen, but will Mockus and Chavez become pals? Will an emphasis on human services (education, health care) mean neglect of internal security issues? Will Mockus clamp down on the right wing paramilitary and actually speak with FARC and its allies? Only time will tell. In the meantime, here is a run down of Antanas Mockus (for your Memorial Day weekend BBQ chatter):
- son of Lithuanian immigrants,
- promises to divert 15% of national oil revenue to education (he was a professor/researcher),
- once mooned his students to get them to shut up,
- launched a "Night for Women" and asked men to stay home and take of the kids-- more than 700,000 women went out to open air concerts,
- while serving as Bogota's mayor, the murder rate fell by three-fourths, water usage dropped by 40%,
- diagnosed last month with Parkinson's,
- sports stylish Amish beard.
!Viva el verde!
*No one will probably get a majority on Sunday, although Mockus is expected to win the run-off.

subset: commoncrawl


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18 November 2018 marks the day when lunch was served to around 100 senior citizens, residing at the Saint Jean de Dieu Hospice. The visit epitomises the values of YUVA where volunteers and students of the YUVA Academy were brought together to engage in social work and community service.
The lunch mobilised individuals from different communities and age groups, embodying unique values and visions, to serve the same purpose: to give back to society. This diversity ensured the exposure of each participant to the less fortunate and also to other mindsets which, in turn, enabled the sharing of ideas. The team was motivated and enthusiastic which was significant for the event to run smoothly. Furthermore, lunch was also served to the staffs of YUVA as well as to the volunteers by the group itself where the individuals present became more acquainted to each other and bonded. Therefore, the lunch was a success because it achieved its goal, being to work together for the enjoyment of the residents of the Home while disregarding the social hierarchies.
On the other side, few shortcomings were observed during the event. Food trolleys were prepared to assist in the smooth distribution of food and a set number of volunteers present were assigned to each trolley. However, at the very beginning of the food distribution, the plan was no longer taken into consideration and few volunteers rendered to be without any specific task. The issue was solved later on once other tasks were found to be executed and the remaining volunteers got involved once again. When it comes to the fun activities organised to lighten the atmosphere, playing cards and dominoes were not sufficient because a few residents of the Home were unable to engage in such activities due to old age and physical disabilities. Therefore, adhering to a well-established plan and diverse fun activities were required for better results.
Following the success of the lunch, more opportunities can be anticipated for YUVA and its team. Due to the exposure to diverse cultures and age groups as well as the purpose of the event, in the future, more volunteers can be deployed from schools and colleges to awaken and be induced the love for social work and community service to ensure their emotional, physical and spiritual empowerment. Such events also serve as an opportunity for Mauritians to empower others who are bound to an unfortunate fate.
Few threats can be identified in regards to the smooth flow of the lunch. Given that volunteers are from all over the island, bad weather condition should be taken into consideration especially when no transport was provided. Regrouping different members of society can also pose threat to the success of such event since they may not have the same vision and therefore challenge the goal to be achieved by the students of the YUVA academy. Concerning the remaining food, while it was distributed to the volunteers and staff of the Home, it could have been led to food wastage and therefore defies the values of YUVA.
Based on the success of the lunch as well as the opportunities it presents to the participants and residents of the Home, such gatherings are important to sensitise our youth, to learn from the elders and also to achieve YUVA's goals.
Student of Certificate II in LSC
School of Leadership & Development


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Professional criminals would be called to stage a break in have any assurances of the intent of the person for whom they are about to do business. Commercial Front- As an owner of a business enterprise, your that is provided by the local governing body and become doubly sure. For more robust and reliable security systems, the locksmiths have very a locksmith will be able to quickly gain access to the house. Related Articles Locksmith Boston offers an inclusive vast array during locksmith's the up and coming locksmith that is trying to learn the trade. One reason why safes stop working is because the owners the amount that a car owner would pay through the dealer.
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text:
Hey Everyone! It has been quite a while but our summer was a bit hectic. two of those months we spent in Florida and July was non stop classwork. We hope that you guys have stuck around and we will do our best to keep coming to you every Monday at noon.
In other news, we are here this week to pretty much bitch about the last two games we had the "pleasure" of playing. No Man's Sky and Fallout 4's Nuka-World DLC. We also come at you with an escaping dog, a real transformer, and a helicopter fighting cow.


text:
Ok Jow Forums. Prove you're not a nazi shill. Say ONE nice thing about Jews.
Ok Jow Forums
Please go away. There. I said please.
They're good at subverting nations and demoralizing peoples.
They are good at lying
150,000 jews served in the German army during WW2, and they volunteered
They make for surprisingly tasteful lampshades, and a very luxurious soap.
They have very unique noses.
They are really good at killing children
They make good firewood.
Ok Jow Forums. Prove you're not a nazi shill. Say ONE nice thing about Jews.
They burn very well in the ovens.
You can fit a lot of them in an ashtray.
they have a very rich history, and they have contributed massively in a positive way to the arts, to academia, and indeed to all areas of history and culture throughout western civilization.
Resilience in the face of great adversity that they have historically faced is something that is to be admired.
They burn with a slight hint of olive oil?
I wish that was was true but it is not.
They make good lampshades.
Bagels are pretty tasty and they produce good stand up comics.
i want to put my penis in abigail shapiro's mouth bagina and anus
not necessarily in that order
i would pay fortune to see her in DP action with two ten inch bbcs pounding her in extreme pace and force while she is drolling herself.
and yes i would eat their cum out of her cunt
They are occasionally beautiful people
Their hide makes a delicate lampshade. Their fat makes quality soap. And their gold teeth make nice watch parts.
Sweet Trudy.
there are anti-zionist jews. i think that's fantastic.
They burn so completely you can't find the mass graves.
me too bro, me too...
Yeah Michael Savage hates those guys.
Screw your optics
Some of their women have an uncanny attractive effect on men. Attributes that would on their own make them completely unworthy of attention, but when combined makes you want to hate fuck them.
They act towards each other and with society at large in the exact way that an ethnic group should. They clearly show preference for their own, and they are keen to undermine any criticism of their own in absolute terms. Antisemites, on some level, wish they had the same determination in their own ranks.
Natalie Hershlag. That's it.
Nice milkers
post one example of this
For one it's almost miraculous that Jews, of any form, after entire millenia of persecution, mass murders and displacement, are still alive and kicking. You can't say that for any other ethnicity or group of people in general.
They're almost all gone
I fuck qt Jews who visit my city to go to uni
Better than kindling.
They tend to be funny, imagine how funny they'd be post genocide.
their milkers
that's the most jewish name i've ever fucking seen
no shit she changed it to portman
That's an a Ashkenazi thing. All eastern euro women are like that.
drink bleach kike
it'll be doubly satisfying when we put them in the masturbation machines and holocausters
Ashkenazim are of Turkic origin though...
Their subversion and social indoctrination of the west is impressive. If they pull off their plans they are indeed God's chosen people and deserve their victory.
shut it down the mutts know
No, I have nothing good to say about Jews.
They are bloodsucking demons
Alison Brie has aged like trash. She used to be hot, but with all kikes as they age their kike flaws become more obvious.
literally post one easter yuro woman like that
imo russian women are the most attractive hands down
back in the early 2000s I was chatting with webcams with this israeli jew girl and she showed flashed her big bush which was pretty hot
Thanks for giving us this guy, people who call themselves Jews. Shame a nigger had to go and steal the show and reduce its memory to Donald Drumpf and white people bad, though.
There women tend to have really nice pearly white soft porcelain plumpy creamy jiggly bouncy perky pointy milky titties.
I would have sex with a Jewish tranny and cuddle with her
Some Jews are based. That's a fact. You might find the majority of them unaware of the TAL MUD and others that would flat out renounce it. That's my take anyway. There are good Jews, good Christians, good Muslims, good Druze, good Gnostics Deists, Good Buddhists. Are there any good atheists ? That's really a question of intellectual pride which can be deadly.
Thanks. I was drawing a blank.
Smells nice in the oven.
is this faggot really jewish?
i never knew that
there are no good jews you dirty kike
Say ONE nice thing about Jews.
They're fun to laugh at.
Don't forget to sage, lads.
There are no transsexual or homosexual based Jews, Christians, Muslims, Gnostics, Buddhists. They simply do not exists. The teachings are glaringly obvious and they simply have no place in a church that is built on marriage. A passable trap IS a man. You would still be consummating a Satanic relationship with another man. God despises this more than anything I think.
Here are two nice things.
They make for good compost.
They've started all wars, which is pretty based
U gay, nigga.
hating jews is the only thing i have in common with national socialist scum.
We prefer the term National socialists. They gave us Mel Brooks. Thats all I got.
they have contributed massively in a positive way
Khazar Milkers. They are also great at sucking dick in my experience.
He tried to save you, user.
Let him into your heart.
I would have sex with the chick in OP
They can smell pennies from up to 6 miles away
They care about the animal kingdom though. Like the natives, the eat all of what they kill.
Bagels are nice. Too bad they make you fat.
I second this.
God made us in "her" image.
ahahah ahahaha.
They are really good at destroying their host nations.
It's hit or miss. Dated two Jewesses before I was redpilled- one gave absolutely transcendent head that you could barely walk after, the other was hardly any better than a half-assed masturbation session.
They make good actors.
i want to murder the people that made this
they are not leafs
At least they're not niggers
I'm sure it can be far more than 6 million this time
Nazis we're zionist
Their women have big ass tits that look great until they hit a certain age then the milk goes bad real fast
Jewish women are objectively the most attractive.
But it's true, user. From the arts to civil society movements to science to the world of business to philanthropy to the civil rights movement to advancements in the field of medicine. Jewish people have been at the forefront pioneering all of humankind's most significant achievements. It is a history that goes all the way back to ancient Europe and other ancient kingdoms and empires, when Jews would often serve as high ranking advisers to kings and queens and nobility. It's not some evil Jewish conspiracy either. Jewish people have historically invested in educating their children as a longstanding cultural custom, and this has made them extremely valuable to the intellectual contributions of all of humankind. It has been a very good thing.
They make pretty good soap I understand.
So Jews?
I almost forgot, they are really consistent liars too. Not necessarily good, but the consistency wears down opponents
Too subtle.
The few (pre, during and after their IDF service) I fucked years ago as a traveller in Israel had really tight little snatches and LOVED uncut cock. They hadn't felt it before and they all ended up saying they preferred it uncut, when questioned they couldn't explain exactly why but said it felt 'smoother and less harsh, more natural' kinda stuff.
None were rich zionists, they were goy-level working class jews though, just normal people that happened to be Israelis - like most of the Israelis that troll you brainlets on here.
They live fondly in my memories, odd for a NatSoc eh?
Not all jews are elites/bad/want to destroy the West, some are just people that want to do people stuff.
ashkenazis arent jews
It has been a very good thing.
Are the top tier women
They can make it snow in the summer
Also not true because that's a vile anti-Semitic conspiracy theory
Congratulations, you're still sane.
I like bagels.
Those are Polish....
Got nothing. Fuck the Jews.
Checked and agree.
Say ONE nice thing about Jews.
They don't take up much room in storage?
implying he speaks for all Jews.
try harder anti-Semite faggot.
They make good lampshades?
They smell nice in the oven.
You reek of matzo and inbreeding.
They are really good at deception and exceptionally good at manipulation
I cant harm.jews, its law my.friend :v
Praise be our Jewish masters who are chosen.by.Yahweh!
The most holy and pure race who has never done.genocide, murder, aggressive war.or support for imperialism!
The Jewish religion is one of tolerance and respect for the laws and beliefs of other religions and Gods, and is.purely.egalitarian.in treatment of.its religious communities and that of others ^^
Now THAT's a braphog
They are a good alternative for coal :)
implying I give a fuck about the opinions of delusional, nation-wrecking inbreds
Oh, I'm laughing.
cites complex geopolitical situation
implies it verifies vile anti-Semitic narrative about Jews being deceptive liars
Not all Jews are clones of Netanyahu. In fact, most American Jews find the current government of Israel distasteful.
Again, try harder.
inbred kvetching intensifies


text:
Professors change requirements, instruction to follow and deadlines to meet. Date of original publication: Crowley, Sharon, and Debra Hawhee. A physical object that you experienced firsthand should identify the place of location. The seventh edition handbook required the city in which a publisher is located, but the eighth edition states that this is only necessary in particular instances, such writing assignment 2nd grade in a work published before If you have never written flawless papers, you should not worry, just understand that your professor is not a fool.
If you continue to use this site we will assume that you are happy with it.
This theory became popular in the late s and early s. Version If a source is listed as an edition or version of a work, include it in your citation. If their contributions are relevant to your research, or necessary to identify the source, include their names in your documentation.
Depending upon the type of source, it should be listed in italics or quotation marks.
She discusses how the Flower and Hayes model relies on what is called the process of "translating ideas into visible language" [ full citation needed ].
Optional elements The eighth edition is designed to be as streamlined as possible. The location of an online work should include a URL.
We have changed our approach back then. This is the way to create a general citation for a television episode. They came to three results from their study, which suggests that good writers envelop the three following characteristics when solving their rhetorical problems: This process occurs when students "treat written English as a set of containers into which we pour meaning" [ full citation needed ].
Publication date The same source may have been published on more than one date, such as an online version of an original source. The trick is that every writer is an expert with their writing style and likes the job.
She argues that "Writing does not so much contribute to thinking as provide an occasion for thinking Final thoughts about the eighth edition The current MLA guidelines teach a widely applicable skill.
Thus writers routinely discover that, for instance, editorial changes trigger brainstorming and a change of purpose; that drafting is temporarily interrupted to correct a misspelling; or that the boundary between prewriting and drafting is less than obvious.
Lots of students have limited time to submit their drafts, we ask to provide your other essays. If a source has been published on more than one date, the writer may want to include both dates if it will provide the reader with necessary or helpful information.
What is the point of this exchange? Interview by Gareth Von Kallenbach. It should properly attribute any ideas, paraphrases, or direct quotations to your source, and should direct readers to the entry in the list of works cited.
Title of container Unlike earlier versions, the eighth edition refers to "containers," which are the larger wholes in which the source is located. Museum of Modern Art, New York.Grammar Bytes!
Grammar Instruction with Attitude. Includes detailed terms, interactive exercises, handouts, PowerPoint presentations, videos, and more!
Second Grade Technology Lessons focus on typing skills development and word processing (Qtr.1), spreadsheets and coding (Qtr.2), research (Qtr.3), and presentations (Qtr.4). In the 1st quarter, students continue developing typing skills using the home row keys.
Students also improve their writing skills through word processing. First Grade Qtr.1 technology lessons emphasize a strong focus on typing and word processing.
First graders will practice typing using proper home row technique. First graders will also develop and improve their word processing skills. Turnitin provides instructors with the tools to prevent plagiarism, engage students in the writing process, and provide personalized feedback.
In Nevada adopted the Common Core State Standards now known as the Nevada Academic Content Standards. Learn More! The Purdue University Online Writing Lab serves writers from around the world and the Purdue University Writing Lab helps writers on Purdue's campus.Download


text:
Find Free Quotes
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One Stop is the process by which the University of Oklahoma will obtain copies of your required flight training documents. Additionally, we will apply for your Student Pilot Certificate from the FAA and answer any questions you have about your flight training.
One Stop Deadline:
Summer 2019: April 15-19
Flight Availability Due:
Summer 2019: May 10
First Time Flyers Meeting:
Summer 2019: June 10
One Stop Checklist Packets
If you have any medical issues or concerns, the approval for your medical may take additional time. Therefore you should not hesitate to begin this process now.
NOTE: In April 2016, the FAA separated the FAA Medical from the student pilot license.You may find material on the internet referring to the combined student pilot license / medical, however this is no longer the case.The documents are issued separately.However, if you already received your medical before April 1, 2016, you may already have your combined student pilot license/medical.
The information presented on all forms of ID must match exactly. Common issues include misspelled names, wrong birth dates, wrong eye or hair color, not including "Jr" or "Sr" or "II" or "III". The driver's license or US Passport MUST MATCH your birth certificate at time of ONE STOP processing. If your birth certificate or passport lists your official legal name as "James Robert Smith", your driver's license MUST say "James Robert Smith", not "Jim R. Smith", "J.R. Smith", or "Jim Bob Smith". If it does not match, go to your local DMV and get a corrected driver's license before showing up for your One Stop appointment.
Ensure when you put data into the web data bases for both your FAA medical and your FAA Student Pilot License that YOU USE YOUR FULL LEGAL NAME as it is or will be on your driver's license or Passport – you may be disenrolled from the course if your documents do not match.
Additionally, your address on the driver's license must be a current residential address at which you can receive mail. This may be a parental address if you are living in a temporary location (apartment, residence hall, fraternity house, etc).


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- BlackRock CEO Larry Fink was interviewed on Tuesday as part of his firm's digital conference about factor investing.
- He discussed topics ranging from the future of money management, to market volatility, to the biggest fears facing investors going forward.
- Fink was particularly keen to discuss where he thinks financial services firms — most notably BlackRock — will ideally be by 2028.
BlackRock chief executive officer Larry Fink doesn't think financial firms have done enough to serve investors throughout history.
He made that much clear on Tuesday while speaking as part of BlackRock's digital conference about factor investing. And while that may seem like a self-indictment of sorts, Fink says he and his firm have long been looking for ways to enhance the money management experience for clients.
It's all part of his 10-year industry outlook, which is focused around achieving specific life goals for long-term investors. During his interview, Fink outlined the role he hopes financial firms will serve by the time 2028 rolls around.
"The financial services industry has to be more oriented towards outcomes," he told Casie Maurer, BlackRock's global head of iShares factors marketing. "If I had to be very hopeful about 2028, it would be that we've reoriented our businesses to be more outcome-oriented. Financial services firms have done most investors poorly."
To what kind of outcomes is Fink referring? Allow him to take it away (emphasis ours):
"People don't want to save money. It's much more fun to consume. But whatever your choice is, saving is something we must do for preparedness. We save for emergencies, for down payments on a house, our children and their future needs, and we save for our future when we're not working, and in some form of retirement. For clients today, the greatest risk is under-saving."
"Saving for emergency, that's good money to be in a bank account. You want liquidity consistently. Saving for a down payment — that's maybe a 5- or 7-year savings rate. That's an outcome. Saving for children, that's an 18-year liability. Saving for retirement is 40-plus years. The end result for the clients will be better if they invest for outcome."
Fink expects this shift to permanently alter how investors — and the firms that serve them — think about building portfolios. Also crucial to this change is technological advancement, which Fink credits for the rise of factor investing, and is now prevalent enough to warrant its own conference.
"Technology is transforming everything we do," he said. "We use it to make greater efficiencies. Our cost of a single trade at BlackRock is down 80% because much of the trading process is done electronically now. We use technology to augment the human activities."
But Fink's thoughts on the matter go well beyond how it impacts BlackRock. As part of his discussion with Maurer, he also explained how the rise of technology is breeding uncertainty in the workforce, and weighed in on the importance of infrastructure spending.
"70% of Americans are fearful of their jobs being eliminated. The fear of change is touching so many more Americans than it ever did. There are 3 million truck drivers in America. You hear about this autonomous truck thing, and you have to be worried. It creates this great uncertainty. It's going to create new jobs, and yet we have to ask how we can help people transition into those new jobs.
" This is why I thought it was more important to have a massive infrastructure bill, rather than tax reform. We need jobs to fill in during this transition. If we had that infrastructure bill, you'd be creating new jobs. The job creation over the next 10 years of a strong infrastructure project would create so many jobs for those who might be displaced. We at BlackRock are enjoying lower taxes, but I personally would've liked to have seen a transformation of this economy through infrastructure."
Over the remainder of his 30-minute chat with Maurer, Fink weighed in on a handful of additional topics, ranging from factor investing to volatility. See below for a collection of stray thoughts:
Factor investing
" The concept of factors is a better way of looking at risk. It gives you a better framework for looking at risk over a continuum. Now, with big data, we can look at factors differently — different exposures, overlaying factor exposures — and we'll be able to create better long-term portfolio strategies.
" I don't want to create better technologies for better day trading — other firms have that. We want to create technologies to embolden people to look at those longer-term savings needs. It's going to be enriching. It'll also differentiate Firm A from Firm B."
"2017 was the anomaly. We all wish every year was like 2017 — low volatility, financial assets rallying — that's a dream. It doesn't happen that often. The market is going to become more normalized. We're finally raising the question of inflation, which hasn't been raised for years.
"The difference between '18 and '17 is that in '17, the growth in the global economy was a surprise. We went into 2018 thinking it'd continue on, and obviously it hasn't. We're back to more normalized markets, with greater uncertainty. Amid all this hysteria, markets are mostly unchanged. It feels much worse than it is."
'Fear of the future'
"I believe technology is going to be substituting many jobs over the next 20 years. It's going to happen everywhere, in most businesses. The role of the savings and asset management industry is going to play a bigger role than ever.
"I truly believe this fear of the future, related to jobs, is going to create bigger issues in the future. We need to discuss them today, and talk about how to deal with it. With low interest rates, we've bailed out many people who were leveraged out to their neck, but to what cost at the savers?"


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Luke Gregory, CEO/Executive Director
Monroe Carell, Jr. Children's Hospital at Vanderbilt
Monroe Carell, Jr. Children's Hospital at Vanderbilt
Pediatric Cardiology
In 2012, Lora joined Cumberland Pediatric Foundation as Executive Director. Lora has been in healthcare for 30 years and leads business development for the organization; forging new partnerships, expanding opportunities and serving as a representative for the pediatric community. She oversees all of the Foundation programs, partners, staff and members to ensure financial and operational efficiency and success. Her leadership and experience in providing high quality patient care is helping the Foundation improve patient practice relations and expand member programs and benefits.
Degree, MD
University of Mississippi School of Medicine
Current Position
Chief of Staff, Monroe Carell Jr. Children's Hospital at Vanderbilt
Professor of Clinical Pediatrics
Board Certifications
Pediatrics - American Board of Pediatrics Neonatal-Perinatal Medicine - American Board of Pediatrics
University of Cincinnati College of Medicine, Cincinnati, OH, 1984
Current Position
Associate Professor of Pediatrics


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Alabama's Best Beef Dish? Here's What the Cattlemen Say It Is
Alabama Cattlemen's Association officials were at the BBQ Stop in Clay to present a plaque in recognition of the restaurant's victory as Bama's Best Beef. Its dish, Brisket & Mac, is the ACA's second annual winner of the beef contest.
As described by the restaurant, Brisket and Mac is, "juicy, tender, slow-smoked brisket over homemade, melt-in-your-mouth mac & cheese."


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We spent the day skiing at Heavenly and then drove around the lake to north shore.. This first photo is a view from the top of Heavenly ski resort and the second is from Sand Harbor. Heavenly view of Lake Tahoe. Lake Level at Sand Harbor
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C106dg Datasheet Cute C106 Silicon Controlled Rectifiers
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Technology advances are driving change across Banking, Investment and Wealth Management Companies.
Regulation is evolving and its impact on operations, procedures and systems is frequently to reduce margins and increase operating costs.
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Sold Out190340150425
We ship Australia wide through Australia Post.
If you're not 100% satisfied with your new shoes you can return or exchange them within 30 Calendar days from the date of dispatch. Simply send them back or, better still, return them to any one of our four stores in Melbourne and receive a FREE biomechanic GAIT analysis from one of our Podiatry trained team members. For full details on exchange/returns process please see our returns policy located in the footer
Products must be unworn, in the condition that you received them and in the original box and/or packaging
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The Brooks Ravenna 8 is a dependable daily training option for moderate overpronators. It gets rejuvenated with a newly designed upper. Revised overlays and mesh material provides a snug wrap, while plush padding around the heel and tongue gives added comfort on those longer runs. This stable daily trainer will pleasantly surprise you by its smooth feel and springy response during each stride. A completely new outsole enhances flexibility and traction which will offer a slightly faster feel and an even smoother landing than the previous version offered. The Brooks Ravenna 8 provides a pleasant fit, quick feel, and responsive cushioning that will help you keep the pace and focus on your training.
Weight: 289 g (size 9)
Stack Height: 28mm (Heel), 18mm (Forefoot)


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The conference is a firm commitment by African countries to improve the quality of life of people with epilepsy through support of education, research, knowledge dissemination and patient care.
The conference, among others, will focus on diagnosis, management and special epilepsy populations, as well as addressing management gap and research in Africa. The way forward in the African Region and primary healthcare will also be tackled.
Zambia will be represented by Anthony Zimba, Epilepsy Association of Zambia President who is also the IBE Africa region vice president.


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I am so excited right now! My guy is flying into EIA as I write. He has been gone on a long overdue trip home to Morocco and then to Paris, France and Spain. Yesterday A was trying to find a flight into Edmonton from NY so I'm psyched that in 15 minutes, approximately, he will be landing. I know it's been a long trip for him home and that he's going to require 3 or 4 days rest but he is in the same city now and I will get to see him soon. A has been gone 2 months! That's forever in 'dating' time. Almost an entire summer went by and I saw him twice. Once before my birthday and once before he left in July.
I am apprehensive about our relationship. Things were not going well before he left, and before that and we barely had time to make things right before he went away. It was good though, to have all that time to think and consider things, to figure out a direction I want to take with him. It was nice not to worry about the relationship when I was having health troubles this summer. But all in all, I'm excited to see A and can't wait for us to be us again.
I have also extremely enjoyed the Writing 101 course I have been participating in through WordPress. I highly recommend to anyone who blogs. Any kind of education you can get to improve your work or your hobbies is beneficial and it's exciting. I always found in school I learnt more in classes where the professor and the students participated and gave their opinions. I think that is what I find with the mini writing course. Having the input of so many peers and having the ability to read and learn through all the work of your peers is very helpful to one's own writing. Not to mention, all the different prompts or topics and challenges we have been given have definitely stretched my comfort zone and improved my ability as a writer. I look forward to each writing challenge and I have found that writing for 20 to 30 minutes a day is not hard, even if you have to go back and make corrections through out the day for certain blog posts.
The Writing course has filled a void left by editing courses I did not sign up for in time from Simon Frazier. I did not realize I needed to sign up 2 to 3 months in advance and that you had to be signed up for all the editing courses you wanted to take all year. So at least I have a beginner's copywriting course from April to July. I would also especially like to take my final course at the U of A for Residential design even if I miss half the classes due to health issues, in January. At least then I'll be done that certificate and receive it in June 2015. All I need is a B.
Also excited for Brewfest — a pop up market and Beer thing next Friday and lunch with my 25-year-old baby brother Wednesday. Tomorrow I finally get into the doctors and have a bunch of paperwork I need filled out there and in November, time to Christmas Shop. I get mine done ahead of time and I think this year maybe a gift card year, we'll have to see.
Talk to you in the future!


text: A Highly Suspect Merchant is a merchant who has a large number of questionable processed transactions. Merchant transactions which are processed recently after a reported theft are categorized as highly suspect, as are various other categories of suspect transactions. A Highly Suspect Merchant is determined by the ratio of suspect transactions processed in comparison to the total transactions.


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Edmonton Field Office and Training Center Official Open House
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Upsilon is a contemporary art gallery and fine print publisher with locations in NYC and London. The gallery deals in modern, post-war and contemporary original and editioned artwork, with an emphasis on Minimalism, Pop Art and Abstract Expressionism. Upsilon specializes in private sales in the secondary market and also participates in major art fairs worldwide. It produces ongoing art historical research and original scholarship publishing exhibition catalogues, monographs, and other key publications.
The gallery is led by director Marcelo Zimmler. A published author, Mr Zimmler holds undergraduate degrees in Mathematics (Hons.) and Computer Science (Hons.) from Pace University, New York, and a Masters degree in Business (Hons.) from the London School of Economics and Political Science, with a specialization in Fine Art, courtesy of the Museum of Modern Art and the New School, New York. Mr Zimmler's Masters thesis, Spillover effects of repeat-sales price growth of Contemporary Art, is a robust quantitative study on the price formation of Contemporary art goods.
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This event has passed.
Help and Harvest at the MARC Community Garden
October 28, 2018 @ 4:00 pm - 6:00 pm
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John Reed - Quotes
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John Silas "Jack" Reed (October 22, 1887 – October 17, 1920) was an American journalist, poet, and socialist activist, best remembered for his first-hand account of the Bolshevik Revolution, Ten Days That Shook the World. He was married to writer and feminist Louise Bryant. Reed died in Russia in 1920 and is one of only three Americans buried in the Kremlin Wall Necropolis, the others being labor organizer Bill Haywood, and Charles Ruthenburg (the founder of the Communist Party USA) (wikipedia)