Surrogate Antibodies That Specifically Bind and Neutralize CCL17 But Not CCL22

The chemokines CCL17 (TARC) and CCL22 (MDC) function through the same receptor, CCR4, but have been proposed to differentially affect the immune response. To better understand the role of the individual ligands, a panel of rat anti-mouse CCL17 surrogate antibodies was generated that can be used to differentiate CCL17 and CCL22 function in vitro and in vivo. We have successfully identified a panel of neutralizing antibodies by screening hybridomas for the ability to inhibit CCL17-mediated calcium mobilization. Chemotaxis in response to CCL17 is also inhibited, providing further evidence that the antibodies in this panel are antagonistic. Using a recombinant cell line expressing human CCR4, we show that the antibodies block ?-arrestin recruitment as evidence that the antibodies are specifically blocking CCL17 signaling through CCR4. The antibodies within this panel inhibit calcium mobilization with varying potency in the calcium flux assay, having apparent IC50 ranging from approximately 1 to >400?ng/mL. Although both CCL17 and CCL22 function through CCR4, only a single antibody was identified as having detectable binding to CCL22. This panel of CCL17-specific antibodies provides tools that can be used to differentiate CCL17 and CCL22 function through CCR4 interaction in vitro and in vivo.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140164/1/mab.2012.0112.pd

Profiling Critical Cancer Gene Mutations in Clinical Tumor Samples

Background: Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting. Methodology: We developed and implemented an optimized mutation profiling platform (“OncoMap”) to interrogate ∼400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact. Conclusions: Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of “actionable” cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents

Profiling Critical Cancer Gene Mutations in Clinical Tumor Samples

BACKGROUND: Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting. METHODOLOGY: We developed and implemented an optimized mutation profiling platform ("OncoMap") to interrogate approximately 400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact. CONCLUSIONS: Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of "actionable" cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents

Memory, History, and the Journey West in the Twentieth-century Novel

Memory, History, and the Journey West in the Twentieth-Century American Novel brings together two important preoccupations of twentieth-century literary, historical and social discourses: psychoanalytic approaches to memory and the mythology of the American frontier. Through an examination of four classic American novels, Willa Cather\u27s My Ántonia, F. Scott Fitzgerald\u27s The Great Gatsby, Robert Penn Warren\u27s All the Kings Men, and Jack Kerouac\u27s On the Road, my project explores the connection between traveling west and returning to the origins of the self through memory. By examining the impulse for westward travel in such varied novels which span a time period of approximately forty years, I argue that our nation\u27s history is analogous to personal memory and that a return to the personal and national past is entwined with a journey across continental space. One of my primary frames of reference is Frederick Jackson Turner\u27s 1893 essay The Significance of the Frontier in American History. The perspective that Turner presents enables me to establish an alternative understanding of place of origins in America and to suggest that the West represents a direction of the national desire to discover and create, a direction of potential but also a primitive source of our national character. Additionally, Freudian, Jungian and Bergsonian theories, my other primary frames of reference, allow me to develop my argument that movement, particularly westward movement, which ties Americans to a cultural and social past, also provides access to a more deeply rooted personal past. My study also explores the idea that the quintessential American character is a traveler and examines the novels\u27 treatments of the immigrant tradition in America and how this crucial aspect of American life shapes ideas about personal and national identity

Engagement of two distinct binding domains on CCL17 is required for signaling through CCR4 and establishment of localized inflammatory conditions in the lung.

CCL17 (TARC) function can be completely abolished by mAbs that block either one of two distinct sites required for CCR4 signaling. This chemokine is elevated in sera of asthma patients and is responsible for establishing inflammatory sites through CCR4-mediated recruitment of immune cells. CCL17 shares the GPCR CCR4, with CCL22 (MDC) but these two chemokines differentially affect the immune response. To better understand chemokine mediated effects through CCR4, we have generated chimeric anti-mouse CCL17 surrogate antibodies that inhibit function of this ligand in vitro and in vivo. The affinities of the surrogate antibodies for CCL17 range from 685 pM for B225 to 4.9 nM for B202. One antibody, B202, also exhibits weak binding to CCL22 (KD∼2 µM) and no binding to CCL22 is detectable with the second antibody, B225. In vitro, both antibodies inhibit CCL17-mediated calcium mobilization, β-arrestin recruitment and chemotaxis; B202 can also partially inhibit CCL22-mediated β-arrestin recruitment. Both B202 and B225 antibodies neutralize CCL17 in vivo as demonstrated by reduction of methacholine-induced airway hyperreactivity in the A. fumigatus model of asthma. That both antibodies block CCL17 function but only B202 shows any inhibition of CCL22 function suggests that they bind CCL17 at different sites. Competition binding studies confirm that these two antibodies recognize unique epitopes that are non-overlapping despite the small size of CCL17. Taking into consideration the data from both the functional and binding studies, we propose that effective engagement of CCR4 by CCL17 involves two distinct binding domains and interaction with both is required for signaling

Early Lifestyle Intervention for Obesity Prevention in Pediatric Survivors of Acute Lymphoblastic Leukemia

Patients with pediatric acute lymphoblastic leukemia (ALL) experience rapid weight gain during treatment and increases in weight are maintained throughout treatment and beyond. Without prompt interventions, altered dietary and physical activity behaviors may become difficult to reverse, contributing to obesity risk long-term. Fifteen children, aged 3–9 years, diagnosed with pediatric ALL who were on maintenance therapy or within two years of treatment completion (mean BMI percentile: 70.4th) and one parent from each family, were enrolled into a 12-week lifestyle intervention delivered remotely through web-based sessions and phone calls with a lifestyle coach. Outcomes were assessed at baseline and end of the intervention. Thirteen of the 15 enrolled families (86.7%) completed the intervention. Parents reduced the “pressure to eat” feeding practice (change in mean score: −0.60, 95% CI: −1.12 to −0.07; p-value = 0.03) post intervention. Children increased the consumption of milk (0.54 serving/d, 0.02 to 1.07; p-value = 0.04) and percent of calories from protein (2.54%, 0.22 to 4.87%; p-value = 0.04) and reduced the consumption of potatoes (−0.16 serving/d, -0.30 to −0.03; p-value = 0.02). No significant changes were observed for children’s levels of physical activity, BMI, or waist circumference. Results from this pilot support the feasibility and preliminary efficacy of early lifestyle intervention among pediatric ALL survivors

Treatment with B225 anti-CCL17 antibody inhibits inflammation and goblet cell metaplasia in the lung of A. fumigatus-sensitized and challenged mice.

<p>Histology of whole lung sections from day 7 post-conidia challenged mice treated with antibodies at 200 µg/kg. Panels A-C are H/E-stained and panels D-F are PAS-stained. In panels D and F, <i>black arrows</i> highlight goblet cells and <i>red arrows</i> point to the mucus released from these cells in the airways. Samples are from groups as follows: A and D are from isotype control treated; B and E are from B225 treated; C and F are from B202 treated mice. Representative photomicrographs of lung sections from n = 5 mice per group are shown. Original magnification was 200×.</p

In vivo neutralization of CCL17 alters whole lung cytokine levels in A. fumigatus-sensitized and challenged mice.

<p>Cytokine and chemokine levels in homogenized whole lung samples from asthmatic mice at day 7 after conidia challenge. Bioplex analysis was used to measure cytokine and chemokine levels in these samples. Data are expressed as pg/mg for isotype control treated (<i>white bars</i>), B225 treated (<i>gray bars</i>) and B202 treated (<i>black bars</i>) Data are mean ± SEM, n = 5 mice per group. *P<0.05 compared with methacholine-challenged isotype control treated group as indicated (ANOVA and Student-Newman-Keuls Multiple Comparison post Test).</p

Antibodies B202 and B225 inhibit β-arrestin recruitment mediated by CCL17 interaction with CCR4.

<p>The human CCR4 β-arrestin reporter cell line was incubated with mouse CCL17 then assayed for the ability to mobilize β-arrestin as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081465#s2" target="_blank">materials and methods</a>. Mouse CCL17 is able to trigger β-arrestin recruitment by signaling through the human CCR4 receptor in a dose-dependent manner (A). The anti-mouse CCL17 specific antibody, B225, can inhibit mouse CCL17-induced β-arrestin recruitment (B). The dual reactive antibody, B202, strongly inhibited mCCL17 induced β-arrestin recruitment (C) while partially inhibiting mCCL22 induced β-arrestin recruitment (D). In panels C and D: B202 (<i>circles</i>); positive controls mAbs <u>(</u><i>inverted triangles</i>); isotype control (<i>open diamonds</i>); no antibody (<i>dotted line</i>); media only control (<i>solid line</i>). Data are expressed as Relative Light Units (RLU) to quantitate β-arrestin recruitment which is directly proportional to luminescent activity. Antibody dose curve starts at 1 µM with a 1∶2 dilution series (mCCL22 at 20 nM; mCCL17 at 30 nM). Antibodies were tested 3 times using CCL17 and twice using CCL22; shown here are representative data from one experiment.</p