Inhibition of weak-affinity epitope-IgE interactions prevents mast cell degranulation

Development of specific inhibitors of allergy has had limited success, in part, owing to a lack of experimental models that reflect the complexity of allergen-IgE interactions. We designed a heterotetravalent allergen (HtTA) system, which reflects epitope heterogeneity, polyclonal response and number of immunodominant epitopes observed in natural allergens, thereby providing a physiologically relevant experimental model to study mast cell degranulation. The HtTA design revealed the importance of weak-affinity epitopes in allergy, particularly when presented with high-affinity epitopes. The effect of selective inhibition of weak-affinity epitope-IgE interactions was investigated with heterobivalent inhibitors (HBIs) designed to simultaneously target the antigen- and nucleotide-binding sites on the IgE Fab. HBI demonstrated enhanced avidity for the target IgE and was a potent inhibitor of degranulation in vitro and in vivo. These results demonstrate that partial inhibition of allergen-IgE interactions was sufficient to prevent mast cell degranulation, thus establishing the therapeutic potential of the HBI design

A heterobivalent ligand inhibits mast cell degranulation via selective inhibition of allergen-IgE interactions in vivo

Current treatments for allergies include epinephrine and antihistamines, which treat the symptoms after an allergic response has taken place; steroids, which result in local and systemic immune suppression; and IgE-depleting therapies, which can be used only for a narrow range of clinical IgE titers. The limitations of current treatments motivated the design of a heterobivalent inhibitor (HBI) of IgE-mediated allergic responses that selectively inhibits allergen-IgE interactions, thereby preventing IgE clustering and mast cell degranulation. The HBI was designed to simultaneously target the allergen binding site and the adjacent conserved nucleotide binding site (NBS) found on the Fab of IgE Abs. The bivalent targeting was accomplished by linking a hapten to an NBS ligand with an ethylene glycol linker. The hapten moiety of HBI enables selective targeting of a specific IgE, whereas the NBS ligand enhances avidity for the IgE. Simultaneous bivalent binding to both sites provided HBI with 120-fold enhancement in avidity for the target IgE compared with the monovalent hapten. The increased avidity for IgE made HBI a potent inhibitor of mast cell degranulation in the rat basophilic leukemia mast cell model, in the passive cutaneous anaphylaxis mouse model of allergy, and in mice sensitized to the model allergen. In addition, HBI did not have any observable systemic toxic effects even at elevated doses. Taken together, these results establish the HBI design as a broadly applicable platform with therapeutic potential for the targeted and selective inhibition of IgE-mediated allergic responses, including food, environmental, and drug allergies

Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability

This paper combines two techniques—mass spectrometry and protein charge ladders—to examine the relationship between the surface charge and hydrophobicity of a protein (bovine carbonic anhydrase II; BCA II) and its rate of amide hydrogen-deuterium (H/D) exchange. Mass spectrometric analysis indicated that the sequential acetylation of surface lysine-$\epsilon$-NH$_{3}$$^{+}$ groups—a type of modification that increases the net negative charge and hydrophobicity of the surface of BCA II without affecting its 2° or 3° structure—resulted in a linear increase in the total number of backbone amide hydrogen that are protected from exchange with solvent (2 h, pD 7.4, 15 ºC). Each successive acetylation produced BCA II proteins with one additional hydrogen protected after two hours in deuterated buffer (pD 7.4, 15 ºC). NMR spectroscopy demonstrated that these protected hydrogen atoms were not located on the side chain of the acetylated lysine residues (i.e., lys-$\epsilon$-NHCOCH$_{3}$). The decrease in rate of exchange associated with acetylation paralleled a decrease in thermostability: the most slowly exchanging rungs were the least thermostable (as measured by differential scanning calorimetry). The fact that the rates of H/D exchange were similar for perbutyrated BCA II (e.g., [lys-$\epsilon$-NHCO(CH$_{2}$)$_{2}$CH$_{3}$]$_{18}$) and peracetylated BCA II (e.g., [lys-$\epsilon$-NHCOCH$_{3}$]$_{18}$) suggests that the charge is more important than the hydrophobicity of surface groups in determining the rate of H/D exchange. These kinetic electrostatic effects could complicate the interpretation of experiments in which H/D exchange methods are used to probe the structural effects of non-isoelectric perturbations to proteins (i.e., phosphorylation, acetylation, or the binding of the protein to an oligonucleotide or another charged ligand or protein).Chemistry and Chemical Biolog

Driver and Consequences of Multichannel Shopping

Previous research has investigated what happens when customers start utilizing more than a single sales channel (i.e., become multichannel). This research stream has identified two key consequences of multichannel usage. First, Shankar et al. (2003) and Hitt and Frei (2002) determine that customers using an internet channel in addition to the traditional brick-and-mortar channel are more loyal than customers who use a single channel. Sousa and Voss (2004) explain that these higher customer retention rates are because of increased coordination between channels; the coordination among channels increases customer satisfaction, which improves retention rates. Second, Neslin et al. (2006), Thomas and Sullivan (2005), Kumar and Venkatesan (2005), Venkatesan et al. (2007), Ansari et al. (2008), and Kushwaha and Shankar (2008) determine that on average multichannel customers spend more than single channel customers. Although plenty of research exists about multichannel customer management, there is relatively little known about the drivers that induce customers to adopt a new channel. Additionally, previous research has mainly focused on the short term effects and has not attempted to quantify, if any, the long-term effects of multichannel usage. This dissertation examines multichannel customers' decisions. Specifically, I address the following questions: (1) What factors lead customers to adopt new sales channels? and (2) What is the long-term effect of multichannel shopping on customers' spending? The first essay investigates the drivers of new sales channel adoption. In this essay, I propose a conceptual framework grounded in diffusion theory, and test this framework on longitudinal data from a major catalog company using a discrete-time, hazard model. This essay contributes to the marketing literature by providing empirical evidence that social influence impacts the timing of new channel adoption. I find that longer tenured customers are more eager to adopt a new channel and less impacted by social influence. I also find that customers adopt a physical store at a faster rate than an Internet store. Moreover, social influence and customer tenure play more important roles when customers adopt an Internet channel than a brick-and-mortar channel. In contrast, marketing activities play a more important role in customers' adoption of the physical store than in the customer's adoption of the internet channel. These new findings have implications for identifying early adopters and accelerating the diffusion of a new channel. The second essay is the first study to look at how multichannel shoppers' spending pattern changes over time, and is distinctive from past research which examines multichannel customers' spending only in the short term. For this study, I examine longitudinal data from a major U.S. retailer. My empirical analysis is likely to be affected by self-selection bias because heavy users may self-select themselves into using more than one channel. To control for such bias, I combine different panel data econometrics techniques with the propensity score matching method. The results provide empirical evidence that multichannel customers increase their spending when they initially start to use a new channel. In the long run, however, I find that the difference between multichannel and mono-channel customers' spending disappears. The findings have implications for predicting revenue streams from multichannel customers over time. Methodologically, this study is the first to combine dynamic panel data estimation with the propensity score matching. In addition, several papers in social sciences rely on aggregate level data (for example, zip code level demographics from U.S. Census), to create matched pairs. These papers are criticized as some scholars (Gensler et al., 2012) argue that zip code level data do not provide sufficient information to construct functional matched pairs. To address this issue, I create matched pairs based on U.S. Census data and household level data. The findings show that the estimates obtained by both matching procedures are exceptionally similar results

Using Fluorous Amino Acids to Modulate the Biological Activity of an Antimicrobial Peptide

No AbstractPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58035/1/370_ftp.pd

Designer covalent heterobivalent inhibitors prevent IgE-dependent responses to peanut allergen

Allergies are a result of allergen proteins cross-linking allergen-specific IgE (sIgE) on the surface of mast cells and basophils. The diversity and complexity of allergen epitopes, and high-affinity of the sIgE-allergen interaction have impaired the development of allergen-specific inhibitors of allergic responses. This study presents a design of food allergen-specific sIgE inhibitors named covalent heterobivalent inhibitors (cHBIs) that selectively form covalent bonds to only sIgEs, thereby permanently inhibiting them. Using screening reagents termed nanoallergens, we identified two immunodominant epitopes in peanuts that were common in a population of 16 allergic patients. Two cHBIs designed to inhibit only these two epitopes completely abrogated the allergic response in 14 of the 16 patients in an in vitro assay and inhibited basophil activation in an allergic patient ex vivo analysis. The efficacy of the cHBI design has valuable clinical implications for many allergen-specific responses and more broadly for any antibody-based disease

Covalent Heterobivalent Inhibitor Design for Inhibition of IgE-Dependent Penicillin Allergy in a Murine Model

Drug allergies occur when hapten-like drug metabolites conjugated to serum proteins, through their interactions with specific immunoglobulin E (IgE), trigger allergic reactions that can be life-threatening. A molecule termed covalent heterobivalent inhibitor (cHBI) was designed to specifically target drug-hapten specific IgE to prevent it from binding drug-haptenated serum proteins. cHBI binds the two independent sites on a drug-hapten specific antibody and covalently conjugates only to the specific IgE, permanently inhibiting it. The cHBI design was evaluated via ELISA to measure cHBI-IgE binding, degranulation assays of rat basophil leukemia (RBL) cells for in vitro efficacy, and mouse models of ear swelling and systemic anaphylaxis responses for in vivo efficacy. The cHBI design was evaluated using two seperate models: one specific to inhibit penicillin G reactive IgE, and another to inhibit IgE specific to a model compound, dansyl. We show that cHBI conjugated specifically to its target antibody and inhibited degranulation in cellular degranulation assays using RBL cells. Furthermore, cHBIs demonstrated in vivo inhibition of allergic responses in both murine models. We establish the cHBI design to be a versatile platform for inhibiting hapten/IgE interactions, which can potentially be applied to inhibit IgE mediated allergic reactions to any drug/small molecule allergy