Advances in Treatments and Animal Models of Peanut Allergy

Food allergies are a growing health concern affecting approximately 6-8% of the US population. In particular, peanut allergy has an estimated prevalence of greater than 1% of the population and is uncommonly outgrown, making it a life-long disease. Ingestion of allergens can lead to a variety of allergic symptoms ranging from hives or gastrointestinal symptoms to constriction of the airways and anaphylactic shock. Because there is currently no FDA-approved treatment for food allergy, these patients are managed with education and strict allergen avoidance. However, even with the most careful avoidance, accidental ingestion does occur and can lead to life-threatening anaphylaxis. As a result, treatment options are needed. Treatments currently under investigation in clinical trials include peanut oral immunotherapy (OIT), sublingual immunotherapy (SLIT), and epicutaneous immunotherapy (EPIT), though mechanisms of these therapies remain unclear. While results from these trials are promising, limitations include daily dosing, adverse effects, and limited long-term efficacy after therapy is discontinued. Thus, there remains an urgent need for improved therapy options. The work in this dissertation provides the foundation for future drug discovery. First, IgG-mediated basophil inhibition was elucidated as a mechanism of OIT and SLIT and was shown to be associated with long-lived protection. Understanding this mechanism further may result in a targeted therapy option. Separately, a therapy targeting inhibitory receptors on antigen-specific B cells was developed for the prevention of sensitization in a mouse model of peanut allergy. Unfortunately, understanding of food allergy etiology and advances in treatment options has been limited by the lack of an animal model that accurately recapitulates the human disease. Here, we describe the use of the genetically diverse Collaborative Cross to identify CC027/GeniUnc as a more relevant mouse strain that exhibits a severe reaction following oral sensitization and challenge. Together, this work provides a platform for better understanding the mechanisms of food allergy and its treatments, as well as the development of new therapies.Doctor of Philosoph

Necrotizing enterocolitis: Bench to bedside approaches and advancing our understanding of disease pathogenesis

Necrotizing enterocolitis (NEC) is a devastating, multifactorial disease mainly affecting the intestine of premature infants. Recent discoveries have significantly enhanced our understanding of risk factors, as well as, cellular and genetic mechanisms of this complex disease. Despite these advancements, no essential, single risk factor, nor the mechanism by which each risk factor affects NEC has been elucidated. Nonetheless, recent research indicates that maternal factors, antibiotic exposure, feeding, hypoxia, and altered gut microbiota pose a threat to the underdeveloped immunity of preterm infants. Here we review predisposing factors, status of unwarranted immune responses, and microbial pathogenesis in NEC based on currently available scientific evidence. We additionally discuss novel techniques and models used to study NEC and how this research translates from the bench to the bedside into potential treatment strategies

Current Options for the Treatment of Food Allergy

Food allergy is increasing in prevalence; as a result, there is intense focus on developing safe and effective therapies. Current methods of specific immunotherapy include oral, sublingual, and epicutaneous, while nonspecific methods that have been investigated include: Chinese herbal medicine, probiotics, and anti-IgE antibodies. Although some studies have demonstrated efficacy in inducing desensitization, questions regarding safety and the potential for achieving immune tolerance remain. Although some of these therapies demonstrate promise, further investigation is required before their incorporation into routine clinical practice

Intestinal bacteria are necessary for doxorubicin-induced intestinal damage but not for doxorubicin-induced apoptosis

Doxorubicin (DOXO) induces significant, but transient, increases in apoptosis in the stem cell zone of the jejunum, followed by mucosal damage involving a decrease in crypt proliferation, crypt number, and villus height. The gastrointestinal tract is home to a vast population of commensal bacteria and numerous studies have demonstrated a symbiotic relationship between intestinal bacteria and intestinal epithelial cells (IEC) in maintaining homeostatic functions of the intestine. However, whether enteric bacteria play a role in DOXO-induced damage is not well understood. We hypothesized that enteric bacteria are necessary for induction of apoptosis and damage associated with DOXO treatment. Conventionally raised (CONV) and germ free (GF) mice were given a single injection of DOXO, and intestinal tissue was collected at 6, 72, and 120 h after treatment and from no treatment (0 h) controls. Histology and morphometric analyses quantified apoptosis, mitosis, crypt depth, villus height, and crypt density. Immunostaining for muc2 and lysozyme evaluated Paneth cells, goblet cells or dual stained intermediate cells. DOXO administration induced significant increases in apoptosis in jejunal epithelium regardless of the presence of enteric bacteria; however, the resulting injury, as demonstrated by statistically significant changes in crypt depth, crypt number, and proliferative cell number, was dependent upon the presence of enteric bacteria. Furthermore, we observed expansion of Paneth and goblet cells and presence of intermediate cells only in CONV and not GF mice. These findings provide evidence that manipulation and/or depletion of the enteric microbiota may have clinical significance in limiting chemotherapy-induced mucositis

Non-Enzymatic Decomposition of Collagen Fibers by a Biglycan Antibody and a Plausible Mechanism for Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory and destructive joint disorder that affects tens of millions of people worldwide. Normal healthy joints maintain a balance between the synthesis of extracellular matrix (ECM) molecules and the proteolytic degradation of damaged ones. In the case of RA, this balance is shifted toward matrix destruction due to increased production of cleavage enzymes and the presence of (autoimmune) immunoglobulins resulting from an inflammation induced immune response. Herein we demonstrate that a polyclonal antibody against the proteoglycan biglycan (BG) causes tissue destruction that may be analogous to that of RA affected tissues. The effect of the antibody is more potent than harsh chemical and/or enzymatic treatments designed to mimic arthritis-like fibril de-polymerization. In RA cases, the immune response to inflammation causes synovial fibroblasts, monocytes and macrophages to produce cytokines and secrete matrix remodeling enzymes, whereas B cells are stimulated to produce immunoglobulins. The specific antigen that causes the RA immune response has not yet been identified, although possible candidates have been proposed, including collagen types I and II, and proteoglycans (PG's) such as biglycan. We speculate that the initiation of RA associated tissue destruction in vivo may involve a similar non-enzymatic decomposition of collagen fibrils via the immunoglobulins themselves that we observe here ex vivo

The role of human milk nutrients in preventing necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is an intestinal disease that primarily impacts preterm infants. The pathophysiology of NEC involves a complex interplay of factors that result in a deleterious immune response, injury to the intestinal mucosa, and in its most severe form, irreversible intestinal necrosis. Treatments for NEC remain limited, but one of the most effective preventative strategies for NEC is the provision of breast milk feeds. In this review, we discuss mechanisms by which bioactive nutrients in breast milk impact neonatal intestinal physiology and the development of NEC. We also review experimental models of NEC that have been used to study the role of breast milk components in disease pathophysiology. These models are necessary to accelerate mechanistic research and improve outcomes for neonates with NEC

USP6 oncogene promotes Wnt signaling by deubiquitylating Frizzleds

Ubiquitin-specific protease 6 (USP6) is a deubiquitylase that is overexpressed by chromosome translocation in two human neoplasms, aneurysmal bone cyst and nodular fasciitis. The relevant substrates of this ubiquitin-specific protease are not clear. Here, we identify the Wnt receptor Frizzled (Fzd) as a key target of the USP6 oncogene. Increased expression of USP6 increases the membrane abundance of Fzd, and hence increases cellular sensitivity to Wnts. USP6 opposes the activity of the ubiquitin ligase and tumor suppressor ring finger protein 43 (RNF43). This study identifies a new mechanism for pathological Wnt pathway activation in human disease and suggests a new approach to regulate Wnt activity therapeutically

Differential Roles of the PKC Novel Isoforms, PKCδ and PKCε, in Mouse and Human Platelets

Background Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation. Methodology/Principal Findings In this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor. Conclusions These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI

Influence of telopeptides on the structural and physical properties of polymeric and monomeric acid-soluble type I collagen

Currently two factors hinder the use of collagen as building block of regenerative devices: the limited mechanical strength in aqueous environment, and potential antigenicity. Polymeric collagen is naturally found in the cross-linked state and is mechanically tougher than the monomeric, acid-soluble collagen ex vivo. The antigenicity of collagen, on the other hand, is mainly ascribed to inter-species variations in amino acid sequences of the non-helical terminal telopeptides. These telopeptides can be removed through enzymatic treatment to produce atelocollagen, although the effect of this cleavage on triple helix organization, amino acidic composition and thermal properties is often disregarded. Here, we compare the structural, chemical and physical properties of polymeric and monomeric type I collagen with and without telopeptides, in an effort to elucidate the influence of either mature covalent crosslinks or telopeptides. Circular dichroism (CD) was used to examine the triple helical conformation and quantify the denaturation temperature (Td) of both monomeric collagen (36.5 °C) and monomeric atelocollagen (35.5 °C). CD measurements were combined with differential scanning calorimetry (DSC) in order to gain insight into the triple helix-to-coil thermal transition and shrinkage temperature (Ts) of polymeric atelo collagen (44.8 °C), polymeric collagen (62.7 °C), monomeric atelo collagen (51.4 °C) and monomeric collagen (66.5 °C). Structural and thermal analysis was combined with high pressure liquid chromatography (HPLC) to determine the content of specific collagen amino acidic residues used as markers for the presence of telopeptides and mature crosslinks. Hydroxylamine was used as the marker for polymeric collagen, and had a total content of 9.66% for both polymeric and polymeric atelo collagen; tyrosine was used as the marker for telopeptide cleavage, was expressed as 0.526% of the content of polymeric collagen and the partially-reduced content of 0.39% for atelocollagen