Protostrongylus stilesi, the parenchymal lungworm, has been attributed an important role in the widespread respiratory diseases of the Rocky Mountain bighorn sheep (Ovis canadensis canadensis). This study was conducted to delineate some of the immunologic and non-specific interactions between parasite and host. A procedure was developed for- the immunochemical quantitation of the ovine immunoglobulins IgG(1), IgG(2) and IgM and for the semi-quantitative analysis of IgA. This technique obviated, the preparation of highly specific antisera required for single radial immunodiffusion. The lungworm did not appear to be a significant part of the total antigenic environment of infected animals since no relationship between immunoglobulin levels and parasite activity was detected. Genetic influences were suggested in the levels of IgG(2) which remained at a relatively constant level characteristic of individual animals. The seromucoids were evaluated as correlates of the inflammatory reaction to parasitism. These proteins were useful in detecting changes in parasite activity and bacterial infection. Gastrointestinal disorders associated with severe scouring were accompanied by the disappearance of circulating orosomucoid (α-1 acid glycoprotein). The loss of this low molecular weight protein appeared to be related to vascular leakage. Lungworm infections induced the appearance of homocytotropic antibodies which could be detected in vitro by their ability to sensitize peripheral polymorphonuclear leucocytes for adherence to the larval cuticle. The elution of this antibody from sensitized cells and the inhibition of the adherence reaction with specific antiserum suggested that the reaction was mediated at least partly by IgG(1). The adherence reaction was used to assay homocytotropic activity of serum from infected animals (washed-cell test). This test was correlated with the ability of sera to sensitize skin for anaphylaxis. The effective hypersensitive response, accounting for both sensitizing and blocking activity, was determined by exposing normal cells to larvae in a medium containing serum (decomplemented-serum test). The results of this test paralleled inflammatory changes in parasitized animals, monitored by the levels of serum orosomucoid. Using this method, levels of homocytotropic and blocking antibodies were measured throughout the annual parasite cycle. Preliminary observations indicated that immunogenic inflammation, associated with the "spring rise" and "self cure," resulted from a shift in a dynamic balance between competing antibodies rather than a proportionate increase in homocytotropic activity. The response of peripheral lymphocytes to the presence of larvae or larval extracts was cursorily examined. Although ovine lymphocytes did not respond well enough in culture to draw definitive conclusions, the presence of larval extracts appeared to have a detrimental effect on cell survival and transformation. Even in the presence of EDTA, minute amounts of antibody sensitized lymphocytes for adherence to the larval cuticle. Sera from a wide variety of sources had an inhibitory effect on this reaction. Treatment of larvae with neuraminidase and high concentrations of NaCl engendered improved adherence. This suggested that Protostrongylus had adopted mechanisms similar to the trophoblast or the neoplastic cell which allegedly evade immunologic recognition with a strongly anionic coat. These aspects are discussed in terms of the possible role of Protostrongylus in respiratory diseases of the bighorn sheep, and the exploitation of molecular mechanisms in the control of parasitic diseases. / Land and Food Systems, Faculty of / Graduate

Class of 2017 Abstract / Objectives: Milk protein allergy is estimated to affect 1.2% to as much as 17% of people of all ages. Advair® Diskus® (FP/SX) utilizes lactose as an excipient which limits the utility of this product for this population. Furthermore, Advair® Diskus® is formulated as an interactive physical mixture via a micronization process. Alternatively, spray dried engineering achieves narrow particle size distribution, allowing greater deposition in the targeted respiratory bronchioles. The purpose of this dry powder inhaler (DPI) study was to conduct an in vitro comparative analysis of the aerodynamic performance of a co-spray dried lactose-free formulation of FP/SX with a mannitol excipient as a molecular mixture versus the Advair® Diskus® 250/50 (FP/SX) interactive physical mixture product. Methods: Utilizing mannitol as an excipient, a co-spray dried FP/SX powder was prepared using the Buchi Mini-Spray Dryer B-290 under closed system configuration. The resulting feed solution was spray dried at pump rates of 25%, 50%, and 100% with all other parameters remaining constant (aspiration, atomization rate, nitrogen gas rate). The primary outcome measure, aerodynamic performance, was assessed using the Copley Next-Generation Impactor (NGI). NGI data for the DPIs was used to calculate mass median aerodynamic diameter (MMAD), geometric standard deviation (GSD), and fine particle fraction (FPF) of each powder, including the Advair® Diskus®. Residual water content was quantified by Karl Fischer titration. Particle characteristics were visualized by scanning electron microscopy. Results: FPF, MMAD, and GSD were calculated from NGI data; Wolfram Alpha software was used to calculate MMAD and GSD. T-test regression was used for comparative analysis of spray-dried and Advair® Diskus® powders. MMAD for each spray dried sample was analyzed using a t-test regression against the MMAD values from the Advair® Diskus®. Using aerodynamic analysis studies triplicated for each powder, there was no significant difference between the spray dried powder and Advair® Diskus® for MMAD and GSD (p-values >0.05). The 50% and 100% pump rate samples had similar FPF to the Advair® Diskus® (p-values >0.05). However, the 25% pump rate sample had a significantly improved FPF compared to the Advair® Diskus® (p <0.01). Conclusions: A co-spray-dried lactose-free formulation of FP/SX with a mannitol excipient demonstrated similar aerodynamic performance to the Advair® Diskus® which consists of a physical mixture of two drugs with lactose. Of significance, 25% pump rate spray-dry conditions demonstrated an improved FPF compared to the Advair® Diskus®.

Introduction: Fungi are common aeroallergens responsible for at least 3% – 10% of allergic diseases worldwide, with the proportion hugely variable in different populations. Treatment is complicated by viable nature and disease causing ability of the allergen and is often only palliative. Thus, this study aimed to serve as a pilot investigation to design novel anti-allergy therapeutics to cure allergy at the molecular level. It investigates the effect of wild type fungal peptides and corresponding variant peptides on allergy associated immunological responses – cellular and cytokine based – to use such variant peptides to cause the delicate shift from an allergic to a normal immune response. Further, the study explores the role of bioinformatics in investigating allergy and designing novel therapeutics. Methods: This study used ProPred, a bioinformatics software, to predict wild type peptides from selected allergens of Aspergillus fumigatus and Alternata alternaria for a target population. These were then modified to generate single amino acid variants. Both these peptide sets were tested to compare the cellular and cytokine patterns they generated in sensitised (n = 3) and healthy volunteers (n = 3) to check for anti-allergy responses that may be exerted by certain variants. The recruited population was also subjected to skin prick testing (SPT, n = 46) to check for co-sensitisations patterns and HLA typing (n = 40) to evaluate ProPred accuracy for peptide prediction. This study also attempted an in silico search for unknown Penicillium chrysogenum allergens by comparing known Penicillium and A. fumigatus allergens to identify probable agents of co-sensitization. Results: Of the wild type and variant peptides tested in this study, one variant peptide – peptide 1.1v from Asp f 2 – was successfully identified to change the cellular and cytokine profile to promote an anti-allergic response when compared to its corresponding wild type form (1.1o). This candidate is a good target for further investigation for use in peptide immunotherapy. Further, 8 shared allergens between A. fumigatus and P. chrysogenum were identified that may possibly be agents of co-sensitization between these species. SPT results indicated maximum subject co-sensitization between A. fumigatus and Candida albicans and P. chrysogenum. HLA typing results demonstrated the efficiency of ProPred to be 96.29%, thus implying that bioinformatics can effectively be used to study allergy in this novel manner. Conclusion: This study has demonstrated that variant peptides with a single amino acid change can cause the delicate shift from an allergic to a healthy immune response in sensitised subjects. This approach – in combination with other allergy associated factors such as epitope specificity for HLA types and inherent co-sensitization patterns in a population – can effectively be used to design peptide candidates for immunotherapy to target allergy at the molecular level. With promising results obtained in this pilot study, this approach guarantees further investigation in immunotherapy. This study has also demonstrated that bioinformatics can be effectively used to design and execute allergy studies in a targeted and inexpensive manner.

The Interleukin (IL)-17 cytokine family, expressed by T helper (Th)17 cells and γδ T cells, plays pivotal roles in adaptive immune responses. They have been implicated in autoimmune and allergic diseases as well having roles in bacterial and fungal clearance. Importantly, IL-17 producing γδ T cells have been shown to be critical for the development of adaptive Th17 responses in the murine model of multiple sclerosis, experimental autoimmune encephalomyelitis. Interestingly, natural ligands of the aryl hydrocarbon receptor (AhR) are known to influence the development of Th17 cells. It has been shown previously that prolonged topical exposure of mice to the contact allergen 2-4 dinitrochlorobenzene (DNCB) or to the respiratory sensitiser trimellitic anhydride (TMA) causes the preferential development of a preferential T helper (Th)1 or Th2 response, respectively. The presence of the novel IL-17 family cytokines and their cellular source was investigated following both single and prolonged exposure to allergen. Exposure only to the contact allergen DNCB resulted in up-regulation of the expression of IL-17 by dermal γδ T cells. It was shown also that topical application of a range of contact allergens and of the respiratory allergen TMA resulted in IL-17 expression by γδ T cells in the lymph node draining the site of exposure. However, differential kinetics were observed between the two classes of allergens. Exposure to the contact allergens resulted in rapid expression of IL-17 within 6-16 h, whereas the respiratory allergen displayed considerably delayed kinetics, with maximal levels detected 48 h post exposure. Treatment with DNCB only was shown to be associated with the development of Th17 cells following prolonged exposure to chemical allergen. Thus DNCB provoked a Tc1/Th1/Th17 profile, in contrast prolonged exposure to TMA resulted in a very selective Th2 cytokine pattern. The influence of γδ T cells on polarised responses to chemical allergens was investigated also using γδ T cell knockout mice; here the adaptive Th17 response induced by DNCB was completely abrogated. These data demonstrate that the absence of IL-17 production by γδ T cells during the early innate immune response affects the subsequent adaptive Th17 response stimulated by chemical contact allergens. Finally, the importance of the affinity of the AhR for endogenous ligands during in vitro Th17 polarisation was assessed. Using three strains of mice with differential AhR affinities the contribution of ligation of these receptors in Th17 cell development was investigated. In all three strains AhR ligation was required for optimal polarisation of Th17 cells, even in strains that are reported to express a low affinity receptor. These data suggest that across a range of receptor affinities, including low affinity receptors analogous to that of humans, endogenous AhR ligands may play a major role in driving Th17 cell differentiation, regardless of receptor phenotype.

M.Tech. / Cat allergy can be defined as an abnormal immune response by the body to cat dander or cat saliva, which is considered to be a harmless environmental agent. In individuals who display a hypersensitivity to cats, exposure can manifest in a complex of symptoms which can negatively influence their normal day to day functioning. Conventional treatment for cat allergy comprises of antihistamines and decongestants, which have many adverse effects and in many patients prolonged use can result in the development of a tolerance to that particular drug. To date no research has been conducted on the homoeopathic complex of Cat saliva 9cH and Histaminum 9cH for treating the symptoms of cat allergy. The aim of this study was to determine the efficacy of Cat saliva 9cH and Histaminum 9cH in treating the symptoms of cat allergy, by use of an allergy skin prick test and a symptom score card. The complex of Cat saliva 9cH and Histaminum 9cH was prepared according to homoeopathic principles and prescribed according to isopathic principles. Thirty participants between the ages of 18 and 45 both male and female, who all had a positive test result for an allergy skin prick test specifically to cat allergen, were selected for this four week, double blind, placebo-controlled study. During the conduction of the skin prick test the wheal diameter, flare reaction and degree of itchiness were measured at the beginning of the study (week 1) and at the end of the study (week 4). The participants were randomly divided into the experimental and control group. The control group received the placebo and the experimental group received the homoeopathic complex of Cat saliva 9cH and Histaminum 9cH. Participants were asked to take two tablets sublingually in the morning and two tablets at night for the duration of the study period (4 weeks). Each participant received a score card which rated the severity, frequency and duration of their symptoms and was completed at the end of each week. The results were statistically analysed using the Mann-Whitney U Test, the Friedman-K Related Samples Test, the Wilcoxon Signed Ranks Test, Independent-Samples T-Test and descriptive statistics. The results showed that the iv homoeopathic complex of Cat saliva 9cH and Histaminum 9cH had improved all symptoms in the experimental group, however when compared to the control group was only found to be statistically significant in improving the severity of the symptom “runny itchy stuffy nose”, the frequency of the symptoms “red itchy eyes”, “runny itchy stuffy nose” and “sneezing”, as well as improving the duration of the symptom “runny itchy stuffy nose”.

Identification et caractérisation d'une population d'ILC2 associée à la sévérité de la rhinite allergique et de l'asthmeTrois catégories de cellules lymphoïdes innées (innate lymphoid cells, ILC) ont été décrites récemment sur la base de leurs phénotypes et leurs caractéristiques fonctionnelles : les ILC1, ILC2 et ILC3. Les ILC2 semblent avoir un rôle pro-inflammatoire important dans l’allergie en raison de leur capacité à produire de grandes quantités de cytokines TH2.Pour mieux comprendre le rôle de ces cellules dans l’allergie respiratoire, nous avons comparé les ILC sanguines de patients atteints d’une rhinite allergique associée ou non à un asthme, à celles de sujets non allergiques. Cette étude révèle de multiples différences fonctionnelles entre les ILC circulantes de sujets sains et allergiques. Notamment, la fréquence d’ILC2 exprimant le récepteur aux chimiokines CCR10 est augmentée dans le sang de patients asthmatiques sévères.CCR10 pouvant permettre le recrutement des ILC vers les organes cibles, le rôle des ILC2 CCR10+ dans la physiopathologie de l’asthme a été étudié. Leur présence dans les poumons humains a été observée. Des analyses fonctionnelles et phénotypiques ont révélé que cette sous-population cellulaire était peu activée mais présentait une plasticité leur conférant des caractéristiques proches des ILC1. La déplétion de ces cellules dans un modèle murin d’asthme allergique aggrave l’hyperréactivité bronchique.Les travaux de cette thèse documentent le rôle des ILC dans l’asthme. En particulier, la fréquence sanguine d’ILC2 CCR10+ augmente avec la sévérité de la maladie. Les résultats obtenus dans les modèles animaux suggèrent que ces cellules auraient un rôle bénéfique dans le contrôle de l’asthme. La voie du CCR10 pourrait représenter une nouvelle cible pour le développement de traitements innovants contre l’asthme ou une source prometteuse de biomarqueurs. / Identification and characterization of an ILC2 subset linked to allergic rhinitis and asthma severityInnate lymphoid cells (ILCs) have been classified into ILC1, ILC2 and ILC3 subsets based on their respective phenotypes and functions. Considering the strong ability of ILC2s to produce TH2 cytokines, these cells likely play a significant role in allergic diseases.To better understand the role of these cells in respiratory allergies, we compared blood ILCs from allergic patients with or without asthma to non-allergic individuals. Together our results show multiple functional differences between ILC from allergic and healthy subjects. In particular, ILC2s expressing the chemokine receptor CCR10 are specifically enriched in the blood of patients with severe allergic asthma.Considering that CCR10 could allow the recruitment of ILCs to target organs, the role of CCR10+ ILC2s in asthma physiopathology has been studied. This ILC2 subtype is present in human lungs. Functional and phenotypic analyses revealed that these cells are less activated than other ILC2s and show ILC1-like properties. CCR10+ ILC2s depletion in a mouse model of allergic asthma exacerbate airway hyperreactivity.Together, this work documents the role of ILCs in asthma. Specifically, circulating CCR10+ ILC2 frequency increases with asthma severity. The results obtained in mouse models suggest that these cells could have a beneficial role in asthma control. CCR10 pathway could represent a new target to elaborate breakthrough treatments against asthma or a source of promising biomarkers.

No description available.

Exercise-induced anaphylaxis (EIA) is a rare severe disease in which patients express asthmatic and allergic symptoms. Little is known about EIA and its pathology. This manuscript presents an hypothesis that combined hormonal (estrogen and progesterone) contraceptive use and stress during puberty alter the immune system and predispose the adult female to EIA. Presented here is what is known about asthma, a much more common disease, and a pilot, experimental paradigm in which EIA is induced in Syrian hamsters. Asthmatic and allergic cases are much more prevalent in pubescent and adult females than in adult males. Women express higher levels of lung inflammation at stages of their lives when estrogen and progesterone levels are at their lowest (i.e., the follicular phase of the menstrual cycle and menopause). Combined birth control pills have been utilized by doctors to treat asthmatic women. Contraceptive treatments maintain steady levels of estrogen and progesterone throughout the menstrual cycle. It is hypothesized that if female hamsters are given constant levels of hormones as well as ovalbumin and exercise challenges during puberty and then have the hormones taken away during adulthood, they will produce abnormal lung sounds and corresponding pathological histology. To test this hypothesis, female Syrian hamsters were treated with ovalbumin, exercise challenge, both and none (the control). They were also treated to maintain constant levels of estrogen and progesterone during puberty. Although the results were inconclusive, the model may demonstrate that constant ovarian hormones, ovalbumin sensitization, and exercise challenges permanently strain the immune system of females in adulthood.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Asthma is a chronic inflammatory lung disease with intermittent flares predominately mediated through memory T cells. The majority of the T cells in tissues such as the lung are tissue-resident memory (Trm) cells, defined as cells that maintain long-lasting presence in the tissue and have rapid functional recall following challenge. Allergen-specific CD4 T helper cells that secrete the cytokine IL-9 have been shown to be a necessary component of asthma pathogenesis. However, the precise characterization and function of IL-9-secreting CD4+ cells (Th9 cells) are unknown. Here we demonstrate that IL-9 production is progressively lost in Th9 cells over several rounds of culture and that environmental cues dictate the instability or effector function of the Th9 phenotype. We show Th9 cells are long-lived tissue-resident cells with the capacity to rapidly respond to secondary allergen challenge causing allergic airway disease (AAD). We found in a memory model of Aspergillus fumigatus challenge, Th9 cells maintain tissue residency throughout a 12-week period of antigen-free rest. Additionally, we demonstrated increased frequency of IL-9-producing cells and quantity of IL-9 upon rechallenge, characteristic of a secondary response. Antibody blockade of IL-9 immediately prior to the recall challenge significantly reduced overall allergic lung inflammation, suggesting that IL-9 plays an obligate role in the allergic memory response following pulmonary allergen challenge. The protection afforded by IL-9 antibody blockade was not seen in a chronic model asthma-like disease demonstrating IL-9 has a specific role in allergic memory responses. Interestingly, IL-9-secreting cells have a polyfunctional multi-cytokine phenotype demonstrating a highly pathogenic state that we reproduced in culture. These observations suggest that IL-9 from Trm cell populations and Th9 cells play a novel role in allergen recall responses and are potential therapeutic targets for patients suffering from chronic intermittent asthma. / 2022-05-05