Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003
Performing Department
Dept: Food Sciences
Non Technical Summary
Cassandra Suther is a PhD candidate at the University of Massachusetts Amherst. She splits her time between two labs, Dr. Moore's at the University of Massachusetts, and Dr. Zhou's at University of Connecticut Health. The projectincludes a truly interdisciplinary training opportunity, with an equally interdisciplinary associated scientific project.The candidate will be trainedin ideas and techniques not typically found in food science or medical departments alone to advance her career goals, along with mentoring in her pursuit of a career in institutional or governmental human health research. This will include 15hrs ofworkshop, short courses, seminars and technical talks, conference attendance, updated IDP and frequent meetings with mentors and collaborators.A research project is also included to further her proficiency as a young scientist.Food allergies are increasing at an alarming rate, with 10% of Americans effected. Some reports suggest infection with enteric viruses could result in effects that enhance immunological tolerance of potential allergens. Human norovirus is the leading cause of foodborne illness and has been associated with immunological changes in the gut that could promote tolerance to allergens. Similarly, it has been demonstrated to interact with host commensal bacteria; however, the effect norovirus infection directly has on the immune system and microbiota with respect to food allergies has not been directly investigated. Using bothnorovirus and food allergy mouse models, we plan to investigate the effects norovirus has on both the development and severity of food allergies.Aim 1 will include inoculation of norovirus before the food allergies has been sensitized (development). Aim 2 will observe the effect of inoculation of norovirus after sensitization has occurred (severity). Elucidating the relationship between norovirus, food allergies and the gut microbiome could better inform treatment and understanding of food allergy.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
This projects provides an opportunity for Cassandra to strengthen her unique backgrounds, works, and interests to address national health challenges in nutrition and health through her continuing education and future career through an interdisciplinary research project. The overall goal of this project is to train the candidate through mentoring, career, and research objectives.Mentoring and career training We propose to train Cassandra in ideas and techniques not typically found in food science or medical departments alone to advance her career goals, along with mentor in her pursuit of a career in institutional or governmental human health research.Main objects to be observed in her mentoring/career training will include-15hrs (7-8hrs halfway through the project) of workshops, short courses, and technical talks given at both UMass and UConn.1-2 conferences attended per year.An updated IDP once per year.Yearly meeting with committee members.Weekly meeting with each mentorMonthly meetingwith both mentorsBoth mentors will assist the student with job prospects, including advice, information on different branches (industry, government, or academia) and help with applications, cover letters, presentations, and interviews.Research PlanThe goal of this project is to elucidate the relationship between norovirus, food allergies and the gut microbiome to better inform treatment and understanding of food allergy.The following objectives will be used to measure progress on the research project- Well-established model and positive controls (Y1)Completion of Aim 1's data collection. (Y1)Completion and conclusion of the Aim 1. (Y1-2)Completion of Aim 2's data collect. (Y2)Completion and conclusion of the Aim 2. (Y2)The manuscripts preparation. (Y2)
Project Methods
Objective 1 will cover the sensitization period of a food allergy. This is an important part of allergy prevention, as allergic reactions cannot occur without first sensitization. We will do this by simultaneously inoculating mice with murine norovirus (D -2) while sensitizing them with ovalbumin (OVA).Objective 2 will interrogate the effect norovirus has on an already established OVA allergy. We will do this by simultaneously inoculating mice with murine norovirus (D14) while challenging them with ovalbumin (OVA).Both objectives use similar techniques, sampling, and analysis. The main different is the timing of norovirus infection. As such, methods below are for both. Mice: C3H/HeJ female mice (8 weeks old) will be used, as this strain and sex is commonly used in food allergy model. The gut microbiome may be transferred from mother to offspring in mice colonies, so to increase similarities between microbiome, mice and their bedding will be mixed for two weeks following the start of the experiments.Food allergy and murine norovirus models: For sensitization, 5 mg of OVA with 10 μg per mouse of cholera toxin, as adjuvant, will be used via intragastric gavage on day 0 and day 7. On day 14, 10 mg of only OVA will be gavaged into mice, divided into 2 doses at 30 to 40-minute intervals, with sampling within 1 hour. An acute norovirus strain, MNV-1, will be used as previously reported31. Mice will be infected via oral gavage with 3×107 plaque forming units in 250μl PBS.Antibiotic procedure: For mice in groups with a depleted microbiome, an antibiotic cocktail of Bacitracin 0.5 mg/ml, Neomycin 2 mg/ml, Vancomycin 0.2 mg/ml, and Pimaricin 1.2 μg/ml will be prepared and gavaged into mice six times over a two week time prior to murine norovirus infection or OVA sensitization..Sample collection: Stool for microbiome sequencing and viral titer will be collected at baseline, 3 days after MNV-1 infection, after the total sensitization period, at the end of the experiment and stored in -80 before DNA or RNA extraction. Diarrhea patterns and weight loss will be measured. Viral load will also be determined 3 days post inoculation and before challenge (to verify a cleared infection). Blood samples, via cheek bleed, will be taken on day 7 and then on day 14 before and after the challenge for IgE measurements. Anaphylactic symptoms, via histamine levels, anaphylactic scoring, and temperature, will be taken following challenge. Th2 cytokine and Treg measurements will be reported via flow cytometry for each blood sampling time point. Intestines will be harvested and sent to the histology core location on UConn Health campus for inflammation scoring.Microbiome and SCFA measurement. Stool samples will be sent for 16s sequencing with a subset of samples being subjected to metagenomic sequencing. Total genomic DNA will be extracted from stool. For 16s analysis, the V1 to V3 regions of 16S rRNA gene will be amplified (primers 27F and 534R32), barcoded, and then sequenced on the Illumina Miseq (2 × 300 bp) and Hiseq (2 × 250 bp), available at UMass and UConn sequencing cores. Nine types of SCFAs (acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic, caproic, and heptanoic acid) will be quantified at all time points by gas chromatography (GC)-mass spectrometry (MS) at the Host-Microbiome Center at Brigham & Women's Hospital.Analysis plan. To investigate a correlation between the microbiome data, norovirus infection, and allergic sensitization/reaction, we will apply linear regression models, after adjustment for gender, weight, and possible litter effects. For microbiome taxa data, we will only focus on the bacteria with relative abundances of >0.5% to decrease the possibility of high false discovery rate. Power Calculation: Diversity is a common parameter that measures microbial community complexity. Sample size of 10 per group will have a >90% power to detect the microbiome difference as described above using a two-sided Wilcoxon rank-sum test assuming a significance level of 0.05.Interpretation of Results: Lower levels of allergic immune marks, including IgE, cytokine measurements, histamine, temperature, anaphylactic scoring, and inflammation scores on gut tissues will provide evidence that virus/host interactions or secondary changes to bacteria (which 16s/SCFA analysis will show us) may affect allergic sensitization.The completion of ourmilestones will be used a measurement of our effots-Well-established model and positive controls (Y1)Completion of Aim 1's data collection. (Y1)Completion and conclusion of the Aim 1. (Y1-2)Completion of Aim 2's data collect. (Y2)Completion and conclusion of the Aim 2. (Y2)The manuscripts preparation. (Y2)