Shreffler:Notebook/Milk Project

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Project Description/Abstract
Mechanistic Studies of patients enrolled in the baked milk study.

Team

 * Lara Ford
 * Alyssa Chase

Subjects

 * 180 total subjects
 * 120 active subjects
 * 60 control subjects

Time Points

 * 0 months (baseline)
 * 3 months
 * 6 months
 * 12 months
 * 18 months

Mechanistic Assays

 * Basophil Activation
 * RNA Extraction and RT-PCR (48 hour culture)
 * T Reg Studies (7 day culture)
 * CD25 Depletion Experiments (7 day culture)

Work Flow

 * 0 months (baseline): Basophil Activation, RNA Extraction and RT-PCR (48 hour culture), T Reg Studies (7 day culture)
 * 3 months: Basophil Activation, RNA Extraction and RT-PCR (48 hour culture), T Reg Studies (7 day culture)
 * 6 months: Basophil Activation, T Reg Studies (7 day culture), CD25 Depletion Experiments (7 day culture)
 * 12 months: RNA Extraction and RT-PCR (48 hour culture), T Reg Studies (7 day culture)
 * 18 months: Basophil Activation, T Reg Studies (7 day culture), CD25 Depletion Experiments (7 day culture)

Baseline (0 Month) Visits

 * At each baseline study visit, basophil activation, characterization of T-helper phenotype (48 hour culture) and frequency of milk-specific regulatory T cells (CD4+ CD25+ T reg) (7 day culture) will be assessed.
 * Assays performed with each sample (active and controls) will include:
 * Basophil Activation
 * RNA Extraction and RT-PCR (48 hour culture)
 * T Reg Studies (7 day culture)
 * Each subject will have two green top tubes (approximately 18 mL) of heparinized whole blood drawn for the mechanistics studies.
 * 3 mL of whole blood will be used for the basophil activation assay.
 * Remaining 15 mL of whole blood will be used to derive PBMCs for 48 hour and 7 day cultures (RNA Extraction/RT-PCR and T Reg Studies).
 * RNA Extraction/RT-PCR: 8 x 106 PBMCs
 * T Reg Studies: 8 x 106 PBMCs

Future Considerations

 * A subset of samples will undergo:
 * CD25 Depletion Experiments (7 day culture) – goal is to have data on approximately 30 subjects with an equal number in each randomization group (slow escalation [15] vs. rapid escalation [15])
 * In order to perform the CD25 Depletion experiment on a particular sample, you will need an additional 30 x 106 PBMCs.
 * CD25+ pre-depletion: 1 x 106 PBMCs (for flow)
 * CD25- post-depletion: 1 x 106 PBMCs (for flow)
 * CD25 Depletion Experiment: 28 x 106 PBMCs (a 30% recovery of CD25- cells will ensure 8 x 106 PBMCs for 7 day culture.)

* 15:43, 11 November 2009 (EST):

 * reviewed BM001 and BM068 T reg data
 * some samples have used Aqua live/ dead and some Violet live/ dead
 * will run side by side violet and aqua stained cells
 * reviewed to ensure good FoxP3 staining
 * Plan to acquire 10 more, talk again by phone in two weeks.

*15:02, 26 October 2009 (EDT):
Met with Lara
 * Discussed plan to pursue FAAN grant application -- Deadline 12/1
 * Training plan
 * Agreed that training plan could build on Lara's strengths of epi/ math and still complement the cellular immunology
 * Investigate R course
 * Investigate Clinical Research program
 * Research plan
 * Agreed to focus on basophil assay (perhaps t cells)
 * Could get T cell data in time for grant if Alyssa and Emily run
 * Statement
 * Draft by first week of November

*18:02, 31 July 2009 (EDT):
Met with Lara & Alyssa '''Next meeting afternoon of 8/3 or 8/4? -- should address plan for acquisition of T cell data at that time'''
 * Lara has reviewed clinical database report
 * several instnaces noted of apparent protocol deviations or missing data
 * Lara will pull charts and review/ update
 * Lara to present to division lab meeting on 8/18
 * Lara will update database, update serology data
 * Alyssa and Lara will complete primary (FlowJo) analysis of basophil data acquired to date
 * Wayne will write/ edit R script to compile that data
 * Wayne suggested that Lara organize monthly(?) meeting with Beth, Jessica, Alyssa, Lara, Wayne to provide feedback on study progress, data integrity, etc.