Shreffler:Notebook/Milk Project

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Contents

Project Description/Abstract

Mechanistic Studies of patients enrolled in the baked milk study.

Team

Experimental Overview for Mechanistic Assays

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).
      1. RNA Extraction/RT-PCR: 8 x 106 PBMCs
      2. 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.
      1. CD25+ pre-depletion: 1 x 106 PBMCs (for flow)
      2. CD25- post-depletion: 1 x 106 PBMCs (for flow)
      3. CD25 Depletion Experiment: 28 x 106 PBMCs (a 30% recovery of CD25- cells will ensure 8 x 106 PBMCs for 7 day culture.)

Meeting Notes

* 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

  • 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.

Next meeting afternoon of 8/3 or 8/4? -- should address plan for acquisition of T cell data at that time

Analyses/ Presentations

Image:Example.jpg


References

  1. Nowak-Wegrzyn A, Bloom KA, Sicherer SH, Shreffler WG, Noone S, Wanich N, and Sampson HA. . pmid:18620743. PubMed HubMed [Nowak-Wegrzyn]
  2. Wanich N, Nowak-Wegrzyn A, Sampson HA, and Shreffler WG. . pmid:19348919. PubMed HubMed [Wanich]
All Medline abstracts: PubMed HubMed

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