Transcript CR003ms-MNM

• Would be constructed for submission to the Journal of Clinical
Investigation, Impact Factor = 15; (Alternative, J. Exp. Medicine)
• Calvano, Lowry, and Xiao would put together the first draft (S &
S, Intro & Methods, Tables 1-2, Fig 1, 11 Jul 05; LLM revision,
15 Jul 05)
• Thrust would be an extension of the CR001 Nature publication, with
further development of the hypotheses put forth in that manuscript
• Would be a set up for, or simultaneously with the G029 and G035
manuscripts, and thus the focus on specific functional modules
needs to be considered in the context of the changes observed in
the trauma patients
• Results would be an overall description of the genomic responses by
the three cell types (and mixed cell populations) by clustering,
following by Venn diagrams showing overlap in probe sets that
changed in response to LPS, followed by pathway analysis of
common and uniquely expressed genes
• The lack of complete 2 hr data, and the reduced enrichment of
monocytes at 6 hrs would require that less emphasis be placed on
the timing of changes, and more on the qualitative aspects of the
changes
• The dominant means of extracting biological information from the
data set would be by pathway analysis, rather than by canonical
analysis
• Looking first at the subset of 132 genes whose expression
changed concordantly, Wenzhong and Steve would look to
see if there were common pathways that were affected in all
three populations (Wen, gene list and pathways, 12 Aug 05)
• A similar nonstatistical approach would be employed using
the "Magic Cell". Dr. Moldawer would obtain from
Wenzhong the gene lists, and would create "Magic PMNs",
"Magic Monocytes" and "Magic Lymphocytes" (Wen, gene
list, 10 Aug 05)
• Wen should create from the overall interactome revealed in the
CR001 Nature paper, individual interactomes based on the PMN,
MO and T-cell data. The functional modules (9-10) identified in the
CR001 paper should be re-explored using the cell specific gene
expression data
• Unique genes whose expression varied in specific cell populations
would also be subjected to pathway analysis with the intent being
to identifyi unique functional modules
• For the T-cell specific pathways, Carol could provide unique
insights into potential functional consequences for the changes in
this cell population
Table 1. Differential cells counts over time to demonstrate the
endotoxin-induced changes in lymphocytes, monocytes, and
granulocytes
Table 2. Leukocyte enrichment as assessed by flow cytometry.
Time
After
Endotoxin
0 Hrs
2 Hrs
6 Hrs
RossetteSep T-cells
95 ±81,2
96 ±62
86 ±32
RossetteSep Monocytes
80 ±53
QNS4
67 ±93
Ficoll-Dextran Granulocytes
96 ±15
96 ±25
99 ±15
Enrichment Method
1Data
represent the Mean ±SEM; 2percent side scatter low, CD2 high;
3percent side scatter intermediate, CD33 high; 4quantity not sufficient
for microarray analysis due to monocytopenia (see Table 1); 5percent
side scatter high, CD66b high
Lysis
-2
2 6h
Neutrophil Monocyte
-2 2 6h
-2 6
h
Figure 1
T cell
-2 2 6h
CR003 Cell Specific
Response
Tcells
Monocytes
1834
3064
Unique
1256
171
Unique
1750
152
255
991
Unique
4070
Neutrophils
5468
Significant Genes (FDR < 0.01)
Common Genes of N/M/T cells at 6 hrs
Number of probe sets at 1% FDR:
82 down, 50 up
Number of probe sets at 5% FDR:
336 down, 144 up
Number of probe sets at 10% FDR:
621 down, 260 up
Figure 2
HLA II
CR003 lysis
CR003
Neutrophil
CR003 MO
CR003 T