Biology Reference
In-Depth Information
restoration of tissue homeostasis. To this end, rapid clearance of
Ad from circulation by Kupffer cells may have a protective role
against the dissemination of Ads to lymphoid organs, therefore
reducing systemic infl ammation. In several gene therapy clinical
trials, serum levels of IL-6, IL-10, and IL-1 were elevated [
7
-
10
]
after systemic Ad administration at high doses (2 × 10
12
to 6 × 10
13
virus particles). However the role of these cytokines in the initia-
tion of an immediate innate immune response remains unclear.
A histological evaluation of tissues, including lung, liver, and
spleen, revealed areas of leukocyte and neutrophil infi ltration as
well as infarcts ([
11
,
12
] and our unpublished observation), indi-
cating that most tissues in the body are involved in the infl amma-
tory response to Ad after its intravascular administration. It has
been widely accepted for a long time that Ad-mediated liver
damage plays a central role in the pathogenesis of acute systemic
infl ammation caused by intravenous Ad administration. Specifi cally,
it has been found that the activation of MIP-2 chemokine is critical
for neutrophil attraction to liver tissue, and the inactivation of
MIP-2 with an anti-MIP-2 antibody ameliorates liver pathology
after intravenous Ad administration [
12
].
In this chapter, we focus on the analysis of infl ammatory cyto-
kine and chemokine activation using ProteomeProfi ler™ antibody
arrays (R&D Systems, Minneapolis, MN). We successfully used
Mouse Cytokine Array panel A (Cat. No. ARY006) for simultane-
ous qualitative evaluation of expression for 40 different infl amma-
tory cytokines and chemokine in mouse tissues, serum, and
plasma. Here, we also describe the approach to adapt this assay for
quantitative analyses of the amounts of select infl ammatory cyto-
kines and chemokines in mouse spleen after intravenous Ad
administration [
6
,
13
].
2
Materials
1. Phosphate-Buffered Saline (PBS), 20 mM NaCl, 2.68 mM
KCl, 10 mM Na
2
PO
4
, and 1.76 mM KH
2
PO
4
(pH 7.4).
2. PBS with protease inhibitors: 10
2.1 Standard
Qualitative Cytokine
and Chemokine
Analysis
μ
g/mL Aprotinin, 10
μ
g/mL
Leupeptin, and 10
μ
g/mL Pepstatin.
3. Triton
®
X-100.
4. Pipettes and sterile pipette tips.
5. Gloves.
6. Deionized water.
7. Rocking platform shaker.
8. Table-top microcentrifuge.
9. A plastic container with the capacity to hold 50 mL (for wash-
ing the arrays).
