Biomedical Engineering Reference
In-Depth Information
and procollagen I and III and decreases fibrosis in a mouse model of bleomycin-
induced lung injury.
137
These cytokines, IFN-γ, IL-12, and IL-18, have limita-
tions in vivo at moderate to high doses that can cause adverse effects
138
limiting
their therapeutic applications.
Using the cloned cytokine complementary deoxyribonucleic acid (cDNA)
under the regulation of a constitutive promoter, these have been applied as a
means of boosting in vivo production of specific cytokines. For example, IFN-γ
and IL-12 have proven effective as prophylactics and adjuncts in therapy against
diverse human diseases.
139
The transfer of an expression of IL-12 gene in mouse
airway cured airway eosinophilia and immunoglobulin E (IgE) synthesis.
140
Many other benefits from the use of IFN-γ, IL-12, and IL-18 gene therapy have
been shown in different animal models
141,142
but the use of lipofectamine to
transduce plasmids in mice are not directly applicable to humans because of
issues of toxicity.
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Kong and collaborators
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investigated the mechanism of cIFN-mediated
immunomodulation using the mouse OVA-allergic asthma model. The sum-
mary of some of the studies they performed are given below.
7.4.1 Protocol for the Preparation of Chitosan IFN-
γ
pDNA
NPs and Green Fluorescent Protein Test of Expression
In this study, Kong and collaborators,
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used mouse IFN-γ cDNA that was
cloned in the mammalian expression vector pVAX (Invitrogen, San Diego, CA)
which was complexed with chitosan as previously described.
143
The stepwise
process is as follows.
(1)
Plasmids in 25 mM Na
2
SO
4
and chitosan (Vanson, Redmond, WA) that
was dissolved in 25 mM Na acetate, pH 5.4, to a final concentration of
0.02% were separately heated for 10 min at 55 °C.
(2)
After heating mix the chitosan and the DNA (plasmid-encoding green
fluorescent protein [pEGFP]).
(3)
Vortex the mixture vigorously for 20-30 s.
(4)
Store at room temperature until use.
(5)
Take the Transmission electron microscopy (TEM) and Dynamic Light
Scattering (DLS) to verify that the NMs are 200-300 nm diameter.
(6)
Administer 10 µg of pEGFP intranasally to mice.
(7)
After 1, 3, and 7 days, euthanize the mice.
(8
) Collect the lungs and fix in buffered formalin.
(9)
Embed the whole lungs in paraffin, section, and examine for GFP by fluo-
rescent microscopy.
(10
) Estimate the approximate percentage of GFP-positive cells.
In the same study, mice were allergen-sensitized by intraperitoneal injection
of 50 µg of OVA (ovalbumin) adsorbed to 2 mg of alum which was followed
by an intranasal challenge with 50 µg of OVA after 1 or 2 weeks.
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Boosting
