Biomedical Engineering Reference
In-Depth Information
•
Synthesis of TNF-α (tumor necrosis factor alpha) and an oxygen intermediate such as nitric
oxide by macrophages activated by IFN-γ (interferon gamma)
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Activation of NK (natural killer) cells by IL-2 (interleukin 2)
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Adsorption of an antibody against tumor cell antigens targeting macrophages and cytotoxic
T lymphocytes.
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Activation of CD8+ T lymphocytes by an MHC(major histocompatibility complex) -
mediated cell contact mechanism between antigen-presenting cells (APCs) and CD8 cells
Several kinds of immunotherapy protocol are available both in human and veterinary
medicine [4, 5, 6, 7]. Heat shock proteins (HSPs) such as gp96 or HSPs70 which are synthesized
by the cells submitted to stress are advantageous vaccination adjuvants due to their chaperone
properties and their role in antigen presentation [8]. As chaperone molecules, almost all the
cell peptides are associated with these proteins and HSP purification provides a fingerprint of
the cell's protein synthesis [9]. This is particularly useful for cancer cells which synthesize
numerous abnormal proteins during their natural progress [10]. These cells being genetically
unstable, their abnormal protein synthesis differs from patient to patient and during the course
of the disease. The HSPs and their associated peptides (AAPs) have special receptors (CD91)
on dendritic cells which allow the internalization of the AAPs and their modification in order
to be expressed at the surface of class I HLA proteins on the cell membrane, triggering
activation of the CD8 T cells if they are abnormal [11].
Cancer cells are stressed by the mechanical and metabolic characteristics of the tumour. They
synthesize many HSPs [12]. We thus isolated these proteins in order to make an autologous
vaccine against the tumour. The HSPs were purified using a hydroxylapatite powder (HA)
column. The powder carrying the HSPs was then injected subcutaneously to stimulate the
immune system's response to the tumour.
Purification of HSPs using classical way is long and tedious. The use of hydroxylapatite
powder allows a much faster purification process. Hydroxylapatite chromatography has been
described by Tiselius in the early seventies. Hydroxylapatite chromatography is an adsorption
chromatography. The adsorption mechanism is very poorly understood. The surface of the
material is occupied bu Ca
++
and PO
4
-
. These ions are supposed to interact with the chemical
groups of the proteins. However, post synthesis treatment such as sintering or spray- drying
process modify the physico-chemical properties of the material surface. Furthermore, the
interaction of the material surface with biological fluids triggers epitaxial growth of carbonated
apatite at the surface of the material [13].
It was decided that the proteins purified will be injected carried by the particles for several
reasons: the hydroxylapatite particles have been described as vaccine adjuvant, they are
phagocytosed by the APCs and can deliver the proteins directly in the APCs, they trigger an
afflux of APCs at the injection site [14]. Most of the adjuvants used in antiinfectious vaccines
are nano or microparticular.
The aims of this study was to check the feasibility of this protocol using HA-particles with
dogs suffering of high grade lymphoma and to know if secondary effects were detected.
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