Abstract
ABSTRACT
Dendritic cells (DCs) are professional antigen-presenting cells (APC) that can initiate immune response by priming naïve lymphocytes and polarizing them to give rise to Th1 or Th2 type responses.
In humans and other species, DCs progenitor cells, such as CD14+ monocytes, can be induced to differentiate in culture into DCs in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL4).
DCs circulate in the peripheral immune system as immature cells, they are able to uptaka antigens (Ag) through phagocytosis and endocytosis by specific endocytic receptors (mannose receptor (MMR)). After capture of the Ag, DCs are activated and migrate more efficiently to lymph nodes, going through a maturation phase.
Mature DCs (mDCs) are less capable of uptaking Ag, but are able to activate and polarize the cellular T immune response. Because Th1-type immune response have been connected to success in combating viral infections, a promising therapeutic application of DCs would be their differentiation in vitro following reinfusion into the host to boost an immune response.
Immunoterapy using ex vivo generated DCs has recently been shown to enhance protective immunity against simian immunodeficiency virus infection in macaques and in human immunodeficiency virus-infected humans (HIV).
Clinical studies have been performed in humans using tumor antigen-loaded
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Abstract
DCs generated ex vivo and reinfused into the same individual to induce an immune response against the malignant tumor.
Feline immunodeficiency virus (FIV) infection in cats has long been studied as a model for human AIDS, but very little is known on the role of DCs in this particular context. For generated DCs from peripheral blood monocytes, are estimated three differentes methods: purification by Percoll gradient, purification by MaCS and at last colture of cells adhering to plastic for 24h.
The purity and the cells numbers obtained on a Percoll gradient and the difficulty to differentiate monocytes purificated by maCS in medium containing 3% autologous plasma, led us to use adherence to plastic as a better method to generate DCs from feline PBMC.
Monocytes, derived fromperipheral blood, were test adherence to plastic for 24h in medium containing 3% autologous plasma, after several attemps done to better times for adhering cells (3, 6, 12, and 24h), and to tests better serum supplements and its concentration, in order to obtain the higher number of DCs.
To make sure that feline cells would make contact with as few foreign antigens as possible during in vitro culture, the use of FCS was avoided by using autologous plasma and only carrier-free feline recombinant cytokines were used when available.
DCs were generated from peripheral blood monocytes in the presence of feline IL4 and GM-CSF and after five days of colture,their maturation was induced alternatively with lipolisaccarid (LPS), human recombinant tumor necrosis factor (TNFα), Poly(I:C), activated feline platelets, CD40 ligand (CD40L) and
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Abstract
human recombinant interferon α (IFNα), because these stimula were proved to induce to maturation in DCs from other species.
After 48h, their CD14, CD1a, MHC class II, and B7.1 surface expression was analysed in parallel with their ability to uptake Ag by pinocytosis and endocytosis by MMR or prime mixed leukocyte feline reaction.
In the present thesis’s work, we are estabilished the optimal conditions to colture sizeable numbers of feline DCs for use in immunotherapy/vaccination.
Besides assess in our experiments what LPS resulted to be the best stimulus to induce efficient maturation of feline monocyte-derived DCs. Finally we set parameters predicting mDC ability to induce an a better immune response in order to establish a protocol to use feline DCs in vivo in vaccine/immunotherapy experiments. These parameters include MHC class II molecules, already expressed on iDCs (immature dendritic cells) and ten times higher on mDCs, and B7.1 expressed only on mDCs.
However, the capacity of DCs to be the best inductors of MLR is the best correlate for DCs maturation maturation.
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