The innate or non-specific immune system provides immediate defence reactions against invading pathogens. Granulocytes, monocytes/macrophages, NK-cells, mast cells and dendritic cells are important cellular constituents of the innate immune system.
Cells of the innate immune system do not possess antigen-specific receptors but instead recognize conserved molecules expressed by large numbers of microorganisms. These conserved molecules are referred to as pathogen-associated molecular patterns or PAMPS and the corresponding receptors on cells of the innate immune system are called pattern-recognition receptors or PRRs.
Toll-like receptors (TLR) are an example for PRRs. TLRs recognize different types of molecules expressed by bacteria, viruses, fungi and protozoa. Extensive research has allowed correlating TLRs with the molecules they recognize: e.g. the endotoxin LPS, which is widely expressed by gram negative bacteria, is recognized by TLR 4, while unmethylated CpG-DNA sequences, which distinguishes bacterial DNA from mammalian DNA, is recognized by TLR-9.
FOCUS Immunology has established a complete range of assays to functionally analyse the triggering of TLRs and the subsequent activation of the respective cell.
Ligands for the 9 well characterized human TLRs (TLR1 – TLR9) are used to stimulate Peripheral Blood Mononuclear Cells (PBMC) in vitro and analyze the secretion of the cytokine TNF-alpha.
This assay allows analyzing the effect any compound, whether NCE or NBE, may have on TLR-mediated stimulation of cells of the innate immune system.
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The advent of biologicals has brought new challenges to drug development. Proteins with immunomodulatory activity such as monoclonal antibodies directed against major switches of the immune system or proteins such as receptor antagonists or agonists may have dramatic effects on immune cell function.
The recent past has shown just how dramatic these effects may be for the well-being of – in this case – volunteers in a clinical study.
The ensuing investigation of this incident resulted in committee recommendations among which the use of human ex vivo / in vitro cultures had a prominent place. This is based on the rationale that biologicals are very often species-specific and thus traditional animal models are in all likelihood not adequate to investigate effects on the immune system.
FOCUS Immunology offers a range of assays to assess the effects of biologicals on different aspects of immune cell activity, which allow a comprehensive analysis of possible immunomodulatory activities.
Specifically, the following assays are provided by FOCUS Immunology : (i) cytokine release, (ii) cell proliferation, (iii) expression of activation markers, (iv) oxidative burst reaction, (v) chemotaxis, (vi) NK cell activity, (vii) cytotoxicity (CDC, ADCC), (viii) NO production, (ix) histamine release, (x) changes in the frequency of defined cell populations.
It may be advisable to test not only resting cells but also cells after suboptimal stimulation in order to account for activation processes which may not suffice by themselves to activate resting cells but may strongly augment activation processes occurring independently.
It also may be advisable to test different means of presenting the tested biological to the cells, from soluble to different types of surface-bound presentation.
There is a medical need for immunostimulatory compounds. In particular novel cancer therapies aim at boosting the immune system to attack and destroy tumors in afflicted patients. CpG-oligos are one result of these research and development efforts.
FOCUS Immunology has established in vivo as well as ex vivo assays to assess the immunostimulatory capacity of compounds.
The ex vivo models provided by FOCUS Immunology allow to explore stimulatory activities at the level of proliferation, cytokine secretion and expression of activation markers in isolated lymphoid or myeloid cells. Defined cell populations may be purified from peripheral blood and used in the respective assay systems. The read-out systems may be at bulk culture level or single cell level.
FOCUS Imunology’s in vivo model for immunostimulation testing uses a suboptimal immunization schedule in mice. Animals are immunized with selected peptides in the presence or absence of the test-compound and at specified points in time the extent of the ensuing immune response is analyzed by measuring the frequency of antigen-specific responding cells upon in vitro restimulation with peptide in the EliSpot assay and by quantifying IFN-gamma from the serum of animals.
The specific or adaptive immune system comprises T and B lymphocytes. FOCUS Immunology provides assays to analyze the activity of these cells.
Cytokine secretion or proliferation is assessed in bulk cultures, or alternatively at the single cell level by single cell analysis techniques. The latter techniques offered by FOCUS Immunology are EliSpot or ICS (Intracellular Cytokine Staining) to analyze cytokine secretion on the single cell level or the flow cytometry-based CFSE assay to determine proliferation of individual T or B cells.
Prior to analysis the cells may be stimulated polyclonally by using PHA, PMA + Ionomycin, or antibodies directed against CD3 and CD28. Or the cells may be stimulated in an antigen-specific manner by using CMV (Cytomegalovirus) antigens and Tetanus Toxoid.
For many of the assays it is possible to select different cell populations to start with such as whole blood, purified peripheral blood mononuclear cells (PBMC), or selected lymphoid subpopulations such as B cells, T cells, CD4+cells or regulatory T cells and so on.
Specifically, FOCUS Immunology performs the following tests to determine effects of compounds on cells of the specific immune system:
(i) Proliferation assays to determine the proliferation rate of T or B lymphocytes. Upon antigen-specific or non-specific stimulation, cell proliferation will be determined either in bulk cultures by metabolic assays (MTS or ATP-dependent) or by measuring DNA synthesis (BrdU incorporation).
Alternatively, cell proliferation may be determined on the single cell level using the CFSE assay. Hereby, cells are labelled with CFDA-SE (carboxyfluorescein diacetate succinimidyl ester), a cell-permeable, non-fluorescent compound, which is converted by intracellular esterases to the fluorescent and non-membran-permeable CFSE (carboxyfluorescein succinimidyl ester). This molecule is highly stable and is distributed during cell division evenly to the daughter generations. Thus, flow cytometry allows to track proliferation of stimulated lymphocytes through consecutive generations.
(ii) Flow cytometry assays to analyze functional states of lymphocytes. T and B lymphocytes are not only characterized by lineage-specific markers such as CD3 for T cells and CD19 or CD20 for B cells but also by a continuously growing panel to distinguish subpopulations. CD4 and CD8 for helper and cytotoxic T cells and CD4,CD25 for regulatory T cells are just a few examples.
Additionally, cells of the lymphoid lineage acquire specific activation markers upon stimulation, which are not expressed on resting cells. Among these are CD69, CD25 and CD86.
A combination of lineage and activation markers allows analyzing the functional states of lymphoid cells on the single cell level, which thus allows analyzing the effects test-compounds on the functionality of lymphoid subpopulation.
FOCUS Immunology uses a dual-laser FACSCalibur from BD Biosciences to analyze up to four different markers in parallel allowing a detailed analysis of cell surface and/or intracellular markers.
(iii) Analysis of cytokine induction. Upon antigen-specific or non-specific stimulation cytokine production will be measured using either of three assays:
(a) ELISA, to determine the total amount of cytokine(s) secreted into the cell culture medium,
(b) EliSpot, to determine the frequency of cells secreting (a) particular cytokine(s), and
(c) Intracellular Cytokine Staining (ICS), to measure multiple cytokines and correlate their expression on the single cell level with lineage markers.
Friday, September 4, 2009
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