The aim of FOCUS Immunology’s “Immune Screening” testing package is to find out whether novel or known compounds affect basic immune functions; this may alert early on in development to potential side effects in clinical studies and on the other hand it may pave the way towards definition of new indications.
To this end FOCUS Immunology provides a selection of assays comprised in the “Immune Screening Package”. This package is purposefully set to address the “major” aspects of immune cell activation but this package may at any time be complemented by more refined assays from the the “Specific Immune System Package”, the “Innate Immune System Package”, or the “Biologicals (of higher risk) Package”.
Specifically, the following assays are offered to screen for possible immuno-modulatory effects of known or to be developed compounds:
Cell proliferation: Upon stimulation, cell proliferation is determined either in bulk cultures by metabolic assays or by measuring DNA synthesis (BrdU incorporation). Alternatively, cell proliferation can be determined on the single cell level by using a fluorescence-based assay (CFSE assay).
Cytokine release: Upon stimulation cytokine production can be measured using either of three assays: (a) ELISA, to determine the total amount of cytokine(s) secreted into the cell culture medium (link to List), (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.
Expression of activation markers: Immune cells are not only characterized by cell-type-specific markers such as CD3 for T cells, CD19 or CD20 for B cells or CD14 for monocytes, but also by a continuously growing panel of CD markers to distinguish subpopulations. Examples are CD4 and CD8 for helper and cytotoxic T cells and CD4 + CD25 for regulatory T cells. Additionally, cells of the lymphoid lineage acquire specific activation markers upon stimulation, which are not expressed on resting cells. Examples for activation markers of T cells are CD69 and CD86.
The combination of lineage and activation markers in one assay (“multi-color flow cytometry”) allows analyzing the functional states of immune cells on the single cell level. This provides refined information on the effects test-compounds on the functionality of immune cell subpopulations.
FOCUS Immunology operates 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.
Cytokines are important mediators of immune cell functionality and may strongly influence the outcome of an immune response. Accordingly, it is important to know whether a compound alters the production and secretion of cytokines partaking in an immune response.
FOCUS Immunology has established ex vivo assays in order to detect effects of compounds on cytokine secretion. To this effect, whole blood cultures or purified blood cells are suboptimally stimulated in order to activate both lympoid and myeloid cells in the presence or absence of the test-compound.
Suboptimal stimulation is used in order to test the compounds in the linear range of the dose-response-curve of the stimulant and which allows the detection of compound-mediated effects.
At termination of the culture, the following cytokines are determined from the supernatant by ELISA: IL-6 and TNF-alpha as prototype pro-inflammatory cytokines, IL-8 as mediator of chemotaxis, IL-2 as a central regulator of immune cell functions, IFN-gamma for its involvement in anti-viral immune responses and IL-10 as the major immunosuppressive cytokine.
FOCUS Immunology’s cytokine package was designed to keep the assay concise and to answer many questions with a reasonable use of resources.
It is of course possible to add or leave out any cytokine for your specific project.
We have experience in analyzing a great many more than the above listed cytokines and are also willing to establish any assay for which reagents are available or if the need is dire enough to develop our own reagents.
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.
See also:
Expert Report – you can obtain your free expert report here.
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.
Friday, September 4, 2009
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