In the workshop the central role of so-called “multiple cytokine secreting cells” (also termed “multiple cytokine producing cells”) in the pharmaco-dynamics of immuno-modulatory compounds and associated adverse events was pointed out.

As the name implies, these cells are characterized by the production of several cytokines in any single cell and thus contrast cells in which only one or two cytokines may be detected. In other words: “Multiple Cytokine Secretion” / “Multiple Cytokine Production” is a functional characteristic of a given cell and pretty much every T cell may become such a multiple cytokine secreting cell upon activation. Multiple cytokine secretion is a temporary state, only as long as the cell is being activated.

Physiological stimulation of T cells to multiple cytokine secretion is required for host defense and tissue remodeling. However, aberrant or un-intentional stimulation under pathological conditions or by (over-dosing of) immuno-modulatory compounds may result in severe pathologies, such as the above-mentioned “Cytokine Release Syndrome“.

The analysis of multiple cytokine secreting cells requires sophisticated multi-parameter assays, like multiplex immunological assays for measuring cytokine profiles in cellular secretions under various stimulation conditions. Ultimately, it requires multi-color intracellular cytokine staining by flow cytometry, which allows multiple cytokine determinations at the single level.

FOCUS Immunology is experienced in providing state-of-the-science analyses for multiple cytokine secreting cells under GLP conditions. If you are interested in learning more about FOCUS Immunology’s experience and offers or if you want to discuss your specific experimental needs, please feel free to contact
Dr. Eddy Bruyns, Head of FOCUS Immunology Laboratory via e-mail eddy.bruyns@focus-cdd.com or via telephone +49 6221 64935124.

EMEA workshop: “In vitro cytokine release assays to predict Cytokine Release Syndrome: the current-state-of-the science”

The members of the SOCS family of molecules are important negative feedback regulators in adaptive and innate immune responses, while IL-17 producing CD4+ cells, the so-called “Th17 cells”, play a central role in the development of autoimmune diseases.

From their findings the authors conclude as follows: “Based on the described immunomodulatory mechanisms, good safety profile and oral biovailabalility, statins represent a promising therapeutic approach for multiple sclerosis and other chronic inflammatory diseases.”

Along the lines of this study by Zhang and colleagues, FOCUS Immunology is prepared to provide state-of-the-art services for pharmacodynamic (PD) analyses of immuno-modulatory compounds under GLP conditions.

If you are interested in learning more or in discussing your specific experimental needs, please feel free to contact Dr. Eddy Bruyns, Head of FOCUS Immunology Laboratory via e-mail eddy.bruyns@focus-cdd.com or via telephone +49 6221 64935124.

Source
Zhang et al.
Simvastatin Inhibits IL-17 Secretion by Targeting Multiple IL-17-Regulatory Cytokines and by Inhibiting the Expression of IL-17 Transcription Factor RORC in CD4+ Lymphocytes
The Journal of Immunology 2008; 180; 6988-6996

So-called “Suppressors of Cytokine Signaling (SOCS)” are important molecular players in this negative feedback regulation. 7 members of the SOCS molecular family have been described so-far. The SOCS family members 1 and 3 are of particular importance, since they are involved in adaptive and innate immune responses.

SOCS-1 and -3 are induced by LPS stimulation via the Toll-like Receptor-4 (TLR-4). While SOCS-1 directly targets the downstream signaling molecules of TLR4, SOCS-3 regulates the secondary effects of many LPS-induced cytokines. E.g. IL-1R signalling pathways are negatively regulated by SOCS-3.

In adaptive immune responses SOCS-1 and -3 play a role in the regulation of T cell activation, proliferation and diferentiation.
Recently, SOCS molecules were found to be central elements in the pharmacodynamics of the immuno-modulatory compound simvastatin and probiotic lactobacilli.

FOCUS Immunology is prepared to provide state-of-the-art services for pharmacodynamic (PD) studies for immunomodulatory compounds under GLP conditions. This includes studies on the regulation of intra- and extracellular mediators of inflammationand immunity.

If you are interested in learning more about FOCUS Immunology’s experience and offers or if you want to discuss your specific experimental needs, please feel free to contact Dr. Eddy Bruyns, Head of FOCUS Immunology Laboratory via e-mail eddy.bruyns@focus-cdd.com or via telephone +49 6221 64935124.

Source
Dimitriou et al.
Putting out the fire: coordinated suppression of the innate and adaptive immune systems by SOCS1 and SOCS3 proteins
Immunological reviews 2008, Vol. 224: 265-283

More information on SOCS1: Wikipedia on SOCS1
More information on SOCS3: Wikipedia on SOCS3

In a recent publication Lee and colleagues explored whether probiotics induced anti-inflammatory properties through induction of SOCS.

Helicobacter pylori (H. pylori) or lipopolysaccharides derived thereof were found to increase the expression of pro-inflammatory cytokines in a human gastric cell line. Pre-treatment of the cells with probiotic bacteria of the Lactobacillus group reduced the H.Pylori-induced expression of these cytokines.

When the underlying molecular mechanisms were explored in more detail, the administration of probiotics was found to increase the expression of SOCS-2 and SOCS-3.

The authors conclude that the “Anti-inflammatory signals of SOCS … might be a key anti-inflammatory mechanism of probiotics …”

FOCUS Immunology is prepared to provide state-of-the-art servcies for pharmacodynamic (PD) studies for immunomodulatory compounds under GLP conditions.

If you are interested in learning more about FOCUS Immunology’s experience and offers or if you want to discuss your specific experimental needs, please feel free to contact Dr. Eddy Bruyns, Head of FOCUS Immunology Laboratory via e-mail eddy.bruyns@focus-cdd.com or via telephone +49 6221 64935124.

Source
Jeong Sang Lee et al.
Anti-inflammatory actions of probiotics through activating suppressor of cytokine signaling (SOCS) expression and signaling in Helicobacter pylori infection: A novel mechanism
Journal of Gastroenterology and Hepatology 25 (2010) 194-202

The development of new biologicals is associated with specific challenges. Some relate to the immunogenic and immune-modulatory properties of biologicals. Some keywords are „immunogenicity testing”, “cytokine storm”, “species-specificity of ligand-receptor interactions and down-stream signaling”.

In view of these challenges FOCUS has developed specific testing packages with an emphasis on immuno-modulatory activities of novel drug candidates.

The following testing packages have recently been defined:

1. Specific immune system package
2. Innate immune system package
3. Immune activity screening package for novel and established compounds
4. Cytokine modulation package for immuno-modulators, esp. immuno-suppressive compounds
5. Immuno-stimulation package for immune-modulators, vaccines and oncology drugs
6. Biologicals testing package, esp. for new biological entities (NBEs)

A comprehensive summary can be found in the white paper to be downloaded here.

Source: Cormac Sheridan, „Fresh from the biology pipeline-2009“ Nature Biotechnology Volume 28 Number 4 April 2010

Modern cell biological techniques allow to distinctively address the different activation pathways by specific stimulators and inhibitors, be it by specific antibodies against cell surface receptors (e.g. anti-CD3, or anti-CD28) or be it by chemical reagents (e.g. phorbol myristate acetate or ionomycin).

These sophisticated stimulation protocols can then be combined with appropriate analytical technologies to assess the phenotype and function of the activated cell populations, like expression of cell surface activation markers, secretion of cytokines, or proliferation.
FOCUS Immunology operates these technologies and tailors their use to the specific needs of a study protocol.

To learn more about FOCUS Immunology’s expertise and service offer in T cell biology contact us at immunology@focus-cdd.com or contact directly our Head of FOCUS Immunology Dr. Eddy Bruyns at eddy.bruyns@focus-cdd.com.

FOCUS Immunology laboratory is part of the Clinical Research Organisation (CRO) FOCUS-CDD GmbH. FOCUS Immunology provides immunological biomarker services under GLP and non-GLP conditions for the development of novel medicines.

FOCUS Immunology has established state-of-the art protocols for the phenotypic and functional analysis of NK cells.

For further information on our expertise in NK cell biology please contact us at immunology@focus-cdd.com or contact directly our Head of FOCUS Immunology Dr. Eddy Bruyns at eddy.bruyns@focus-cdd.com.

FOCUS Immunology laboratory is part of the Clinical Research Organisation (CRO) FOCUS-CDD GmbH. FOCUS Immunology provides immunological biomarker services under GLP and non-GLP conditions for the development of novel medicines.

Together with clinical institutions of the Heidelberg Medical Centre FOCUS Immunology has established processes that allow to use purified LPL for cell biological and mode-of-action studies.

For further information on our expertise and service offer in LPL biology please us at immunology@focus-cdd.com or contact directly our Head of FOCUS Immunology Dr. Eddy Bruyns at eddy.bruyns@focus-cdd.com.

FOCUS Immunology laboratory is part of the Clinical Research Organisation (CRO) FOCUS-CDD GmbH. FOCUS Immunology provides immunological biomarker services under GLP and non-GLP conditions for the development of novel medicines.

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.

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.