TECHNOLOGY

THE PRINCIPLE

The contemporary theory of the immune response states that the immune system is not programmed to respond to foreign particles, as it was conceived for many years during the self-nonself era of immunology. What turns the immune system on and off is a very important question in understanding disease and pathology. The condition of the tissue is very central to understanding homeostasis, tolerance and the immune response. The central dogma of immunology is that the immune system receives signals to respond from the ‘tissue’ and that the immune system is far less concerned with things that are foreign than with those that do damage.

Tissue Damage and Immune Response

We emphasise on tissue damage as it is the central driver of an immune response whether the damage is an outcome of a drug molecule or a pathogen or endogenous metabolites. The prediction of a drug molecule’s immunogenicity or cytotoxicity to non-target tissue will be best understood at this upstream event.

A case example of severe tissue damage is the incidence of Trovafloxacin, an antibiotic that was withdrawn from the market as it proved to be lethal to some patients, ending in financial loss and lawsuits. Trovafloxacin (sold as Trovan by Pfizer and Turvel by Laboratorios Almirall) was developed as a broad spectrum antibiotic to inhibit the uncoiling of supercoiled DNA in various bacteria. The lethality of the drug was found to be through causing necrosis of hepatocytes leading to liver failure.

Tissue damage that is non-lethal/non-toxic will generate an immune response that may lead to autoimmune pathogenesis. The immune system is programmed to respond to distressed tissue that is associated with an altered microenvironment (protein expression) and is typically followed by inflammation.

Any drug has the potential to cause unwanted tissue damage. The outcome of such damage is the release of intracellular molecules to the immune system. Conventional ‘immunogenicity’ testing considerations are limited to ‘antigenicity’ testing of the drug molecule only. In standard toxicology studies, immune response data such as infiltrations of lymphocytes in tissue sites are ignored as transient immunity and the possibility of disease occurring from chronic immunogenic events is overlooked. It is this broader immunogenicity of a drug that we consider serious and our assays are designed to answer your questions about drug-induced tissue damage and immune response.

Tissue Damage Study involves:
  • In vitro cell systems representing human tissues of drug target sites
  • Drug challenge
  • Assessment of cell damage and cytotoxicity
  • Assessment of immunogenic by products of tissue damage
How Will You Benefit?
  • Determine drug induced damage to non-target tissue
  • Better understand the potential of a drug molecule’s immunogenicity or cytotoxicity to non-target tissue at this upstream event
  • Predict drug-induced secondary pathology
Methods

Cell cultures (with or without tissue damage induction) are assessed for tissue damage in our lab. Methods we currently use are:

  • Microscopy based histochemistry or immunohistochemistry for example H&E staining or Ab-based staining
  • Cell viability assays such as Trypan Blue staining/ AnnexinV and PI staining (flow cytometry)/ Crystal violet staining (absorbence reading and/or microscopy imaging)
  • MTT assay
  • TUNEL assay
  • Oxidative stress assay
  • Cleaved caspase (Western or FC)

Amiodarone is a widely used drug for cardiovascular problems. It is well known to cause interstitial lung disease in patients.

Image above: cell death in human lung cells (A549) treated with Amiodarone (left panel 24 hrs, showing some cells detached; right panel 96 hrs, showing all cells detached and mostly necrotic).

Does this generate an immune response against lung tissue?

A Case Study

EXPERIMENTAL MODEL OF DIABETES THROUGH INDUCED TISSUE DAMAGE

In an in vivo study on autoimmune diabetes we induced targeted cell death of beta cells in pancreatic islets with streptozotocin. In a dose dependent manner the beta cells showed increasing levels of cell death (necrosis), verified by microscopy. The level of tissue damage was correlated with the incidence of diabetes or acute toxicity (total necrosis). The images show clear difference between healthy live and damaged beta cells in microscopy of tissues. Note the disintegration of the nuclei in B.

On a translational level, a similar model can be developed to test the mechanism and impact of a drug in a human in vitro system.