NCL's Immunology team has put together a series of new protocols which probe various immunotoxicology aspects of nanomedicines. The 7 new protocols have been published on the NCL Assay Cascade Protocols webpage and are available for download. They include:
ITA-26: Detection of Intracellular Complement Activation in Human T Lymphocytes
The complement present in plasma is a group of approximately 30 proteins which are produced by the liver. Plasma complement plays an important role in innate immunity and promotes adaptive immunity against pathogens. Activation of plasma complement may occur in response to a drug product, and can result in immediate-type hypersensitivity reactions such as Complement Activation Related Pseudoallergy (CARPA). Recently, the concept of complement has been expanded to include so-called intracellular complement. Activation of intracellular complement in T-lymphocytes has been found to correlate with autoimmune disorders, and there is growing evidence to suggest the involvement of T-lymphocytes in the development of drug-induced hypersensitivity reactions. Therefore, understanding the ability of nanomaterials to activate intracellular complement may aid in establishing long-term safety profiles for these materials.
ITA-31: Detection of Nanoparticle-Mediated Total Oxidative Stress in T-Cells Using CM-H2DC-FDA Dye
ITA-32: Detection of Mitochondrial Oxidative Stress in T-Cells Using MitoSOX Red Dye
ITA-33: Detection of Changes in Mitochondrial Membrane Potential in T-Cells Using JC-1 Dye
Induction of oxidative stress is a common toxicity associated with nanoparticles. Generation of reactive oxygen species (ROS) and its control by antioxidant systems is a part of normal cellular metabolism. However, disturbance of redox homeostasis and accumulation of ROS damages cellular organelles and DNA, eventually leading to cell death, and can activate stress-related pathways to release of pro-inflammatory cytokines. The aim of these protocols is to understand the propensity of nanomaterials to induce total oxidative stress (ITA-31), oxidative stress in the mitochondria (ITA-32), and mitochondrial damage as assessed by change in mitochondrial membrane potential (ITA-33). The combination of mitochondrial membrane potential loss and mitochondrial ROS lead to organelle specific toxicity that ultimately cause cell death.
ITA-34: Detection of Antigen Presentation by Murine Bone Marrow-Derived Dendritic Cells
ITA-35: Antigen-Specific Stimulation of CD8+ T-Cells by Murine Bone Marrow-Derived Dendritic Cells
Nanoparticles have been extensively tested as therapeutic vaccine delivery vehicles for the treatment of cancer. A key effort in this area is the delivery of antigens to antigen-presenting cells (APCs). The set of protocols aim to determine the ability of nanoparticles to deliver antigen to APCs, such that an antigenic class I peptide sequence is properly presented in the context of class I major histocompatibility complex (MHC) molecules (ITA-34), and to measure the antigen-specific T-cell responses that are invoked (ITA-35). Both protocols use the model antigen ovalbumin (OVA) in the context of murine cells, but may be used to infer the ability of the nanoparticle system to deliver other, similar antigens.
ITA-36: Detection of Naturally Occurring Antibodies to PEG and PEGylated Liposomes
Poly(ethylene glycol) (PEG) is commonly used in the pharmaceutical industry to modify recombinant proteins and nanoparticle surfaces to improve hydrophilicity and decrease recognition by the immune system. Despite improved protection from the immune recognition, the immune system is still able to identify these products and mount an antibody response against them. Such immune responses may result in the development of anti-drug antibodies (ADA), and among antibodies specific to biological drug or nanocarrier, include the formation of antibodies to the PEG itself. Several reports suggest the existence of naturally occurring antibodies in the blood of healthy donor volunteers. Although the physiological significance of anti-PEG antibodies is unknown, several studies suggest they may affect the clearance of PEGylated products (e.g., Accelerated Blood Clearance or ABC phenomenon) and contribute to complement activation and other antibody-mediated toxicities. This protocol will enable detection of antibodies reactive to PEG2000, mPEG2000 and PEGylated liposomes, common reagents used for delivery of anti-cancer drugs.
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