An Overview of Peptides and Peptide Pools for Antigen-Specific Stimulation in T-Cell Assays.

The analysis of antigen-specific T-cell responses has become routine in many laboratories. Functional T-cell assays like enzyme-linked-immuno-spot (ELISPOT), which depend on antigen-specific stimulation, increasingly use peptides to represent the antigen of interest. Besides single peptides, mixtures of peptides (peptide pools) are very frequently applied. Such peptide pools may, for example, represent entire proteins (with overlapping peptides covering a protein sequence) or include noncontiguous peptides such as a collection of T-cell-stimulating peptides. The optimum specification of single peptides or peptide pools for T-cell stimulation assays will depend on the purpose of the test, the target T-cell population, the availability of sample, requirements regarding reproducibility, and, last but not least, the available budget, to mention only the most important factors. Because of the way peptides are produced, they will always contain certain amounts of impurities such as peptides with deletions or truncated peptides, and there may be additional by-products of peptide synthesis. Optimized synthesis protocols as well as purification help reduce impurities that might otherwise cause false-positive assay results. However, specific requirements with respect to purity will vary depending on the purpose of an assay. Finally, storage conditions significantly affect the shelf life of peptides, which is relevant especially for longitudinal studies. The present book chapter addresses all of these aspects in detail. It should provide the researcher with all necessary background knowledge for making the right decisions when it comes to choosing, using, and storing peptides for ELISPOT and other T-cell stimulation assays.

Immunosuppression Monitoring-What Clinician Needs to Know?

The liver is well known for its immunotolerance, but rejection without immunosuppression is frequently encountered post liver transplantation, especially in humans.1 Indeed, the amount of immunosuppression required post liver transplant is less compared to other organ transplants like kidney, heart, and intestine.2 Reports of successful weaning of immunosuppression have been reported but are not practiced for fear of unwanted alloimmune response leading to rejection. Life-long immunosuppression is needed in most patients for graft survival but is associated with side effects like renal dysfunction, metabolic abnormalities, or risk of de novo malignancies. Also, the appropriate dose of immunosuppression to achieve adequate graft function and prevention of toxicities is very important. One shoe does not fit all. There are significant individual variations in response and side effect profile. Also, the level of immunosuppression varies with the underlying liver disease like autoimmune disease requires higher immunosuppression. Thus, monitoring the adequate immunosuppression with the minimization of drug toxicity is imperative post-transplant. Unfortunately, the current methods for immunosuppression monitoring rely on testing the immunosuppressive drug levels rather than the immune system activity. We have discussed the concept of alloreactivity, available methods of immunosuppression and drug monitoring and investigational methods in this review.

Gene Therapy for the Treatment of Neurological Disorders: Central Nervous System Neoplasms.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a median survival of 16.2-21.2 months post diagnosis (Stupp et al., N Engl J Med 352(10): 987-996, 2005). Because of its location, complete surgical resection is impossible; additionally because GBM is also resistant to chemotherapeutic and radiotherapy approaches, development of novel therapies is urgently needed. In this chapter we describe the development of preclinical animal models and a conditionally cytotoxic and immune-stimulatory gene therapy strategy that successfully causes tumor regression in several rodent GBM models.