Mechanisms of disease and injury: utilization of mutants, monoclonals, and molecular methods.

Abstract

Rapid advances in our ability to localize and quantify macromolecular changes in health and disease are being brought about by the availability of genetically altered animals (mutants), purified reagents such as monoclonal antibodies, and new molecular methods. Targeted gene deletion (knockouts) and gene insertions (transgenics) in animals can allow identification of the importance and function of macromolecules. Monoclonal antibodies and fluorescent labels coupled with advances in microscopy provide exacting and multi-dimensional information about localization and cellular changes in proteins, carbohydrates, and lipids using immunohistochemistry, fluorescent activated cell sorting, and immunoprecipitation. Similarly, new applications of molecular methods can be used to identify and localize nucleic acids in tissues via in situ hybridization, polymerase chain reaction (PCR), reverse transcription (RT) PCR, differential display RT-PCR, RNase protection assays, and microchip arrays. The ligand for CD40 (CD40L), an important immunoregulatory molecule, is an example of the successful application of mutants, monoclonal antibodies, and molecular methods to cloning and biological characterization of new molecules. CD40L knockout mice, monoclonal antibodies, and several molecular methods were used to identify mutations in CD40L as the genetic basis for hyper-IgM syndrome in humans, to provide new insights into the pathobiology of Pneumocystis carinii infection, and to evaluate CD40L for immunotherapy of tumors and opportunistic infections.