Several groups are investigating the mechanisms of action of therapeutic immunoglobulins (IVIg) in order to improve their use. In vitro models such as CD40-CD154 interaction are necessary to study the physiological re...Several groups are investigating the mechanisms of action of therapeutic immunoglobulins (IVIg) in order to improve their use. In vitro models such as CD40-CD154 interaction are necessary to study the physiological response of human B lymphocytes to IVIg. Human B lymphocytes treated with IVIg triggers a rapid phospho-rylation (<1 h) of extracellular-regulated kinases 1 and 2 (ERK1/2), which subsequently results in increased differentiation and decreased pro-liferation. However, the modulation of human lymphocyte physiology by IVIg is a gradual and cumulative process and requires long-term experimentation. Differentiation of human B lymphocytes into Ig-secreting cells can be evaluated both at the transcription and translation levels. The secretion of immunoglobulins can be assessed using ELISA or ELISPOTS whereas expression of immunoglobulin genes can be measured using semi-quantitative or quantitative PCR methods. We hereby report a comparison of these methods to explain how contradictory observations towards IVIg effects could result from their use. Our results indicate that ELISA and ELISPOTS will provide consistent observations by opposition to real-time PCR quantification. Besides, the reliability of each of these me-thods remained dependent on the stimulation period as well as the preparation of cellular extracts or cell samples following IVIg-treatment.展开更多
文摘Several groups are investigating the mechanisms of action of therapeutic immunoglobulins (IVIg) in order to improve their use. In vitro models such as CD40-CD154 interaction are necessary to study the physiological response of human B lymphocytes to IVIg. Human B lymphocytes treated with IVIg triggers a rapid phospho-rylation (<1 h) of extracellular-regulated kinases 1 and 2 (ERK1/2), which subsequently results in increased differentiation and decreased pro-liferation. However, the modulation of human lymphocyte physiology by IVIg is a gradual and cumulative process and requires long-term experimentation. Differentiation of human B lymphocytes into Ig-secreting cells can be evaluated both at the transcription and translation levels. The secretion of immunoglobulins can be assessed using ELISA or ELISPOTS whereas expression of immunoglobulin genes can be measured using semi-quantitative or quantitative PCR methods. We hereby report a comparison of these methods to explain how contradictory observations towards IVIg effects could result from their use. Our results indicate that ELISA and ELISPOTS will provide consistent observations by opposition to real-time PCR quantification. Besides, the reliability of each of these me-thods remained dependent on the stimulation period as well as the preparation of cellular extracts or cell samples following IVIg-treatment.
