This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Human body integrity is largely based on a healthy immune system fighting pathogens. One key feature of the immune system is expression of memory, i.e. maintaining the ability to fight a pathogen upon its re-encounter effectively, even after years of pathogen absence. Based on that, vaccinations can prevent from severe infections. In pathogen defense, proteins binding specifically to the pathogen (antibodies) play a vital role. Antibodies tag the pathogen and thus cause highly efficient degradation of the pathogen by specialized immune cells or neutralize it. The physical half-life of antibodies is less than 4 weeks. By contrast, specific antibodies induced by vaccination or infection can be readily detected in human blood serum after decades of antigen absence (humoral memory). This suggests that serum antibody levels are regulated at the stage of antibody secreting cells, and their specific precursors (memory B cells). For both cell types, longevity vs. continuous renewal is discussed as means to maintain the entire cell population and its composition, but both concepts were neither proven nor disproven physically. In this project we want to measure 14C content in the DNA of antibody-secreting cells and memory B cells obtained from donors exposed to elevated levels of atmospheric 14C in Europe during the 1960s. Elevated levels of the 14C isotope can still be detected in cells being quiescent since then, revealing their age. 14C analysis will require accelerator mass spectrometry (AMS) ensuring a precise quantitation of very low levels of 14C from minimum DNA samples. In analogy to Spalding et al (Cell, Vol. 122, 133?143, July 15, 2005), this analysis will determine the in vivo age of candidate subsets of antibody-secreting cells and memory B cells and prove or disprove longevity of these cells, arguing in favor or against B cell longevity as a key element of humoral memory. The expected results will define new requirements for the successful development of effective vaccines. AMS will be used in this project to determine the content of 14C in DNA samples isolated from immune cells, thereby revealing their average age. AMS is indispensable as the cell populations to analyze are very rare, thus yielding very small DNA sample size. According to previous studies, the concentration of the 14C within the sample is again very low. Comparable analysis has already been performed at the LLNL and we believe that close cooperation with the resource is necessary to conduct a successful project. Except for the very small sample size, no additional development of methodology appears to be required. The results from 14C analyses will be combined with different results derived from various immunological methods to provide new insight in functionally different effector B cells and mechanisms of humoral memory. AMS results are a vital part of the whole study. Human blood and tissue will be obtained and specific cells will be extracted. From these cells, DNA will be isolated using established protocols compatible with latter 14C quantitation. DNA will be sent to the resource for 14C analysis.
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