Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Y704, Y729, Y744, Y764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation and cell survival. However, it is unclear whether these tyrosines are equally important under more physiological conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated GCSF- R deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (mO), single tyrosine "add back" mutants or wild type (WT) receptors. G-CSFinduced responses were determined in primary colony assays, serial replatings and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Y764 strongly enhanced proliferativeresponses, which was reverted by inhibition of ERK activitity. Y729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing mO gradually dropped compared to WT. The presence of Y729, but also Y704 and Y744, both involved in activation of STAT3, further reduced replating
efficiencies. Conversely, Y764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a >104–fold increase of colony forming cells over mO after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.

Change of perspective: a measurable and desired outcome of chronic disease self-management intervention programs that violates the premise of preintervention/postintervention assessment

Objective: To determine if participants in chronic disease self-management courses have a change of perspective of their health status (a response shift), and if this is measurable with a paper-based questionnaire.

Methods: Nine items were developed to measure potential benefits of self-management courses. These were based on the constructs of a previous questionnaire, the Health Education Impact Questionnaire (HEI-Q). Cognitive interviews elicited spontaneous statements about the reasons for paper-based answers. Sensitivity, specificity, and overall accuracy of items were calculated using the interview as a relative gold standard. Response shift can be negative (i.e., after the course, participants realize that, before the course, they were worse than they thought they were), positive (i.e., participants now realize they were better than they thought they were), or absent (no change).

Results: Interviews (n = 39) reflected that true response shift occurred in approximately half the replies to questionnaire items. Of these, 31% were negative response shift, 20% were positive response shift. Response shift was absent in 32% of replies. Presence or absence of response shift could not be determined in 17% of replies across items. Significant concordance between questionnaires and cognitive interviews (average overall accuracy 0.79) indicated that the HEI-Q Perspective questionnaire detects response shift in participants of self-management courses. The questionnaire revealed that 87% of participants had response shift in at least 1 item.

Conclusion: This study suggests that preintervention/postintervention assessments of interventions such as self-management courses are confounded by a change in perspective of a large proportion of respondents. It also indicates response shift is a valuable outcome of self-management courses that can be measured with a paper-based questionnaire.

Conversation

 'Conversation' was part of Melbourne Urban Screens Festival at Federation Square from 3 - 5 Oct. 2008 and Rotterdam International Film Festival (38th : 2009 : Rotterdam, The Netherlands) from 21 Jan. - 1 Feb. 2009. Both events curated by Mirjam Struppek