Serine-threonine kinases and transcription factors active in signal transduction are detected at high levels of phosphorylation during mitosis in preimplantation embryos and trophoblast stem cells

Serine-threonine kinases and transcription factors play important roles in the G1-S phase progression of the cell cycle. Assays that use quantitative fluorescence by immunocytochemical means, or that measure band strength during Western blot analysis, may have confused interpretations if the intention is to measure G1-S phase commitment of a small subpopulation of phosphorylated proteins, when a larger conversion of the same population of proteins can occur during late G2 and M phases. In mouse trophoblast stem cells (TSC), a human placental cell line (HTR), and/or mouse preimplantation embryos, 8/19 ser- ine-threonine and tyrosine kinases, 3/8 transcription factors, and 8/14 phospho substrate and miscellaneous proteins were phosphorylated at higher levels in M phase than in interphase. Most phosphoproteins appeared to associate with the spindle complex during M phase, but one (p38MAPK) associated with the spindle pole and five (Cdx2, MEK1, 2, p27, and RSK1) associated with the DNA. Phosphorylation was detected throughout apparent metaphase, anaphase and telophase for some proteins, or for only one of these segments for others. The phosphorylation was from 2.1- to 6.2-fold higher during M phase compared with interphase. These data suggest that, when planning and interpreting quantitative data and perturbation experiments, consideration must be given to the role of serine-threonine kinases and transcription factors during decision making in M phase as well as in G1-S phase

Reduced expression of FOXP3 and regulatory T-cell function in severe forms of early-onset autoimmune enteropathy.

International audienceBackground & aims: Little is known about the pathophysiology of early onset forms of autoimmune enteropathy (AIE). AIE has been associated with mutations in FOXP3-a transcription factor that controls regulatory T-cell development and function. We analyzed the molecular basis of neonatal or early postnatal AIE using clinical, genetic, and functional immunological studies.Methods: Gastroenterological and immunological features were analyzed in 9 boys and 2 girls with AIE that began within the first 5 months of life. FOXP3 and IL2RA were genotyped in peripheral blood monocytes. FOXP3 messenger RNA and protein expression were analyzed using reverse-transcription polymerase chain reaction, flow cytometry, and confocal immunofluorescence of CD4(+) T cells. Regulatory T-cell function (CD4(+)CD25(+)) was assayed in coculture systems.Results: AIE associated with extraintestinal autoimmunity was severe and life-threatening; all patients required total parenteral nutrition. Regulatory T cells from 7 patients had altered function and FOXP3 mutations that resulted in lost or reduced FOXP3 protein expression; 2 infants had reduced regulatory T-cell activity and reduced levels of FOXP3 protein, although we did not detect mutations in FOXP3 coding region, poly-A site, or promoter region (called FOXP3-dependent AIE). Two patients had a normal number of regulatory T cells that expressed normal levels of FOXP3 protein and normal regulatory activity in in vitro coculture assays (called FOXP3-independent AIE). No mutations in IL2RA were found. Conclusions: Most cases of AIE are associated with alterations in regulatory T-cell function; some, but not all, cases have mutations that affect FOXP3 expression levels. Further studies are needed to identify mechanisms of AIE pathogenesis

Cost-effectiveness of intestinal transplantation for adult patients with intestinal failure:a simulation study

Background: Home parenteral nutrition (HPN) and intestinal transplantation (ITx) are the 2 treatment options for irreversible intestinal failure (IF). Objective: This study simulated the disease course of irreversible IF and both of these treatments HPN and ITx to estimate the cost-effectiveness of ITx. Design: We simulated IF treatment in adults as a discrete event model with variables derived from the Dutch Registry of Intestinal Failure and Intestinal Transplantation, the Intestinal Transplant Registry, hospital records, the literature, and expert opinions. Simulated patients were enrolled at a rate of 40/mo for 10 y. The maximum follow-up was 40 y. Survival was simulated as a probabilistic function. ITx was offered to 10% of patients with Results: The average survival was 14.6 y without ITx and 14.9 y with ITx. HPN costs were (sic)13,276 for treatment introduction, followed by (sic)77,652 annually. The costs of ITx were (sic)73,000 during the first year and then (sic)13,000 annually. The ICER was (sic)19,529 per life-year gained. Conclusion: Our simulations show that ITx slightly improves survival of patients with IF in comparison with HPN at an additional cost of (sic)19,529 per life-year gained