👤 A Raafat

Concepts surrounding the mechanisms of thrombocytopenia in ITP have shifted from the traditional view of autoantibody mediated platelet destruction to more complex mechanisms in which impaired platelet production, T-cell-mediated effects, and disturbed cytokine profiles play a role. Interleukin 27 (IL-27) plays pleiotropic roles in immunomodulation and autoimmune diseases.We aimed to determine the level of IL-27 in patients with ITP and its relationship to patient and disease characteristics as well as disease chronicity and response to treatment.Sixty childrens with primary immune thrombocytopenia were consequetively enrolled in this study as well as 20 age and sex matched healthy controls.ITP patients had significantly higher levels of IL-27 than controls (770.6 and 373.8 pg/ml, respectively). Patients with acute ITP had the highest levels of IL-27 among patient groups, while patients in remission had the lowest IL-27 levels (860.1and 622.9 pg/ml, respectively). Patients who received IVIG and combined steroids plus IVIG had significantly higher IL-27 levels than others. Patients who received Eltrombopag had significantly lower IL-27 levels than others.IL-27 seems to play a role in pathogenesis of childhood ITP. IL-27 can be used as a predictor for disease occurrence as well as responsiveness to treatment. Show less

The immortal strand theory postulates stem cells protect themselves from DNA replication-associated mutations and subsequent cancer risk through selective segregation of template DNA strands. Stem cells self-renew by asymmetric cellular division. During asymmetric division, stem cells maintain their template DNA strands, while the newly synthesized DNA strands segregate to newly formed daughter cells. Previous studies have demonstrated that self-renewing mammary stem cells originate in the expanding mammary ducts during puberty-associated allometric growth. In this study, we labeled newly forming mammary stem cells with the thymidine analog 5-ethynl-2'-deoxyuridine for 2 weeks during allometric ductal expansion. Cells that incorporate and retain the nuclear label following extended chase periods are termed label-retaining cells (LRCs). A second nuclear label, 5-bromodeoxyuridine, was administered before euthanasia to identify cells traversing the cell cycle. Mammary cells collected following euthanasia were sorted based on nuclear label retention. Members of the Notch and Wnt signaling pathways were found differentially expressed by mammary LRCs. These pathways are involved in the regulation of stem cells in the mouse mammary gland. Upon further analysis, we found that in contrast to non-LRCs, Notch1 and Notch2 are expressed and localized in the nuclei of the LRCs. Expression of Notch-inducible genes, Hes1 and Hey2, was elevated in LRCs. Inhibition of Notch1 by shRNA reduced colony forming potential and label retention by mammary epithelial cells in vitro. These results indicate that genes are differentially regulated in the LRC population of mammary glands and Notch1 mediates asymmetric cell division of mammary progenitor cells. Show less

Notch genes play a critical role in mammary gland growth, development and tumorigenesis. In the present study, we have quantitatively determined the levels and mRNA expression patterns of the Notch receptor genes, their ligands and target genes in the postnatal mouse mammary gland. The steady state levels of Notch3 mRNA are the highest among receptor genes, Jagged1 and Dll3 mRNA levels are the highest among ligand genes and Hey2 mRNA levels are highest among expressed Hes/Hey target genes analyzed during different stages of postnatal mammary gland development. Using an immunohistochemical approach with antibodies specific for each Notch receptor, we show that Notch proteins are temporally regulated in mammary epithelial cells during normal mammary gland development in the FVB/N mouse. The loss of ovarian hormones is associated with changes in the levels of Notch receptor mRNAs (Notch2 higher and Notch3 lower) and ligand mRNAs (Dll1 and Dll4 are higher, whereas Dll3 and Jagged1 are lower) in the mammary gland of ovariectomized mice compared to intact mice. These data define expression of the Notch ligand/receptor system throughout development of the mouse mammary gland and help set the stage for genetic analysis of Notch in this context. Show less

We have identified the transforming acidic coiled-coil protein-3 (Tacc3) as a binding partner for Notch4/Int3 and were able to show that it binds to the intracellular domain (ICD) of all members of the Notch receptor family. Members of the Tacc family reside at the centrosomes and associates with microtubules. Recent studies suggest that Tacc3 also contributes to the regulation of gene transcription. Tacc3 specifically interacts with the Notch4/Int3 CDC10/Ankyrin repeats and to a lesser extent, with residues C-terminal to these repeats in the ICD. Dual label immunofluorescence of mouse mammary tissue shows Tacc3 co-localizes with the Notch3 ICD. Co-immunoprecipitation of endogenous Notch and Tacc3 proteins from NIH3T3 cell extracts, lung and mammary gland confirms that these two proteins interact under physiological conditions. In addition, knock down of Tacc3 in NIH3T3 cells leads to the up-regulation of Hey2, a target gene for Notch signaling. The affinity of Tacc3 binding to Notch4/Int3 ICD is similar to that between Rbpj and Notch4/Int3 ICD. Notch4/Int3 ICD-Tacc3 interaction results in the inhibition of transcription from a Hes1-Luciferase reporter vector in COS-1 cells. The inhibition was reversed in these cells by increasing the levels of Rbpj. Taken together, these results suggest that Tacc3 is a negative regulator of the Notch signaling pathway. Show less

Transgenic mice expressing the Notch 4 intracellular domain (ICD) (Int3) in the mammary gland have two phenotypes: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. We have conditionally ablated the Rbpj gene in the mammary glands of mice expressing whey acidic protein (Wap)-Int3. Interestingly, Rbpj knockout mice (Wap-Cre(+)/Rbpj(-/-)/Wap-Int3) have normal mammary gland development, suggesting that the effect of endogenous Notch signaling on mammary gland development is complete by day 15 of pregnancy. RBP-J heterozygous (Wap-Cre(+)/Rbpj(-/+)/Wap-Int3) and Rbpj control (Rbpj(flox/flox)/Wap-Int3) mice are phenotypically the same as Wap-Int3 mice with respect to mammary gland development and tumorigenesis. In addition, the Wap-Cre(+)/Rbpj(-/-)/Wap-Int3-knockout mice also developed mammary tumors at a frequency similar to Rbpj heterozygous and Wap-Int3 control mice but with a slightly longer latency. Thus, the effect on mammary gland development is dependent on the interaction of the Notch ICD with the transcription repressor/activator Rbpj, and Notch-induced mammary tumor development is independent of this interaction. Show less