A novel high-content immunofluorescence assay as a tool to identify at the single cell level γ-globin inducing compounds

The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ-and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathie

Macrophages support pathological erythropoiesis in Polycythemia Vera and Beta-Thalassemia

Regulation of erythropoiesis is achieved by integration of distinct signals. Among these, macrophages are emerging as erythropoietin-complementary regulators of erythroid development, particularly under stress conditions. We investigated the contribution of macrophages for physiological and pathological conditions of enhanced erythropoiesis. We utilized mouse models of induced anemia, Polycythemia vera and β-thalassemia in which macrophages were chemically depleted. Our data indicate that macrophages contribute decisively for recovery from induced anemia as well as the pathological progression of Polycythemia vera and β-thalassemia by modulating erythroid proliferation and differentiation. We validated these observations in primary human cultures, showing a critical direct impact of macrophages on proliferation and enucleation of erythroblasts from healthy individuals and Polycythemia vera or β-thalassemic patients. In summary, we identify a new mechanism that we named “Stress Erythropoiesis Macrophage-supporting Activity” (SEMA) that contributes to the pathophysiology of these disorders and will have critical scientific and therapeutic implications in the near future

Reversible Disruption of Pre-Pulse Inhibition in Hypomorphic-Inducible and Reversible CB1-/- Mice

Although several genes are implicated in the pathogenesis of schizophrenia, in animal models for such a severe mental illness only some aspects of the pathology can be represented (endophenotypes). Genetically modified mice are currently being used to obtain or characterize such endophenotypes. Since its cloning and characterization CB1 receptor has increasingly become of significant physiological, pharmacological and clinical interest. Recently, its involvement in schizophrenia has been reported. Among the different approaches employed, gene targeting permits to study the multiple roles of the endocannabinoid system using knockout (-/-) mice represent a powerful model but with some limitations due to compensation. To overcome such a limitation, we have generated an inducible and reversible tet-off dependent tissue-specific CB1-/- mice where the CB1R is re-expressed exclusively in the forebrain at a hypomorphic level due to a mutation (IRh-CB1-/-) only in absence of doxycycline (Dox). In such mice, under Dox+ or vehicle, as well as in wild-type (WT) and CB1-/-, two endophenotypes motor activity (increased in animal models of schizophrenia) and pre-pulse inhibition (PPI) of startle reflex (disrupted in schizophrenia) were analyzed. Both CB1-/- and IRh-CB1-/- showed increased motor activity when compared to WT animals. The PPI response, unaltered in WT and CB1-/- animals, was on the contrary highly and significantly disrupted only in Dox+ IRh-CB1-/- mice. Such a response was easily reverted after either withdrawal from Dox or haloperidol treatment. This is the first Inducible and Reversible CB1-/- mice model to be described in the literature. It is noteworthy that the PPI disruption is not present either in classical full CB1-/- mice or following acute administration of rimonabant. Such a hypomorphic model may provide a new tool for additional in vivo and in vitro studies of the physiological and pathological roles of cannabinoid system in schizophrenia and in other psychiatric disorders

Survivin and p53 expression in patients with Crohn's disease: an immunohistochemical study

Crohn’s disease (CD) is characterized by acute phases of patchy transmural inflammation, involving any part of the intestinal tract with intervening periods of remission. It has been well established that patients with Crohn’s disease are at increased risk of developing intestinal adenocarcinoma. The regulation of apoptotic cell death may have an important effect on the pathogenesis and progression of colon cancer. Disturbance of apoptosis is known to be very crucial in neoplastic progression and transformation. Mutations in the proapoptotic and tumor suppressor gene p53 are associated with neoplasia in ulcerative colitis, but little is understood of their significance in Crohn’s disease. Members of the inhibitor of the apoptosis protein (IAP) family including survivin are expressed in many tumors. It is known that well differentiated adenocarcinoma tend to express higher level of survivin than normal mucosa. Expression of survivin was observed in the cytoplasm of adenoma with dysplasia and colonrectal carcinoma cells. In order to explore the role of p53 and survivin as marker of neoplasia in CD patient, the aim of this study was to evaluate, by immunohistochemical analysis, the expression and distribution of p53 and survivin immunoreactive cells in a series of Crohn’s disease bioptic tissues with apparently normal adjacent tissues. The immunohistochemical study showed an increase in p53 and survivin expression. Our results suggest that p53 and survivin overexpression in CD patients may predispose the intestinal mucosa to dysplasia that may progress to a higher grade of neoplasia overtime

β-Globin Gene Cluster Haplotypes in the Corsican and Sardinian Populations

The distribution of β-globin cluster haplotypes has been studied in the populations of Corsica (France) and Sardinia (Italy). The analysis was carried out using five restriction fragment length polymorphism markers on chromosome 11 inside the β-globin cluster using the restriction enzymes HincII and HindIII. The results show a remarkable heterogeneity within the two islands. However, the presence of rare haplotypes common to the most conservative areas (Nuoro and Corte) of the two islands is particularly inter- esting. These data support the hypothesis of a common origin of the popula- tions of Sardinia and Corsica during the middle and upper Paleolithic periods and could be interpreted as a founder effect