Emanuela D'Amore

Characterization of the TGF-β1 signaling abnormalities in the Gata1low mouse model of myelofibrosis.

Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-β1 release. To clarify the role of TGF-β1 in the pathogenesis of this disease, the TGF-β1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-β1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered. David-pathway analyses identified alterations of TGF-β1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1(low) phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-β1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-β1 signaling that may be responsible for MF in Gata1(low) mice and may represent additional targets for therapeutic intervention in PMF.

Rapid diagnosis of avian infectious bronchitis virus by the polymerase chain reaction

Abstract A simple, sensitive and specific polymerase chain reaction (PCR) procedure was developed in order to detect infectious bronchitis virus (IBV) directly in tissue samples. Viral RNA was extracted from allantoic fluids and cell cultures infected experimentally with different strains of IBV and from tissues of naturally infected birds. Viral RNA was then amplified and identified by a nested RT-PCR assay using two sets of primers flanking a well-conserved region of the nucleocapsid gene. The selected IBV nucleocapsid sequence was detected successfully by simple direct electrophoresis of amplified material.

L-DOPA PRELOADING INCREASES THE UPTAKE OF BOROPHENYLALANINE IN C6 GLIOMA RAT MODEL: A NEW STRATEGY TO IMPROVE BNCT EFFICACY

PURPOSE Boron neutron capture therapy (BNCT) is a radiotherapeutic modality based on (10)B(n,alpha)(7)Li reaction, for the treatment of malignant gliomas. One of the main limitations for BNCT effectiveness is the insufficient intake of (10)B nuclei in the tumor cells. This work was aimed at investigating the use of L-DOPA as a putative enhancer for (10)B-drug 4-dihydroxy-borylphenylalanine (BPA) uptake in the C6-glioma model. The investigation was first performed in vitro and then extended to the animal model. METHODS AND MATERIALS BPA accumulation in C6-glioma cells was assessed using radiowave dielectric spectroscopy, with and without L-DOPA preloading. Two L-DOPA incubation times (2 and 4 hours) were investigated, and the corresponding effects on BPA accumulation were quantified. C6-glioma cells were also implanted in the brain of 32 rats, and tumor growth was monitored by magnetic resonance imaging. Rats were assigned to two experimental branches: (1) BPA administration; (2) BPA administration after pretreatment with L-DOPA. All animals were sacrificed, and assessments of BPA concentrations in tumor tissue, normal brain, and blood samples were performed using high-performance liquid chromatography. RESULTS L-DOPA preloading induced a massive increase of BPA concentration in C6-glioma cells only after a 4-hour incubation. In the animal model, L-DOPA pretreatment produced a significantly higher accumulation of BPA in tumor tissue but not in normal brain and blood samples. CONCLUSIONS This study suggests the potential use of L-DOPA as enhancer for BPA accumulation in malignant gliomas eligible for BNCT. L-DOPA preloading effect is discussed in terms of membrane transport mechanisms.

In vivo 19F MRI and 19F MRS of 19F- labelled boronophenylalanine-fructose complex on a C6 rat glioma model to optimize boron neutron capture therapy (BNCT)

Boron neutron capture therapy (BNCT) is a promising binary modality used to treat malignant brain gliomas. To optimize BNCT effectiveness a non-invasive method is needed to monitor the spatial distribution of BNCT carriers in order to estimate the optimal timing for neutron irradiation. In this study, in vivo spatial distribution mapping and pharmacokinetics evaluation of the (19)F-labelled boronophenylalanine (BPA) were performed using (19)F magnetic resonance imaging ((19)F MRI) and (19)F magnetic resonance spectroscopy ((19)F MRS). Characteristic uptake of (19)F-BPA in C6 glioma showed a maximum at 2.5 h after compound infusion as confirmed by both (19)F images and (19)F spectra acquired on blood samples collected at different times after infusion. This study shows the ability of (19)F MRI to selectively map the bio-distribution of (19)F-BPA in a C6 rat glioma model, as well as providing a useful method to perform pharmacokinetics of BNCT carriers.

In vivo 19F MR imaging and spectroscopy for the BNCT optimization

The aim of this study was to evaluate in vivo the boron biodistribution and pharmacokinetics of 4-borono-2-fluorophenylalanine ((19)F-BPA) using (19)F MR Imaging ((19)F MRI) and Spectroscopy ((19)F MRS). The correlation between the results obtained by both techniques, (19)F MRI on rat brain and (19)F MRS on blood samples, showed the maximum (19)F-BPA uptake in C6 glioma model at 2.5h after infusion determining the optimal irradiation time. Moreover, the effect of L-DOPA as potential enhancer of (19)F-BPA tumour intake was assessed using (19)F MRI.

CXCR4-independent rescue of the myeloproliferative defect of the Gata1low myelofibrosis mouse model by Aplidin.

The discovery of JAK2 mutations in Philadelphia‐negative myeloproliferative neoplasms has prompted investigators to evaluate mutation‐targeted treatments to restore hematopoietic cell functions in these diseases. However, the results of the first clinical trials with JAK2 inhibitors are not as promising as expected, prompting a search for additional drugable targets to treat these disorders. In this paper, we used the hypomorphic Gata1low mouse model of primary myelofibrosis (PMF), the most severe of these neoplasms, to test the hypothesis that defective marrow hemopoiesis and development of extramedullary hematopoiesis in myelofibrosis is due to insufficient p27Kip1 activity and is treatable by Aplidin®, a cyclic depsipeptide that activates p27Kip1 in several cancer cells. Aplidin® restored expression of Gata1 and p27Kip1 in Gata1low hematopoietic cells, proliferation of marrow progenitor cells in vitro and maturation of megakaryocytes in vivo (reducing TGF‐β/VEGF levels released in the microenvironment by immature Gata1low megakaryocytes). Microvessel density, fibrosis, bone growth, and marrow cellularity were normal in Aplidin®‐treated mice and extramedullary hematopoiesis did not develop in liver although CXCR4 expression in Gata1low progenitor cells remained low. These results indicate that Aplidin® effectively alters the natural history of myelofibrosis in Gata1low mice and suggest this drug as candidate for clinical evaluation in PMF. J. Cell. Physiol. 225: 490–499, 2010.

Boronophenylalanine uptake in C6 glioma model is dramatically increased by L-DOPA preloading.

One of the main limitations for BNCT effectiveness is the insufficient intake of (10)B nuclei within tumour cells. This work was aimed at investigating the use of L-DOPA as enhancer for boronophenylalanine (BPA) uptake in the C6 glioma model. The investigation was first performed in vitro, and then extended in vivo to the animal model. BPA accumulation in C6 glioma cells was assessed, using radiowave dielectric spectroscopy (RDS), with and without L-DOPA preloading. C6 glioma cells were also implanted in the brain of 25 rats, randomly assigned to two experimental branches: (1) intra-carotid BPA infusion; (2) intra-carotid BPA infusion after pre-treatment with L-DOPA, administrated 24 h before BPA infusion. All animals were sacrificed, and assessment of BPA concentrations in tumour tissue, normal brain, and blood samples was performed using high performance liquid chromatography (HPLC). L-DOPA preloading induced a massive increase of BPA concentration either in vitro on C6 glioma cells or in vivo in the animal model tumour. Moreover, no significant difference was found in the normal brain and blood samples between the two animal groups. This study suggests the potential use of L-DOPA as enhancer for BPA accumulation in malignant gliomas eligible for BNCT.

A rapid serum neutralization test in microplates for the detection of antibodies to hog cholera virus.

The fluorescent antibody serum neutralization (FASN) test for the detection of antibodies to hog cholera virus was developed utilizing 96-well and Terasaki microplates. This microtechnique, especially when performed in Terasaki plates, offers some advantage if compared with conventional FASN in coverslip cell cultures, being easier and more rapid, saving of reagents and allowing simple microscopic observation.

Evidence for Organ-Specific Stem Cell Microenvironments

The X‐linked Gata1low mutation in mice induces strain‐restricted myeloproliferative disorders characterized by extramedullary hematopoiesis in spleen (CD1 and DBA/2) and liver (CD1 only). To assess the role of the microenvironment in establishing this myeloproliferative trait, progenitor cell compartments of spleen and marrow from wild‐type and Gata1low mice were compared. Phenotype and clonal assay of non‐fractionated cells indicated that Gata1low mice contain progenitor cell numbers 4‐fold lower and 10‐fold higher than normal in marrow and spleen, respectively. However, progenitor cells prospectively isolated from spleen, but not from marrow, of Gata1low mice expressed colony‐forming function in vitro. Therefore, calculation of cloning activity of purified cells demonstrated that the total number of Gata1low progenitor cells was 10‐ to 100‐fold lower than normal in marrow and >1,000 times higher than normal in spleen. This observation indicates that Gata1low hematopoiesis is favored by the spleen and is in agreement with our previous report that removal of this organ induces wild‐type hematopoiesis in heterozygous Gata1low/+ females (Migliaccio et al., 2009, Blood 114:2107). To clarify if rescue of wild‐type hematopoiesis by splenectomy prevented extramedullary hematopoiesis in liver, marrow cytokine expression profile and liver histopathology of splenectomized Gata1low/+ females were investigated. After splenectomy, the marrow expression levels of TGF‐β, VEGF, osteocalcin, PDGF‐α, and SDF‐1 remained abnormally high while Gata1low hematopoiesis was detectable in liver of both CD1 and DBA/2 mutants. Therefore, in the absence of the spleen, Gata1low hematopoiesis is supported by the liver suggesting that treatment of myelofibrosis in these animals requires the rescue of both stem cell and microenvironmental functions. J. Cell. Physiol. 223: 460–470, 2010.

Abnormal P-selectin localization during megakaryocyte development determines thrombosis in the gata1low model of myelofibrosis

Abstract Patients with primary myelofibrosis have increased risk for bleeding and thrombosis. It is debated whether propensity to thrombosis is due to increased numbers of platelet microparticles and/or to pathological platelet-neutrophil interactions. Platelet neutrophil interactions are mediated by P-selectin and even though the megakaryocytes of myelofibrosis patients express normal levels of P-selectin, it remains abnormally localized to the demarcation membrane system rather than being assembled into the α-granules in platelets. Mice carrying the hypomorphic Gata1low mutation express the same megakaryocyte abnormalities presented by primary myelofibrosis patients, including abnormal P-selectin localization to the DMS and develop with age myelofibrosis, a disease that closely resembles human primary myelofibrosis. Whether these mice would also develop thrombosis has not been investigated as yet. The aim of this study was to determine whether Gata1low mice would develop thrombosis with age and, in this case, the role played by P-selectin in the development of the trait. To this aim, Gata1low mice were crossed with P-selnull mice according to standard genetic protocols and Gata1lowP-selwt, Gata1lowP-selnull and Gata1WTP-selnull or Gata1wtP-selwt (as controls) littermates obtained. It was shown that platelet counts, but not hematocrit, are reduced in Gata1low mice. Moreover, platelet microparticles are reduced in Gata1low mice and P-selectin positive platelet microparticles were not found. To determine the phenotypic implications of the different mutations, bleeding time was estimated by a tail cut procedure. Mutant mice were sacrificed and presence of thrombosis was determined by immunohistological staining of organs. Gata1low mice with or without the P-selectin null trait had a prolonged bleeding time compared to wild type mice. However, in Gata1low mice significantly higher frequency of thrombotic events was seen in adult and old Gata1low mice compared to Gata1lowP-selnull mice. Thus, presence of the P-selectin null trait rescued Gata1low mice from the thrombotic phenotype, but did not change the level of platelet microparticles. Taken together these data indicate that abnormal localization of P-selectin, induced by the Gata1low mutation, and thus, increased pathological interactions with leucocytes, is responsible for the increased presence of thrombosis seen in these mice.