Michelina Iacovino

PNPLA3 has retinyl-palmitate lipase activity in human hepatic stellate cells

Retinoids are micronutrients that are stored as retinyl esters in the retina and hepatic stellate cells (HSCs). HSCs are key players in fibrogenesis in chronic liver diseases. The enzyme responsible for hydrolysis and release of retinyl esters from HSCs is unknown and the relationship between retinoid metabolism and liver disease remains unclear. We hypothesize that the patatin-like phospholipase domain-containing 3 (PNPLA3) protein is involved in retinol metabolism in HSCs. We tested our hypothesis both in primary human HSCs and in a human cohort of subjects with non-alcoholic fatty liver disease (N = 146). Here we show that PNPLA3 is highly expressed in human HSCs. Its expression is regulated by retinol availability and insulin, and increased PNPLA3 expression results in reduced lipid droplet content. PNPLA3 promotes extracellular release of retinol from HSCs in response to insulin. We also show that purified wild-type PNPLA3 hydrolyzes retinyl palmitate into retinol and palmitic acid. Conversely, this enzymatic activity is markedly reduced with purified PNPLA3 148M, a common mutation robustly associated with liver fibrosis and hepatocellular carcinoma development. We also find the PNPLA3 I148M genotype to be an independent (P = 0.009 in a multivariate analysis) determinant of circulating retinol-binding protein 4, a reliable proxy for retinol levels in humans. This study identifies PNPLA3 as a lipase responsible for retinyl-palmitate hydrolysis in HSCs in humans. Importantly, this indicates a potential novel link between HSCs, retinoid metabolism and PNPLA3 in determining the susceptibility to chronic liver disease.

NLRP7 affects trophoblast lineage differentiation, binds to overexpressed YY1 and alters CpG methylation

Maternal-effect mutations in NLRP7 cause rare biparentally inherited hydatidiform moles (BiHMs), abnormal pregnancies containing hypertrophic vesicular trophoblast but no embryo. BiHM trophoblasts display abnormal DNA methylation patterns affecting maternally methylated germline differentially methylated regions (gDMRs), suggesting that NLRP7 plays an important role in reprogramming imprinted gDMRs. How NLRP7-a component of the CATERPILLAR family of proteins involved in innate immunity and apoptosis-causes these specific DNA methylation and trophoblast defects is unknown. Because rodents lack NLRP7, we used human embryonic stem cells to study its function and demonstrate that NLRP7 interacts with YY1, an important chromatin-binding factor. Reduced NLRP7 levels alter DNA methylation and accelerate trophoblast lineage differentiation. NLRP7 thus appears to function in chromatin reprogramming and DNA methylation in the germline or early embryonic development, functions not previously associated with members of the NLRP family.

Rapid Genetic Modification of Mouse Embryonic Stem Cells by Inducible Cassette Exchange Recombination

Embryonic stem cell (ESC) differentiation is a useful means by which to produce large quantities of cells in vitro representing early stages of embryonic development. A conditional gene expression system allows interrogation of factors at specific time points in the differentiation of ES cells to defined cell types. We have developed a method for rapidly generating conditional inducible murine ES cells by targeting genes into an Inducible Cassette Exchange (ICE) locus. The ICE locus encodes a doxycycline-inducible floxed Cre, which replaces itself with an incoming floxed gene of interest. The derivative cell lines, selected in G418, thus bear doxycycline-inducible transgenes. We provide detailed methods for performing ICE recombination and generating derivative doxycycline-inducible cell lines.

Notch activation is required for downregulation of HoxA3-dependent endothelial cell phenotype during blood formation

Hemogenic endothelium (HE) undergoes endothelial-to-hematopoietic transition (EHT) to generate blood, a process that requires progressive down-regulation of endothelial genes and induction of hematopoietic ones. Previously, we have shown that the transcription factor HoxA3 prevents blood formation by inhibiting Runx1 expression, maintaining endothelial gene expression and thus blocking EHT. In the present study, we show that HoxA3 also prevents blood formation by inhibiting Notch pathway. HoxA3 induced upregulation of Jag1 ligand in endothelial cells, which led to cis-inhibition of the Notch pathway, rendering the HE nonresponsive to Notch signals. While Notch activation alone was insufficient to promote blood formation in the presence of HoxA3, activation of Notch or downregulation of Jag1 resulted in a loss of the endothelial phenotype which is a prerequisite for EHT. Taken together, these results demonstrate that Notch pathway activation is necessary to downregulate endothelial markers during EHT.

430. Antibodies to Recombinant Human α-L-Iduronidase Enhance Uptake into Macrophages in Murine Mucopolysaccharidosis Type I

Mucopolysaccharidosis I (MPS I) is an inherited, and progressive lysosomal storage disease caused by lack or low level of α-L-iduronidase (IDU), altering the catabolism of glycosaminoglycans (GAG) in lysosomes. Some clinical improvement in patients has been observed by enzyme replacement therapy with recombinant human IDU (rhIDU), but studies suggested that anti-rhIDU antibodies reduce the effectiveness of treatment. Most MPS I treated patients develop a humoral immune response against rhIDU. To determine how anti-rhIDU antibodies alter the tissue and cellular distribution of rhIDU in vivo, we sensitized MPS I mice to rhIDU by administering rhIDU via tail-vein injection from 4 to 16 weeks of age. Mice that developed anti-rhIDU antibodies showed a shift in rhIDU uptake preferentially towards tissues with high reticuloendothelial (RE) content (liver, spleen, thymus) vs. low RE content (lung, kidney, heart, brain). Furthermore, in mice with anti-rhIDU antibodies, rhIDU was mainly localized in Kupffer cells, with relatively less available for hepatocytes. In order to further understand how the humoral immune response against rhIDU alters its distribution, we comparedin vitro uptake of rhIDU in the absence or presence of antibodies against rhIDU in MPS I fibroblasts and macrophages. Bone marrow monocytes were harvested from MPS I mice and derived into macrophages. MPS I macrophages were incubated with rhIDU with or without immunized rabbit serum, pre-immune serum, murine Fc receptor blocking agent and mannose 6-phosphate. We evaluated two types of immunized rabbit serum: one that completely abolished rhIDU uptake into human MPS I fibroblasts (inhibiting serum) and one that only partially (30%) inhibited rhIDU uptake into human fibroblasts (partially-inhibiting serum). The uptake of rhIDU in the presence of serum containing rhIDU antibodies was enhanced into MPS I mouse macrophages when both inhibiting and partially-inhibiting serum was applied. Uptake of rhIDU per cell was higher in macrophages when immune serum than was uptake of rhIDU into human fibroblasts normalized per cell. Treatment with mannose 6-phosphate or Fc receptor block partially inhibited the uptake in macrophages. Our study demonstrated that even a low-level humoral immune response against rhIDU, which only partially inhibits rhIDU uptake into fibroblasts in vitro, nevertheless might alter its tissue distribution in vivo. At the cellular level, antibody-positive mice show reduced rhIDU distribution in hepatocytes, while distribution to tissue macrophages is maintained. In vitro, immunized serum reduced rhIDU uptake into human fibroblasts but increased uptake into murine macrophages. Our findings imply that the altered tissue distribution of rhIDU caused by anti-rhIDU antibodies is partly due to reduced uptake into fibroblasts and partly due to enhanced uptake into tissue macrophages. These results imply that functional immune assays of rhIDU uptake in vitro into fibroblasts may not completely predict the impact of the humoral immune response against enzyme replacement therapy.

HoxA3 function during blood development

D in fibrinolytic markers can result in thrombosis and cardiovascμlar problems and anti-retroviral therapy (ART) has been reported to affect the levels of these markers. How long the patients will be exposed to these ART before the effect of the drugs on the fibrinolytic markers become noticeable is not well known. This study was aimed at bridging this knowledge gap. 20 HIV subjects on ART and 20 controls (nonART) were progressively monitored for three months. CD4 T cell count was determined using the Partec Flow cytometry counter while D-dimer, t-PA and PAI-1 parameters were determined using ELISA kits from TECHNOCLONE, Austria. CD4 cell count increased from 192μ/ml at baseline to 323 μl/ml at month 3 among patients on antiretroviral therapy. D-Dimer values decreased from 301.0μl/ml at baseline to 172.0 μl/ml at month 2 and increased significantly to 226.0 μl/ml at the end of the third month. The median baseline value of PA1-1 at the beginning of therapy was 14.0μg/ml, which increased progressively to 18.2μg/ml at the end of the third month. The baseline value of t-PA at the beginning of therapy was 5.15μg/ml. This value progressively declined to 1.10μg/ml at the end of the first month and increased minimally to 1.45μg/ml and 1.5μg/ml at the end of the 2nd and 3rd month respectively. D-dimer was positively and significantly correlated with CD4 cell counts in both ARV and non-ARV patients.(r = -0.304, p<0.01 vs. r= -0.477; p<0.001). t-PA was negatively and significantly correlated with CD4 T lymphocytes in only those undergoing the antiretroviral therapy( r= -0.294, p<0.01). A progressive increase in PAI-1 value and steady decline in t-PA values within three months of commencement of antiretroviral therapy is capable of predisposing the patients to thrombotic disorders earlier than is expected. Pre-thrombotic assessment during therapy is hereby advocated. Zaccheaus A. Jeremiah, J Blood Disord Transfus 2013, 4:5 http://dx.doi.org/10.4172/2155-9864.S1.002O activation of cell survival programs is a classical hallmark of cancer, allowing malignant cells to evade cytotoxic therapies. Although the concept of targeting cell survival in cancer has long been proposed, it has not yet proven clinically successful for the majority of cancers including acute myeloid leukemia (AML). We have identified a novel tyrosine kinase independent signalling mechanism by which diverse growth factor receptors regulate cell survival but not other cellular responses such as proliferation. We show that this “survival only” response is regulated via non‐canonical growth factor receptor pathways that, remarkably, require the protein kinase activity of phosphoinositol 3‐kinase (PI3K) but not its lipid kinase activity. The protein kinase activity of PI3K is constitutively activated in >80% of primary human AML patient samples and selective targeting of this novel PI3K pathway either pharmacologically or by RNAi results in the rapid induction of apoptosis. We therefore screened compound libraries for drugs that target “survival‐only” pathways and have identified the kinase inhibitor, PIK‐75, that is a potent inducer of apoptosis across a panel of 44 genetically diverse primary AML cases (mean IC50=395nM) at concentrations which did not affect the viability of non‐transformed human bone marrow progenitors (p=0.0074). PIK‐75 represents a first‐ in‐class compound that blocks survival signalling in AML cells through dual inhibition of PI3K and the transcriptional kinase, cyclin dependent kinase 9 (Cdk9)(Kd<10nM), thereby transiently blocking RNA Polymerase II‐mediated transcription leading to the rapid loss of the prosurvival protein, Mcl‐1. Our studies reveal a previously unrecognized alliance between PI3K and Cdk9 in promoting oncogenic survival signals and we show that the simultaneous inhibition of PI3K and Cdk9 is highly synergistic not only AML cells (combination index <0.2), but tumor cells derived from multiple myeloma, breast cancer and brain cancer. Importantly, PIK‐75 treatment is well tolerated in mice, results in the significant reduction of leukemic loads in mice engrafted with human AML cells (p=0.0059), and significantly increases their median survival (p=0005). Thus, our studies identify a novel lead compound that selectively targets two distinct and independent regulators of survival in AML. With the limited clinical efficacy reported so far for many kinase inhibitors, further investigation of dual PI3K and Cdk9 inhibitors is warranted. Elizabeth Tegg, J Blood Disord Transfus 2013, 4:5 http://dx.doi.org/10.4172/2155-9864.S1.002A cure rates for acute lymphoblastic leukemia (ALL) in children are high (85~90%), chemotherapeutic drug resistance still remains a major problem and leads to death in 50-95%. Even survived, patients often suffer from late-term secondary toxic effects of current treatments. Therefore, novel therapy modalities with less toxicity are needed to develop for ALL patients. A preclinical model has been established by evaluating new therapies in NOD/SCID or NSG xenograft model engrafted with primary ALL cells. The immunophenotype and morphology of the original patient’s disease can be retained after a serial of passages in this mouse xenograft model. Response to drugs of interests can be monitored by in vivo bioluminescent imaging and accurately reflects the outcome of ALL patients receiving the novel therapy. Interaction between leukemia cells and their microenvironment has been considered as a new therapeutic target. Bone marrow (BM) stromal cells provide chemoprotection to ALL, thus contributing to drug resistance due to the lack of efficacy of current therapies. In fact, more than 80% of the sites of first relapse in childhood ALL is bone marrow with only ~20% of 5-year survival rate. Integrin α4 mediates adhesion of normal and malignant B-cell precursors to BM stromal cells. According to gene expression profile, integrin α4 is overexpressed in ALL patients and inversely correlated with the survival outcome. Therefore, our group tested whether interference with α4-mediated stromal adhesion could be a new ALL treatment strategy. For this purpose, two models of leukemia were used, one genetic (conditional α4 ablation of BCR-ABL1-induced murine leukemia) and one pharmacological using antibody, like Tysabri to target α4 of primary pre-B ALL. Conditional deletion of α4 sensitized murine leukemia cells to chemotherapeutic agent Nilotinib. Adhesion of primary pre-B ALL cells was α4-dependent and inhibiting α4 sensitized primary ALL cells towards chemotherapy, VDL. Combination of chemotherapy with Tysabri prolonged survival of NOD/SCID recipients of primary ALL suggesting adjuvant integrin α4 inhibition as a novel strategy for pre-B ALL. Douglas K. Graham, J Blood Disord Transfus 2013, 4:5 http://dx.doi.org/10.4172/2155-9864.S1.002D development, hematopoietic stem cells (HSC) are generated in the major arteries of the developing embryos. Between E8.5 and E10.5 in the caudal domain of the aorta, specialized endothelial cells, referred as hemogenic endothelium, undergo to structural changes to become blood progenitors and some of them HSC. How those specialized endothelial cells are committed and what regulates their transformation into blood progenitors is mostly unknown. Runx1 transcription factor is essential for blood development and necessary to generate HSC from the vasculature. We have discovered that HoxA3, a gene exclusively expressed in the vasculature of the embryo, controls the endothelial to hematopoietic transition. HoxA3 is expressed in the aorta prior the release of blood progenitors. By using the ES cells differentiation system, we show that HoxA3 upregulation causes blockage of the endothelial to hematopoietic transition, maintaining the cells at the hemogenic endothelium stage.The arrest of blood development is carried out by downregulation of key transcription factor including Gata1, Gfi1B, Ikaros, PU.1, and Runx1. With epistasis analysis we found that HoxA3 repression of Runx1 is sufficient to block the entire blood development process. In fact we found that when Runx1 is expressed in hemogenic endothelium, it promotes blood development by a dual function: it activates blood related transcription factors, and represses endothelial related genes. As a result the hemogenic endothelium can further develop into blood progenitor cells. Michelina Iacovino, J Blood Disord Transfus 2013, 4:5 http://dx.doi.org/10.4172/2155-9864.S1.002D sepsis there is aberrant activation, migration and sequestration of neutrophils in visceral organsthat leads to widespread release of pro-inflammatory mediators contributing to multi-organ failure and death. Interaction of cell surface integrins with their counterpart ligands, results in the adherence of circulating neutrophils and directed migration to infection sites.In our study, we show that administration of a cyclic analog of RGD peptide (Arg-Gly-Asp) significantly reduced the number of tissue-invading neutrophils and the degree of sepsis-induced lethality in mice as compared to control peptide. Secondly, β1 integrin (CD29) and more specifically VLA-3(CD49c/CD29) is dramatically up regulated on a subpopulation of neutrophils isolated from both human septic patients and mouse sepsis models. Compared with the Gr1highCD11bhighVLA-3low granulocyte population, Gr1highCD11bhighVLA-3high cells from septic animals displayed hyper-inflammatory phenotypes.Administration of VLA-3 antagonist peptides and conditional genetic ablation of VLA(3)/integrin from granulocytes also improved survival and bacterial clearance in septic animals. Thus, our results indicate that expression of VLA(3)/integrin is important for modulating neutrophil trafficking during sepsis, and therapeutics specifically targeted against VLA(3)/integrin may be beneficial. Pranita P. Sarangi, J Blood Disord Transfus 2013, 4:5 http://dx.doi.org/10.4172/2155-9864.S1.002M recognition is an inherent cognitive skill in all higher species of living beings. In human life, the skill starts right from the early infancy. It is well presumed that even in distant future; human beings will recognize each other by the facial morphology and would call each other by their names. So it happens in the recognition of blood cell morphology as well in the initial diagnosis of a hematological disorder. The value of morphological evaluation is crucial in the diagnostic workup of leukemia. Even after so much of advancement in the immunophenotypic and molecular characterization of hemopoietic cells in the present era, the WHO consensus on blast cell count in the bone marrow sample strictly remains a visual recognition of the blasts by their morphology [1]. It is reported well in literatures that a specific morphology of blast often coincides with a specific recurrent genetic abnormality. The reason being unknown and performance of morphological skill being subjective to the level of expertise on the part of the morphologist; recognition of these specific morphological features of a blast still uphold its clinical significance for an early diagnosis and timely intervention. Finding of multiple Auer rods in a blast is almost always consistent with t(15;17)(q22;q12); PML-RARA; a consistent morphological signature of a recurrent genetic abnormality in acute promyelocytic leukemia, the recognition of which leads to a timely intervention to prevent complications arising from disseminate intravascular coagulation (DIC) and severe thrombocytopenia. Likewise, a long slender Auer rod with tapering ends facing a nuclear indentation might suggest the molecular event, t(8;21)(q22;q22); RUNX1-RUNX1T1 which bears a good prognosis; presence of a few dysplastic eosinophils in the bone marrow of an acute myeloblastic leukemia is frequently associated with the genetic event, t(9;11)(q22;q23); MLLT3-MLL that has a poor prognosis; etc. This study reveals the consistency of such well recognized morphological features with recurrent genetic abnormalities in acute leukemia as defined by the World Health Organization Classification of leukemia/lymphoma, 2008. This study was carried out amongst 120 cases of newly diagnosed acute leukemia in a regional cancer institute in India. A complete diagnostic work-up included a complete blood count, peripheral blood and bone marrow smears, bone marrow trephine biopsy and touch imprints, cytochemical study with myeloperoxidase and non-specific esterase; multiparametric 2-laser-4-color flow cytometry and multiplex PCR that could detect 28 translocations or chromosomal rearrangements including more than 80 breakpoints of mRNA spice variants. This study demonstrated a significant correlation of underlying recurrent genetic abnormalities in acute leukemia with corresponding, well defined variations in the blast morphology. This study also emphasizes the clinical significance and cost effectiveness of early recognition of such variants of blastic morphology consistent with specific recurrent genetic abnormalities. Biswadip Hazarika, J Blood Disord Transfus 2013, 4:5 http://dx.doi.org/10.4172/2155-9864.S1.002