Development of a mouse model for the t(10:11)(p13;q14) chromosomal translocation associated with acute leukemia in humans

Acute leukemia is associated with a wide spectrum of gross chromosomal rearrangements. These acquired mutations include balanced and unbalanced chromosomal translocations. The analysis of chromosomal translocations has provided much insight into understanding the biology of hematologic malignancies, leading to improved diagnosis and classification, as well as identification of novel therapeutic targets. The rare but recurring chromosomal translocation [t(10;11)(p13;q21)] results in a CALM-AF10 fusion that occurs in patients with both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). CALM-AF10 transgenic mice developed AML with lymphoid features and had Hoxa gene cluster upregulation. In this model, mice developed leukemia after a long latency period with incomplete penetrance. These findings suggest that additional genetic events are needed to complement CALM-AF10 mediated leukemic transformation. Retroviral insertional mutagenesis was used to identify complementary genetic events that might collaborate with CALM-AF10 during leukemic transformation. A cohort of CALM-AF10 mice was infected with the Mol4070LTR retrovirus; by 5.5 months of age, 50% of the transgenic mice developed AML, a clear acceleration of disease onset compared to either wild type littermates injected with the retrovirus or CALM-AF10 mice not injected with the retrovirus. The tumors assayed by Southern blotting for viral integration showed clonal to oligoclonal expansion. Ligation-mediated PCR and sequence analysis of DNA derived from leukemic cells was used to identify potential collaborating genes at the retroviral insertion sites including Evi1, Nf1, kRas, Zeb2, and Mnl. Identification of these genes as a potential collaborating gene with CALM-AF10 supports the emerging paradigm in leukemia biology that predicts that most, if not all leukemic cells must undergo at least two collaborative events to produce a fully transformed cell. One of these events typically leads to impaired differentiation and enhanced renewal of stem cells, whereas the second event leads to increased proliferation and/or decreased apoptosis. It has been shown here that retroviral infection accelerates the onset of acute leukemia, and identified genes that potentially collaborate with the CALM-AF10 fusion gene in the leukemic transformation process. This transgenic murine model serves as a model system for studying leukemogenesis similar to that observed in humans with leukemia

Quantification of HTLV-I proviral load in experimentally infected rabbits

BACKGROUND: Levels of proviral load in HTLV-1 infected patients correlate with clinical outcome and are reasonably prognostic. Adaptation of proviral load measurement techniques is examined here for use in an experimental rabbit model of HTLV-1 infection. Initial efforts sought to correlate proviral load with route and dose of inoculation and with clinical outcome in this model. These methods contribute to our continuing goal of using the model to test treatments that alleviate virus infection. RESULTS: A real-time PCR assay was used to measure proviral load in blood and tissue samples from a series of rabbits infected using HTLV-1 inocula prepared as either cell-free virus particles, infected cells or blood, or by naked DNA injection. Proviral loads from asymptomatically infected rabbits showed levels corresponding to those reported for human patients with clinically silent HTLV-1 infections. Proviral load was comparably increased in 50% of experimentally infected rabbits that developed either spontaneous benign or malignant tumors while infected. Similarly elevated provirus was found in organs of rabbits with experimentally induced acute leukemia/lymphoma-like disease. Levels of provirus in organs taken at necropsy varied widely suggesting that reservoirs of infections exist in non-lymphoid organs not traditionally thought to be targets for HTLV-1. CONCLUSION: Proviral load measurement is a valuable enhancement to the rabbit model for HTLV-1 infection providing a metric to monitor clinical status of the infected animals as well as a means for the testing of treatment to combat infection. In some cases proviral load in blood did not reflect organ proviral levels, revealing a limitation of this method for monitoring health status of HTLV-1 infected individuals

Deletion of PPAR-γ in immune cells enhances susceptibility to antiglomerular basement membrane disease

Activation of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR-γ) has been shown to be immunoregulatory in autoimmune diseases by inhibiting production of a number of inflammatory mediators. We investigated whether PPAR-γ gene deletion in hematopoietic cells would alter disease pathogenesis in the antiglomerular basement membrane (anti-GBM) mouse model. PPAR-γ+/+ and PPAR-γ−/− mice were immunized with rabbit antimouse GBM antibodies and lipopolysaccharide and evaluated for two weeks. Although both the PPAR-γ+/+ and PPAR-γ−/− mice had IgG deposition in the glomerulus and showed proteinuria two weeks after injection, glomerular and tubulointerstitial disease in PPAR-γ−/− mice were significantly more severe compared with the PPAR-γ+/+ animals. We observed that the PPAR-γ−/− mice had decreased CD4+CD25+ regulatory T cells and an increased CD8+:CD4+ ratio as compared with the PPAR-γ+/+ mice, suggesting that PPAR-γ has a role in the regulation of T cells. Furthermore, plasma interleukin-6 levels were significantly increased in the PPAR-γ−/− mice at two weeks as compared with the PPAR-γ+/+ animals. Taken together, these studies show that the lack of PPAR-γ expression enhances inflammatory renal disease in the anti-GBM antibody-induced glomerulonephritis mouse model and suggests targeting PPAR-γ may have therapeutic efficacy

A surety engineering framework to reduce cognitive systems risks.

Cognitive science research investigates the advancement of human cognition and neuroscience capabilities. Addressing risks associated with these advancements can counter potential program failures, legal and ethical issues, constraints to scientific research, and product vulnerabilities. Survey results, focus group discussions, cognitive science experts, and surety researchers concur technical risks exist that could impact cognitive science research in areas such as medicine, privacy, human enhancement, law and policy, military applications, and national security (SAND2006-6895). This SAND report documents a surety engineering framework and a process for identifying cognitive system technical, ethical, legal and societal risks and applying appropriate surety methods to reduce such risks. The framework consists of several models: Specification, Design, Evaluation, Risk, and Maturity. Two detailed case studies are included to illustrate the use of the process and framework. Several Appendices provide detailed information on existing cognitive system architectures; ethical, legal, and societal risk research; surety methods and technologies; and educing information research with a case study vignette. The process and framework provide a model for how cognitive systems research and full-scale product development can apply surety engineering to reduce perceived and actual risks

Identification of a Common Lupus Disease-Associated microRNA Expression Pattern in Three Different Murine Models of Lupus

Recent reports have shown that microRNAs (miRNAs) regulate vital immunological processes and have emerged as key regulators of immune system development and function. Therefore, it is important to determine miRNA dysregulation and its pathogenic contribution in autoimmune diseases, an aspect not adequately addressed thus far.In this study, we profiled miRNA expressions in splenic lymphocytes from three murine lupus models (MRL-lpr, B6-lpr and NZB/W(F₁)) with different genetic background by miRNA microarray assays and Real-time RT-PCR. Despite the genetic differences among these three lupus stains, a common set of dysregulated miRNAs (miR-182-96-183 cluster, miR-31, and miR-155) was identified in splenocytes when compared with age-matched control mice. The association of these miRNAs with the disease was highlighted by our observation that this miRNA expression pattern was evident in NZB/W mice only at an age when lupus disease is manifested. Further, we have shown that the miRNA dysregulation in MRL-lpr mice was not simply due to the activation of splenocytes. By Real-time RT-PCR, we confirmed that these miRNAs were upregulated in both purified splenic B and T cells from MRL-lpr mice. miR-127 and miR-379, which were greatly upregulated in splenocytes from lpr mice, were moderately increased in diseased NZB/W mice. In addition, Real-time RT-PCR revealed that miR-146a, miR-101a, and miR-17-92 were also markedly upregulated in splenic T, but not B cells from MRL-lpr mice.The identification of common lupus disease-associated miRNAs now forms the basis for the further investigation of the pathogenic contribution of these miRNAs in autoimmune lupus, which will advance our knowledge of the role of miRNAs in autoimmunity. Given that miRNAs are conserved, with regard to both evolution and function, our observation of a common lupus disease-associated miRNA expression pattern in murine lupus models is likely to have significant pathogenic, diagnostic, and/or therapeutic implications in human lupus

The efficacy of the ribonucleotide reductase inhibitor Didox in preclinical models of AML.

Acute Myeloid Leukemia (AML) is an aggressive malignancy which leads to marrow failure, and ultimately death. There is a desperate need for new therapeutics for these patients. Ribonucleotide reductase (RR) is the rate limiting enzyme in DNA synthesis. Didox (3,4-Dihydroxybenzohydroxamic acid) is a novel RR inhibitor noted to be more potent than hydroxyurea. In this report we detail the activity and toxicity of Didox in preclinical models of AML. RR was present in all AML cell lines and primary patient samples tested. Didox was active against all human and murine AML lines tested with IC50 values in the low micromolar range (mean IC50 37 µM [range 25.89-52.70 µM]). It was active against primary patient samples at concentrations that did not affect normal hematopoietic stem cells (HSCs). Didox exposure resulted in DNA damage and p53 induction culminating in apoptosis. In syngeneic, therapy-resistant AML models, single agent Didox treatment resulted in a significant reduction in leukemia burden and a survival benefit. Didox was well tolerated, as marrow from treated animals was morphologically indistinguishable from controls. Didox exposure at levels that impaired leukemia growth did not inhibit normal HSC engraftment. In summary, Didox was well tolerated and effective against preclinical models of AML

Gene expression profiling and candidate gene resequencing identifies pathways and mutations important for malignant transformation caused by leukemogenic fusion genes

NUP98-HOXD13 (NHD13) and CALM-AF10 (CA10) are oncogenic fusion proteins produced by recurrent chromosomal translocations in patients with acute myeloid leukemia (AML). Transgenic mice that express these fusions develop AML with a long latency and incomplete penetrance, suggesting that collaborating genetic events are required for leukemic transformation. We employed genetic techniques to identify both preleukemic abnormalities in healthy transgenic mice as well as collaborating events leading to leukemic transformation. Candidate gene resequencing revealed that 6 of 27 (22%) CA10 AMLs spontaneously acquired a Ras pathway mutation and 8 of 27 (30%) acquired an Flt3 mutation. Two CA10 AMLs acquired an Flt3 internal-tandem duplication, demonstrating that these mutations can be acquired in murine as well as human AML. Gene expression profiles revealed a marked upregulation of Hox genes, particularly Hoxa5, Hoxa9, and Hoxa10 in both NHD13 and CA10 mice. Furthermore, mir196b, which is embedded within the Hoxa locus, was overexpressed in both CA10 and NHD13 samples. In contrast, the Hox cofactors Meis1 and Pbx3 were differentially expressed; Meis1 was increased in CA10 AMLs but not NHD13 AMLs, whereas Pbx3 was consistently increased in NHD13 but not CA10 AMLs. Silencing of Pbx3 in NHD13 cells led to decreased proliferation, increased apoptosis, and decreased colony formation in vitro, suggesting a previously unexpected role for Pbx3 in leukemic transformation