The Polo-Like Kinase 1 (PLK1) Inhibitor NMS-P937 Is Effective in a New Model of Disseminated Primary CD56+ Acute Monoblastic Leukaemia

CD56 is expressed in 15–20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56⁺ monoblastic AML (M5a). The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting (MST = 28, 36, 62 days for vehicle, cytarabine and NMS-P937, respectively). Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56⁺ AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML.     

BCR-ABL Promotes PTEN Downregulation in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the t(9;22) translocation coding for the chimeric protein p210 BCR-ABL. The tumor suppressor PTEN plays a critical role in the pathogenesis of CML chronic phase, through non genomic loss of function mechanisms, such as protein down-regulation and impaired nuclear/cytoplasmic shuttling. Here we demonstrate that BCR-ABL promotes PTEN downregulation through a MEK dependent pathway. Furthermore, we describe a novel not recurrent N212D-PTEN point mutation found in the EM2 blast crisis cell line.     

Agonistic Encounters in a Cricket Frog (Acris crepitans) Chorus: Behavioral Outcomes Vary with Local Competition and within the Breeding Season

Male cricket frogs ( Acris crepitans ) gather in breeding choruses and call to attract females. The call also serves to maintain an individual's calling space. When an intruder invades a resident male's space, the resident will display one of three behavior patterns. He will either attack the intruder, abandon calling, or tolerate the intruder's presence and continue calling. We simulated an intruder by broadcasting a stimulus from a speaker and categorized the response of the resident male. We recorded social variables relevant to local competition among males, specifically, local caller density (within 2 m) and the amplitude of the nearest neighbor's calls. In addition we recorded the size of the subject, the amplitude of the stimulus, and the time of night and the season. We used a multivariate approach to assess the relative importance of these variables. Local caller density and nearest neighbor call amplitude were the best predictors of a resident's response to the intruder; higher levels of local competition resulted in more males tolerating the intruder. In addition, behavior changed over the season: males were more likely to abandon or ignore the intruder early in the season and more likely to attack later in the season. No other variables were related to the outcome of the agonistic encounter. This study suggests that the most important variables impinging on a male's decision to fight, flee, or ignore an intruder are influences external to the individuals involved in the conflict.     

Ligand-based histochemical localization and capture of cells expressing heat-stable enterotoxin receptors

The heat stable enterotoxins (ST) of enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by binding specific intestinal receptors. Precise histochemical localization of ST receptors could provide more information about the pathophysiology of secretory diarrhoea and the role of ST receptors in normal biology. To accomplish this, we quantitatively coupled biotin to the N-terminus of ST1b using biotin-X-X-N-hydroxysuccinimide ester. The derivatized toxin (BST) has an apparent K_d of 11.7±10 nM for rat brush border receptors. We used BST in an affinity panning cell-capture system, to validate its ability to discriminate between receptor-positive and receptor-negative cells. Cell lines expressing ST receptors (human colon carcinoma T84, and COS cells transfected with guanylyl cyclase-C (GC-C) ST receptor cDNA) were captured to streptavidin and anti-biotin-coated plates with high efficiency and specificity. This system provides a novel approach to screening cells for the presence of unique ST-binding proteins. BST was then used with streptavidin-gold to demonstrate the cellular topography of ST receptors at the light microscopic level. Villus enterocytes were intensely stained, but only a faint signal was observed in upper crypts of rat small intestine. Thus, a gradient of increasing receptor density was seen as upper crypt cells matured into villus enterocytes. Higher magnification revealed that ST receptors are concentrated at the apical aspect of villus enterocytes. Recently, guanylin, a putative endogenous ligand for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts. Thus, ST receptors, are concentrated in villus entercoytes, while guanylin appears to be produced at the base of the crypts. This topographical arrangement suggests that there are autocrine or paracrine pathways by which ST receptors interact with endogenous ligands.     

In vitro glycation of a tissue-engineered wound healing model to mimic diabetic ulcers

Diabetic foot ulcers are a major complication of diabetes that occurs following minor trauma. Diabetes-induced hyperglycemia is a leading factor inducing ulcer formation and manifests notably through the accumulation of advanced glycation end-products (AGEs) such as N-carboxymethyl-lysin. AGEs have a negative impact on angiogenesis, innervation, and reepithelialization causing minor wounds to evolve into chronic ulcers which increases the risks of lower limb amputation. However, the impact of AGEs on wound healing is difficult to model (both in vitro on cells, and in vivo in animals) because it involves a long-term toxic effect. We have developed a tissue-engineered wound healing model made of human keratinocytes, fibroblasts, and endothelial cells cultured in a collagen sponge biomaterial. To mimic the deleterious effects induced by glycation on skin wound healing, the model was treated with 300 µM of glyoxal for 15 days to promote AGEs formation. Glyoxal treatment induced carboxymethyl-lysin accumulation and delayed wound closure in the skin mimicking diabetic ulcers. Moreover, this effect was reversed by the addition of aminoguanidine, an inhibitor of AGEs formation. This in vitro diabetic wound healing model could be a great tool for the screening of new molecules to improve the treatment of diabetic ulcers by preventing glycation.     

Application of life cycle inventory analysis to fuel tank system design

Life Cycle Assessment is becoming an important tool for guiding environmental design improvements in the automotive industry. This paper reports the life cycle inventory profiles for two fuel tank systems based on a collaborative effort between the National Pollution Prevention Center at the University of Michigan, General Motors Research and Development, and the National Risk Management Research Laboratory of the U.S. Environmental Protection Agency. Two 31 gallon functionally equivalent fuel tank systems used on a 1996 light duty vehicle were investigated: a multi-layer HDPE tank with a steel shield and PVC coated steel straps, and a steel tank with a HDPE shield and painted steel straps. Overall, the HDPE fuel tank system is environmentally preferable to the steel tank system based on the set of inventory results presented in this investigation. The Life Cycle Inventory analysis indicated lower energy burdens for the HDPE tank system and comparable solid waste burdens for both systems. The total life cycle energy consumption for the steel and HDPE tank systems were 4.9 GJ and 3.6 GJ per tank, respectively. The energy consumption and most of the air pollutants inventoried occurred as a consequence of the use phase. The solid wastes were generated primatily during the material production phase for the steel tank (13 kg) and during the end-of-life management phase for the HDPE tank (14 kg). This study also highlights data analysis and modeling challenges, including manufacturing and use phase allocation methods.     

Modulation of biliary lipid secretion by forskolin and cyclic AMP analogues.

Exposure of isolated perfused rat livers to either 100 microM-forskolin, a potent activator of adenylate cyclase, or to 0.5 mM-concentrations of the cAMP analogues chlorophenylthio cAMP (CPTcAMP), dibutyryl cAMP (dbcAMP) and 8-bromo cAMP (8BrcAMP), to provoke increases in intracellular concentrations of cAMP, resulted in marked changes in bile volume and composition. Bile flow reached a peak after 10 min, before declining towards control levels, and an increase in several secretory parameters was also observed at this time. At 20 min, a substantial decrease in the output of both phospholipid and cholesterol was evident, and this suppression of secretion was maintained throughout the remainder of the experiment. The order of effectiveness of the cAMP-elevating agents at decreasing biliary lipid output was CPTcAMP greater than forskolin greater than dbcAMP greater than 8BrcAMP. Biliary output of bile acids was essentially unaltered compared with controls; similarly, no decrease in the secretion of protein and triacylglycerols into the perfusion medium was observed. This suggests that the elevation of intracellular levels of cAMP may cause a selective inhibition of biliary lipid output rather than a more general inhibition of hepatic secretion.     

Restoration of immunity in lymphopenic individuals with cancer by vaccination and adoptive T-cell transfer

Immunodeficiency is a barrier to successful vaccination in individuals with cancer and chronic infection. We performed a randomized phase 1/2 study in lymphopenic individuals after high-dose chemotherapy and autologous hematopoietic stem cell transplantation for myeloma. Combination immunotherapy consisting of a single early post-transplant infusion of in vivo vaccine-primed and ex vivo costimulated autologous T cells followed by post-transplant booster immunizations improved the severe immunodeficiency associated with high-dose chemotherapy and led to the induction of clinically relevant immunity in adults within a month after transplantation. Immune assays showed accelerated restoration of CD4 T-cell numbers and function. Early T-cell infusions also resulted in significantly improved T-cell proliferation in response to antigens that were not contained in the vaccine, as assessed by responses to staphylococcal enterotoxin B and cytomegalovirus antigens (P < 0.05). In the setting of lymphopenia, combined vaccine therapy and adoptive T-cell transfer fosters the development of enhanced memory T-cell responses.