Spontaneous Genomic Alterations in a Chimeric Model of Colorectal Cancer Enable Metastasis and Guide Effective Combinatorial Therapy

Colon cancer is the second most common cause of cancer mortality in the Western world with metastasis commonly present at the time of diagnosis. Screening for propagation and metastatic behavior in a novel chimeric-mouse colon cancer model, driven by mutant p53 and β-Catenin, led to the identification of a unique, invasive adenocarcinoma. Comparison of the genome of this tumor, CB42, with genomes from non-propagating tumors by array CGH and sequencing revealed an amplicon on chromosome five containing CDK6 and CDK14, and a KRAS mutation, respectively. Single agent small molecule inhibition of either CDK6 or MEK, a kinase downstream of KRAS, led to tumor growth inhibition in vivo whereas combination therapy not only led to regression of the subcutaneous tumors, but also near complete inhibition of lung metastasis; thus, genomic analysis of this tumor led to effective, individualized treatment.     

Interleukin-22 is produced by invariant natural killer T lymphocytes during influenza A virus infection: potential role in protection against lung epithelial damages

Invariant natural killer T (iNKT) cells are non-conventional lipid-reactive αβ T lymphocytes that play a key role in host responses during viral infections, in particular through the swift production of cytokines. Their beneficial role during experimental influenza A virus (IAV) infection has recently been proposed, although the mechanisms involved remain elusive. Here we show that during in vivo IAV infection, mouse pulmonary iNKT cells produce IFN-γ and IL-22, a Th17-related cytokine critical in mucosal immunity. Although permissive to viral replication, IL-22 production by iNKT cells is not due to IAV infection per se of these cells but is indirectly mediated by IAV-infected dendritic cells (DCs). We show that activation of the viral RNA sensors TLR7 and RIG-I in DCs is important for triggering IL-22 secretion by iNKT cells, whereas the NOD-like receptors NOD2 and NLRP3 are dispensable. Invariant NKT cells respond to IL-1β and IL-23 provided by infected DCs independently of the CD1d molecule to release IL-22. In vitro, IL-22 protects IAV-infected airway epithelial cells against mortality but has no role on viral replication. Finally, during early IAV infection, IL-22 plays a positive role in the control of lung epithelial damages. Overall, IAV infection of DCs activates iNKT cells, providing a rapid source of IL-22 that might be beneficial to preserve the lung epithelium integrity.     

The Early Activation of Toll-Like Receptor (TLR)-3 Initiates Kidney Injury after Ischemia and Reperfusion

Acute kidney injury (AKI) is one of the most important complications in hospitalized patients and its pathomechanisms are not completely elucidated. We hypothesize that signaling via toll-like receptor (TLR)-3, a receptor that is activated upon binding of double-stranded nucleotides, might play a crucial role in the pathogenesis of AKI following ischemia and reperfusion (IR). Male adult C57Bl6 wild-type (wt) mice and TLR-3 knock-out (-/-) mice were subjected to 30 minutes bilateral selective clamping of the renal artery followed by reperfusion for 30 min 2.5h and 23.5 hours or subjected to sham procedures. TLR-3 down-stream signaling was activated already within 3 h of ischemia and reperfusion in post-ischemic kidneys of wt mice lead to impaired blood perfusion followed by a strong pro-inflammatory response with significant neutrophil invasion. In contrast, this effect was absent in TLR-3-/- mice. Moreover, the quick TLR-3 activation resulted in kidney damage that was histomorphologically associated with significantly increased apoptosis and necrosis rates in renal tubules of wt mice. This finding was confirmed by increased kidney injury marker NGAL in wt mice and a better preserved renal perfusion after IR in TLR-3-/- mice than wt mice. Overall, the absence of TLR-3 is associated with lower cumulative kidney damage and maintained renal blood perfusion within the first 24 hours of reperfusion. Thus, we conclude that TLR-3 seems to participate in the pathogenesis of early acute kidney injury.     

Search for a relationship between molecular anomalies of the mutant erythrocyte pyruvate kinase variants and their pathological expression

Summary Anomalies of the mutant pyruvate kinase variants and clinical symptoms have been compared in 22 unrelated patients with congenital red cell pyruvate kinase deficiency. This study suggests that some characteristics of the mutant enzymes could play a role in the intensity of haemolysis, namely residual activity, affinity for the substrate phosphoenolpyruvate and for the allosteric activator fructose 1,6 diphosphate and inhibition by ATP.     

Muscarinic M2 receptors in acetylcholine-isoproterenol functional antagonism in human isolated bronchus

The muscarinic functional antagonism of isoproterenol relaxation and the contribution of muscarinic M2 receptors were examined in human isolated bronchus. In intact tissues, acetylcholine (ACh) precontraction decreased isoproterenol potency and maximal relaxation (-log EC50 shift = -1.49 +/- 0.16 and E(max) inhibition for 100 microM ACh = 30%) more than the same levels of histamine contraction. The M2 receptor-selective antagonist methoctramine (1 microM) reduced this antagonism in ACh- but not histamine-contracted tissues. Similar results were obtained for forskolin-induced relaxation. After selective inactivation of M3 receptors with 4-diphenylacetoxy-N-(2-chloroethyl)piperadine hydrochloric acid (30 nM), demonstrated by abolition of contractile and inositol phosphate responses to ACh, muscarinic recontractile responses were obtained in U-46619-precontracted tissues fully relaxed with isoproterenol. Methoctramine antagonized recontraction, with pK(B) (6.9) higher than in intact tissues (5.4), suggesting participation of M2 receptors. In M3-inactivated tissues, methoctramine augmented the isoproterenol relaxant potency in U-46619-contracted bronchus and reversed the ACh-induced inhibition of isoproterenol cAMP accumulation. These results indicate that M2 receptors cause indirect contraction of human bronchus by reversing sympathetically mediated relaxation and contribute to cholinergic functional antagonism.     

Discovery and synthesis of tetrahydropyrimidinedione-4-carboxamides as endothelial lipase inhibitors

Endothelial lipase (EL) inhibitors have been shown to elevate HDL-C levels in pre-clinical murine models and have potential benefit in prevention and treatment of cardiovascular diseases. Modification of the 1-ethyl-3-hydroxy-1,5-dihydro-2H-pyrrol-2-one (DHP) lead, 1, led to the discovery of a series of potent tetrahydropyrimidinedione (THP) EL inhibitors. Synthesis and SAR studies including modification of the amide group, together with changes on the pyrimidinone core led to a series of arylcycloalkyl, indanyl, and tetralinyl substituted 5-amino or 5-hydroxypyrimidinedione-4-carboxamides. Several compounds were advanced to PK evaluation. Among them, compound 4a was one of the most potent with measurable EL_HDL hSerum potency and compound 3g demonstrated the best overall pharmacokinetic parameters.     

Cytosine Arabinoside Induces Apoptosis in Cerebellar Neurons in Culture

Abstract: Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that prevents cell proliferation by inhibiting DNA synthesis. We report that AraC kills cultured cerebellar neurons in a concentration-dependent fashion with an EC₅₀ of ∼60 µ M when added shortly after seeding. This cell death has apoptotic features because we observed (1) morphology of apoptotic nuclei as judged by DNA staining with Hoechst 33258, (2) DNA fragmentation with typical ladder pattern on agarose gel, (3) positive nuclear labeling with a specific in situ DNA fragmentation staining, (4) prevention by deoxycytidine (IC₅₀ = 1 µ M ), protein, and RNA synthesis inhibitors, and (5) release of DNA fragments in the incubating medium. We have also observed that several proteins were overexpressed in AraC-treated neurons by two-dimensional polyacrylamide gel electrophoresis. We conclude that AraC induces a signal that triggers a cascade of new mRNA and protein synthesis, leading to apoptotic cell death in cultured cerebellar granule cells.     

Azurocidin, a Natural Antibiotic from Human Neutrophils: Expression, Antimicrobial Activity, and Secretion

The azurophil granules of human PMN contain four antibiotic proteins, the serprocidins, which have extensive homology to one another and to serine proteases. Azurocidin, a member of this family, is a 29-kDa glycoprotein with broad spectrum antimicrobial activity and chemotactic activity toward monocytes. Insect cells transfected with a baculovirus vector carrying azurocidin cDNA produced a recombinant azurocidin protein. We purified the recombinant azurocidin protein from the culture medium of the infected cells and showed that it retained the antimicrobial activity of the native neutrophil-derived molecule. In addition, we present evidence that a 49-amino-acid region of the recombinant azurocidin protein is required for its secretion from insect cells.