Characterization of a New Chronic Lymphocytic Leukemia Cell Line for Mechanistic In Vitro and In Vivo Studies Relevant to Disease

Studies of chronic lymphocytic leukemia (CLL) have yielded substantial progress, however a lack of immortalized cell lines representative of the primary disease has hampered a full understanding of disease pathogenesis and development of new treatments. Here we describe a novel CLL cell line (OSU-CLL) generated by EBV transformation, which displays a similar cytogenetic and immunophenotype observed in the patient’s CLL (CD5 positive with trisomy 12 and 19). A companion cell line was also generated from the same patient (OSU-NB). This cell line lacked typical CLL characteristics, and is likely derived from the patient’s normal B cells. In vitro migration assays demonstrated that OSU-CLL exhibits migratory properties similar to primary CLL cells whereas OSU-NB has significantly reduced ability to migrate spontaneously or towards chemokine. Microarray analysis demonstrated distinct gene expression patterns in the two cell lines, including genes on chromosomes 12 and 19, which is consistent with the cytogenetic profile in this cell line. Finally, OSU-CLL was readily transplantable into NOG mice, producing uniform engraftment by three weeks with leukemic cells detectable in the peripheral blood spleen and bone marrow. These studies describe a new CLL cell line that extends currently available models to study gene function in this disease.     

Viperin Is Induced following Dengue Virus Type-2 (DENV-2) Infection and Has Anti-viral Actions Requiring the C-terminal End of Viperin

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.     

Synthesising 30 Years of Mathematical Modelling of Echinococcus Transmission

Background

Echinococcosis is a complex zoonosis that has domestic and sylvatic lifecycles, and a range of different intermediate and definitive host species. The complexities of its transmission and the sparse evidence on the effectiveness of control strategies in diverse settings provide significant challenges for the design of effective public health policy against this disease. Mathematical modelling is a useful tool for simulating control packages under locally specific transmission conditions to inform optimal timing and frequency of phased interventions for cost-effective control of echinococcosis. The aims of this review of 30 years of Echinococcus modelling were to discern the epidemiological mechanisms underpinning models of Echinococcus granulosus and E. multilocularis transmission and to establish the need to include a human transmission component in such models.

Methodology/Principal Findings

A search was conducted of all relevant articles published up until July 2012, identified from the PubMED, Web of Knowledge and Medline databases and review of bibliographies of selected papers. Papers eligible for inclusion were those describing the design of a new model, or modification of an existing mathematical model of E. granulosus or E. multilocularis transmission. A total of 13 eligible papers were identified, five of which described mathematical models of E. granulosus and eight that described E. multilocularis transmission. These models varied primarily on the basis of six key mechanisms that all have the capacity to modulate model dynamics, qualitatively affecting projections. These are: 1) the inclusion of a ‘latent’ class and/or time delay from host exposure to infectiousness; 2) an age structure for animal hosts; 3) the presence of density-dependent constraints; 4) accounting for seasonality; 5) stochastic parameters; and 6) inclusion of spatial and risk structures.

Conclusions/Significance

This review discusses the conditions under which these mechanisms may be important for inclusion in models of Echinococcus transmission and proposes recommendations for the design of dynamic human models of transmission. Accounting for the dynamic behaviour of the Echinococcus parasites in humans will be key to predicting changes in the disease burden over time and to simulate control strategies that optimise public health impact.     

Accurate and Rapid Identification of the Burkholderia pseudomallei Near-Neighbour,Burkholderia ubonensis,Using Real-Time PCR

Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc), a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown’s medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown’s agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown’s-positive colonies that are not B. pseudomallei.     

Targeting the Tumour Vasculature: Exploitation of Low Oxygenation and Sensitivity to NOS Inhibition by Treatment with a Hypoxic Cytotoxin

Many cancer research efforts focus on exploiting genetic-level features that may be targeted for therapy. Tissue-level features of the tumour microenvironment also represent useful therapeutic targets. Here we investigate the presence of low oxygen tension and sensitivity to NOS inhibition of tumour vasculature as potential tumour-specific features that may be targeted by hypoxic cytotoxins, a class of therapeutics currently under investigation. We have previously demonstrated that tirapazamine (TPZ) mediates central vascular dysfunction in tumours. TPZ is a hypoxic cytotoxin that is also a competitive inhibitor of NOS. Here we further investigated the vascular-targeting activity of TPZ by combining it with NOS inhibitor L-NNA, or with low oxygen content gas breathing. Tumours were analyzed via multiplex immunohistochemical staining that revealed irreversible loss of perfusion and enhanced tumour cell death when TPZ was combined with either low oxygen or a NOS inhibitor. Tumour growth rate was reduced by TPZ + NOS inhibition, and tumours previously resistant to TPZ-mediated vascular dysfunction were sensitized by low oxygen breathing. Additional mapping analysis suggests that tumours with reduced vascular-associated stroma may have greater sensitivity to these effects. These results indicate that poorly oxygenated tumour vessels, also being abnormally organized and with inadequate smooth muscle, may be successfully targeted for significant anti-cancer effects by inhibition of NOS and hypoxia-activated prodrug toxicity. This strategy illustrates a novel use of hypoxia-activated cytotoxic prodrugs as vascular targeting agents, and also represents a novel mechanism for targeting tumour vessels.     

Greater Ethanol-Induced Locomotor Activation in DBA/2J versus C57BL/6J Mice Is Not Predicted by Presynaptic Striatal Dopamine Dynamics

A large body of research has aimed to determine the neurochemical factors driving differential sensitivity to ethanol between individuals in an attempt to find predictors of ethanol abuse vulnerability. Here we find that the locomotor activating effects of ethanol are markedly greater in DBA/2J compared to C57BL/6J mice, although it is unclear as to what neurochemical differences between strains mediate this behavior. Dopamine elevations in the nucleus accumbens and caudate-putamen regulate locomotor behavior for most drugs, including ethanol; thus, we aimed to determine if differences in these regions predict strain differences in ethanol-induced locomotor activity. Previous studies suggest that ethanol interacts with the dopamine transporter, potentially mediating its locomotor activating effects; however, we found that ethanol had no effects on dopamine uptake in either strain. Ex vivo voltammetry allows for the determination of ethanol effects on presynaptic dopamine terminals, independent of drug-induced changes in firing rates of afferent inputs from either dopamine neurons or other neurotransmitter systems. However, differences in striatal dopamine dynamics did not predict the locomotor-activating effects of ethanol, since the inhibitory effects of ethanol on dopamine release were similar between strains. There were differences in presynaptic dopamine function between strains, with faster dopamine clearance in the caudate-putamen of DBA/2J mice; however, it is unclear how this difference relates to locomotor behavior. Because of the role of the dopamine system in reinforcement and reward learning, differences in dopamine signaling between the strains could have implications for addiction-related behaviors that extend beyond ethanol effects in the striatum.