Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.     

Abnormal kinetochore-generated pulling forces from expressing a N-terminally modified Hec1

Background

Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells.

Methodology/Principal Findings

Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells.

Conclusions/Significance

Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore pulling forces that disrupt the fine balance of kinetochore- and centrosome-associated forces regulating spindle bipolarity. Overall, our findings support a model in which centrosome integrity is influenced by the pathways regulating kinetochore-microtubule attachment stability.     

Connective tissue growth factor induction by lysophosphatidic acid requires transactivation of transforming growth factor type β receptors and the JNK pathway

Transforming growth factor β (TGF-β) is a very strong pro-fibrotic factor which mediates its action, at least in part, through the expression of connective tissue growth factor (CTGF/CCN2). Along with these cytokines, the involvement of phospholipids in wound healing and the development of fibrosis has been revealed. Among them, lysophosphatidic acid (LPA) is a novel, potent regulator of wound healing and fibrosis that has diverse effects on many types of cells. We decided to evaluate the effect of LPA together with TGF-β on CTGF expression. We found that myoblasts treated with LPA and TGF-β1 produced an additive effect on CTGF expression. In the absence of TGF-β, the induction of CTGF expression by LPA was abolished by a dominant negative form of the TGF-β receptor type II (TGF-βRII) and by the use of SB 431542, a specific inhibitor of the serine/threonine kinase activity of TGF-βRI, suggesting that CTGF induction is dependent on LPA and requires active TGF-βRs. Moreover, we show that LPA requires Smad-2/3 proteins for the induction of CTGF expression, but not their phosphorylation or their nuclear translocation. The requirement of TGF-βRI for LPA mediated-effects is differential, since treatment of myoblasts with LPA in the presence of SB 431542 abolished the induction of stress fibers but not the induction of proliferation. Finally, we demonstrated that CTGF induction in response to LPA requires the activation of JNK, but not ERK, signaling pathways. The JNK requirement is independent of TGF-βRI-mediated activity. These novel results for the mechanism of action of LPA and TGF-β are important for understanding the role of pro-fibrotic growth factors and phospholipids involved in wound healing and related diseases.     

Use of a novel triple-tracer approach to assess postprandial glucose metabolism

Numerous studies have used the dual-tracer method to assess postprandial glucose metabolism. The present experiments were undertaken to determine whether the marked tracer nonsteady state that occurs with the dual-tracer approach after food ingestion introduces error when it is used to simultaneously measure both meal glucose appearance (R(a meal)) and endogenous glucose production (EGP). To do so, a novel triple-tracer approach was designed: 12 subjects ingested a mixed meal containing [1-(13)C]glucose while [6-(3)H]glucose and [6,6-(2)H(2)]glucose were infused intravenously in patterns that minimized the change in the plasma ratios of [6-(3)H]glucose to [1-(13)C]glucose and of [6,6-(2)H(2)]glucose to endogenous glucose, respectively. R(a meal) and EGP measured with this approach were essentially model independent, since non-steady-state error was minimized by the protocol. Initial splanchnic glucose extraction (ISE) was 12.9% +/- 3.4%, and suppression of EGP (EGPS) was 40.3% +/- 4.1%. In contrast, when calculated with the dual-tracer one-compartment model, ISE was higher (P < 0.05) and EGPS was lower (P < 0.005) than observed with the triple-tracer approach. These errors could only be prevented by using time-varying volumes different for R(a meal) and EGP. Analysis of the dual-tracer data with a two-compartment model reduced but did not totally avoid the problems associated with marked postprandial changes in the tracer-to-tracee ratios. We conclude that results from previous studies that have used the dual-tracer one-compartment model to measure postprandial carbohydrate metabolism need to be reevaluated and that the triple-tracer technique may provide a useful approach for doing so.     

Rapid in vivo transport of proteins from digested allergen across pre-sensitized gut

Although the route of sensitization to food allergens is still the subject of debate, it is generally accepted the gut immune system plays a pivotal role. However, hitherto the transport of allergens across the normal, pre-sensitized gut epithelium remained largely unknown. Our aim was to identify the route through which protein bodies and soluble proteins from digested peanuts penetrated the pre-sensitized gut epithelium in vivo and the specific cell types involved in the transport. Digestion of peanuts released a large number of protein bodies that are exclusively transported across the epithelium by specialized antigen-sampling M cells and delivered to the lymphoid tissue of Peyer's patch. Intracellular transport of soluble protein also occurred almost exclusively via M cells and it was negligible across absorptive enterocytes. We hypothesize that these conditions which are known to favour strongly the induction of immune responses rather than oral tolerance may play a significant role in the genesis of allergic reactions.