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.     

Resource wars and conflict ivory: the impact of civil conflict on elephants in the Democratic Republic of Congo--the case of the Okapi Reserve

Human conflict generally has substantial negative impacts on wildlife and conservation. The recent civil war (1995-2006) in the Democratic Republic of Congo (DRC) resulted in a significant loss of wildlife, including elephants, due to institutional collapse, lawlessness and unbridled exploitation of natural resources such as minerals, wood, ivory and bushmeat. We used data from distance sampling surveys conducted before and after the war in a protected forest, the Okapi Faunal Reserve, to document changes in elephant abundance and distribution. We employed Generalized Additive Models to relate changes in elephant distribution to human and environmental factors. Populations declined by nearly fifty percent coinciding with a major increase in elephant poaching as indicated by reports of ivory trade during the war. Our results suggest that humans influenced elephant distribution far more than habitat, both before and after the war, but post-war models explained more of the variation. Elephant abundance declined more, closer to the park boundary and to areas of intense human activity. After the war, elephant densities were relatively higher in the centre of the park where they were better protected, suggesting that this area may have acted as a refuge. In other sites in Eastern DRC, where no protection was provided, elephants were even more decimated. Post-war dynamics, such as weakened institutions, human movements and availability of weapons, continue to affect elephants. Survival of remaining populations and recovery will be determined by these persistent factors and by new threats associated with growing human populations and exploitation of natural resources. Prioritizing wildlife protection, curbing illegal trade in ivory and bushmeat, and strengthening national institutions and organizations in charge of conservation will be crucial to counter these threats.     

Targeted deletion of integrin-linked kinase reveals a role in T-cell chemotaxis and survival

Integrin-linked kinase (ILK) is a serine/threonine kinase that is important in cell-matrix interactions and cell signaling. To examine the role of ILK in leukocyte trafficking and survival, we generated T cell-specific ILK knockouts by breeding ILK(flox/flox) mice to transgenic mice expressing Cre recombinase under control of the Lck proximal promoter. Thymic T cells from Lck-Cre(+)/ILK(flox/flox) mice had a marked reduction (>95%) in ILK protein levels. Thymic cellularity was comparable in 3- to 4-week-old mice, but a threefold diminution of thymic T cells became evident by 6 to 8 weeks of age in the T cell-specific ILK knockout mice due to increased cell death of double-positive (DP) T cells. Analysis of peripheral T cells by quantitative PCR and by breeding Lck-Cre(+)/ILK(flox/flox) mice to a YFP-transgenic reporter strain demonstrated an approximate 20-fold enrichment of ILK-competent cells, suggesting these cells have a competitive advantage in trafficking to and/or survival in peripheral lymphatic organs. We explored mechanisms related to altered cell trafficking and survival that might explain the decreases in thymic cellularity and enrichment for ILK-competent cells in the spleen and lymph nodes. We observed a >50% reduction in chemotaxis of ILK-deficient T cells to the chemokines CXCL12 (stromal cell-derived factor [SDF]-1alpha) and CCL19 (macrophage inflammatory protein [MIP]-3beta), as well as enhanced apoptosis of ILK-deficient cells upon stress. Signaling studies in ILK-deficient T cells demonstrated diminished phosphorylation of Akt on the activating phosphorylation site, Ser 473, and a concordant decrease in Akt kinase activity following stimulation with the chemokine SDF-1. Rac1 activation was also markedly diminished in ILK-deficient T cells following chemokine stimulation. These data extend the role of ILK to immune-cell trafficking and survival via modulation of Akt- and Rac-dependent substrates, and have implications for cell recruitment in both homeostatic and pathological processes.     

Interactions of caged-ATP and photoreleased ATP with alkaline phosphatase

Photolytic release of ATP from inactive P(3)-[1-(2-nitrophenyl)]ethyl ester of ATP (NPE-caged ATP) provides a means to reveal molecular interactions between nucleotide and enzyme by using infrared spectroscopy. Reaction-induced infrared difference spectra of bovine intestinal alkaline phosphatase (BIAP) and of NPE-caged ATP revealed small structural alterations on the peptide backbone affecting one or two amino-acid residues. After photorelease of ATP, the substrate could be hydrolyzed sequentially by the enzyme producing three Pi, adenosine, and the photoproduct nitrosoacetophenone. It was concluded that NPE-caged ATP could bind to BIAP prior to the photolytic cleavage of ATP and that Pi could interact with BIAP after photolysis of NPE-caged ATP and hydrolysis, yielding infrared spectra with distinct structure changes of BIAP. This suggests that the molecular mechanism of ATP hydrolysis by BIAP involved small structural adjustments of the peptide backbone in the vicinity of the active site during ATP hydrolysis which continued during Pi binding.     

Glutathione preconditioning attenuates Ac-LDL-induced macrophage apoptosis via protein kinase C-dependent Ac-LDL trafficking

Oxidized low-density lipoprotein (ox-LDL) incorporation into intimally resident vascular cells via scavenger receptors marks one of the early steps in atherosclerosis. Cellular apoptotic damage results from two major serial intracellular events: the binding and scavenger receptor-mediated uptake of oxidizable lipoproteins and the intracellular oxidative responses of accumulated lipoproteins. Most molecular approaches to prevent apoptotic damage have focused on singular events within the cascade of lipoprotein trafficking. To identify a multifocal strategy against LDL-induced apoptosis, we evaluated the role of cellular preconditioning by glutathione-ethyl ester (GSH-Et), a native redox regulator, in the prevention of the uptake and apoptotic effects of an oxidizable scavenger receptor-specific ligand, acetylated low-density lipoprotein (Ac-LDL). Our results indicate that GSH-Et-mediated protein kinase C (PKC) pathway modulation regulates Ac-LDL binding and incorporation into GSH-Et preconditioned cells and subsequently delays reactive oxygen intermediate generation and apoptotic conversion. The GSH-Et protective effects on apoptosis and Ac-LDL binding were reversed by calphostin C, a PKC inhibitor, and were accompanied by an increase in PKC phosphorylation. However, the rate of reactive oxygen intermediate accumulation was not increased following calphostin C treatment, suggesting that GSH-Et may play an important nonreactive oxygen-intermediate-based protective role in regulating apoptotic dynamics. Overall, we report on the novel role for GSH-Et preconditioning as a molecular strategy to limit lipoprotein entry into the cells, which presents a proactive modality to prevent cellular apoptosis in contrast with the prevalent antioxidant approaches that treat damage retroactively.     

The Harderian gland of the Mexican volcano mouse Neotomodon alstoni alstoni (Merriam 1898): a morphological and biochemical approach

The Harderian glands of rodents are large intraorbital exocrine glands with histologic organization that varies among mammalian species. Here we describe some ultrastructural and biochemical features of the Harderian gland in the Mexican volcano mouse Neotomodon alstoni alstoni, a species of restricted habitat. The Harderian glands from male and female adult mice were dissected, processed and embedded in Epon 812 for light and electron microscopy studies. Porphyrin and total lipids were biochemically determined. The macroscopic appearance of the Harderian gland is similar in the male and female. The gland is a bilobulate structure, situated in the orbit towards the posterior side of the eyeball, of whitish color and is surrounded by a connective tissue capsule. The male gland is slightly heavier (127 mg) than that of the female (113 mg). The Harderian gland shows a tubulo-alveolar organization and is composed exclusively of one type of secretory cells. No branched duct system within the gland was found. Adrenergic nerves endings and mast cell were observed in the interstices of the alveoli. Male and female glands produce similar levels of porphyrins. Triglyceride levels were significantly higher (P < 0.05) in the female compared to the male. Abundance of lipids could induce corneal lubrication of the Harderian gland which may confer a protective and adaptative function to the volcano mouse in its natural habitat during the dry and cold seasons.