An international trial of quantitative PCR for monitoring Legionella in artificial water systems

Aims: To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae. Methods and Results: Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l^?1) and culture (CFU l^?1) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l^?1 always exceeded CFU l^?1, and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring. Conclusions: Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required. Significance and Impact of the Study: This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection.     

Receptor activator of NF-kB (RANK) expression in primary tumors associates with bone metastasis occurrence in breast cancer patients

Background

Receptor activator of NFkB (RANK), its ligand (RANKL) and the decoy receptor of RANKL (osteoprotegerin, OPG) play a pivotal role in bone remodeling by regulating osteoclasts formation and activity. RANKL stimulates migration of RANK-expressing tumor cells in vitro, conversely inhibited by OPG.

Materials and Methods

We examined mRNA expression levels of RANKL/RANK/OPG in a publicly available microarray dataset of 295 primary breast cancer patients. We next analyzed RANK expression by immunohistochemistry in an independent series of 93 primary breast cancer specimens and investigated a possible association with clinicopathological parameters, bone recurrence and survival.

Results

Microarray analysis showed that lower RANK and high OPG mRNA levels correlate with longer overall survival (P = 0.0078 and 0.0335, respectively) and disease-free survival (P = 0.059 and 0.0402, respectively). Immunohistochemical analysis of RANK showed a positive correlation with the development of bone metastases (P = 0.023) and a shorter skeletal disease-free survival (SDFS, P = 0.037). Specifically, univariate analysis of survival showed that “RANK-negative” and “RANK-positive” patients had a SDFS of 105.7 months (95% CI: 73.9–124.4) and 58.9 months (95% CI: 34.7–68.5), respectively. RANK protein expression was also associated with accelerated bone metastasis formation in a multivariate analysis (P = 0.029).

Conclusions

This is the first demonstration of the role of RANK expression in primary tumors as a predictive marker of bone metastasis occurrence and SDFS in a large population of breast cancer patients.     

A local proinflammatory signalling loop facilitates adverse age-associated arterial remodeling

Background

The coincidence of vascular smooth muscle cells (VSMC) infiltration and collagen deposition within a diffusely thickened intima is a salient feature of central arterial wall inflammation that accompanies advancing age. However, the molecular mechanisms involved remain undefined.

Methodology/Principal Findings

Immunostaining and immunoblotting of rat aortae demonstrate that a triad of proinflammatory molecules, MCP-1, TGF-β1, and MMP-2 increases within the aortic wall with aging. Exposure of VSMC isolated from 8-mo-old rats (young) to MCP-1 effects, via CCR-2 signaling, both an increase in TGF-β1 activity, up to levels of untreated VSMC from 30-mo-old (old) rats, and a concurrent increase in MMP-2 activation. Furthermore, exposure of young VSMC to TGF-β1 increases levels of MCP-1, and MMP-2 activation, to levels of untreated VSMC from old rats. This autocatalytic signaling loop that enhances collagen production and invasiveness of VSMC is effectively suppressed by si-MCP-1, a CCR2 antagonist, or MMP-2 inhibition.

Conclusions/Significance

Threshold levels of MCP-1, MMP-2, or TGF-β1 activity trigger a feed-forward signaling mechanism that is implicated in the initiation and progression of adverse age-associated arterial wall remodeling. Intervention that suppressed this signaling loop may potentially retard age-associated adverse arterial remodeling.     

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.     

The collagen chaperone HSP47 is a new interactor of APP that affects the levels of extracellular beta-amyloid peptides

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive decline of cognitive function that represents one of the most dramatic medical challenges for the aging population. Aβ peptides, generated by processing of the Amyloid Precursor Protein (APP), are thought to play a central role in the pathogenesis of AD. However, the network of physical and functional interactions that may affect their production and deposition is still poorly understood. The use of a bioinformatic approach based on human/mouse conserved coexpression allowed us to identify a group of genes that display an expression profile strongly correlated with APP. Among the most prominent candidates, we investigated whether the collagen chaperone HSP47 could be functionally correlated with APP. We found that HSP47 accumulates in amyloid deposits of two different mouse models and of some AD patients, is capable to physically interact with APP and can be relocalized by APP overexpression. Notably, we found that it is possible to reduce the levels of secreted Aβ peptides by reducing the expression of HSP47 or by interfering with its activity via chemical inhibitors. Our data unveil HSP47 as a new functional interactor of APP and imply it as a potential target for preventing the formation and/or growth amyloid plaques.     

Cannabidiol reduces Aβ-induced neuroinflammation and promotes hippocampal neurogenesis through PPARγ involvement

Peroxisome proliferator-activated receptor-γ (PPARγ) has been reported to be involved in the etiology of pathological features of Alzheimer's disease (AD). Cannabidiol (CBD), a Cannabis derivative devoid of psychomimetic effects, has attracted much attention because of its promising neuroprotective properties in rat AD models, even though the mechanism responsible for such actions remains unknown. This study was aimed at exploring whether CBD effects could be subordinate to its activity at PPARγ, which has been recently indicated as its putative binding site. CBD actions on β-amyloid-induced neurotoxicity in rat AD models, either in presence or absence of PPAR antagonists were investigated. Results showed that the blockade of PPARγ was able to significantly blunt CBD effects on reactive gliosis and subsequently on neuronal damage. Moreover, due to its interaction at PPARγ, CBD was observed to stimulate hippocampal neurogenesis. All these findings report the inescapable role of this receptor in mediating CBD actions, here reported.     

Further support to the uncoupling-to-survive theory: the genetic variation of human UCP genes is associated with longevity

In humans Uncoupling Proteins (UCPs) are a group of five mitochondrial inner membrane transporters with variable tissue expression, which seem to function as regulators of energy homeostasis and antioxidants. In particular, these proteins uncouple respiration from ATP production, allowing stored energy to be released as heat. Data from experimental models have previously suggested that UCPs may play an important role on aging rate and lifespan. We analyzed the genetic variability of human UCPs in cohorts of subjects ranging between 64 and 105 years of age (for a total of 598 subjects), to determine whether specific UCP variability affects human longevity. Indeed, we found that the genetic variability of UCP2, UCP3 and UCP4 do affect the individual's chances of surviving up to a very old age. This confirms the importance of energy storage, energy use and modulation of ROS production in the aging process. In addition, given the different localization of these UCPs (UCP2 is expressed in various tissues including brain, hearth and adipose tissue, while UCP3 is expressed in muscles and Brown Adipose Tissue and UCP4 is expressed in neuronal cells), our results may suggest that the uncoupling process plays an important role in modulating aging especially in muscular and nervous tissues, which are indeed very responsive to metabolic alterations and are very important in estimating health status and survival in the elderly.