Synthesis and propagation of complement C3 by microglia/monocytes in the aging retina

INTRODUCTION Complement activation is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which may be mediated in part by para-inflammatory processes. We aimed to investigate the expression and localization of C3, a crucial component of the complement system, in the retina during the course of aging. METHODS SD rats were born and reared in low-light conditions, and euthanized at post-natal (P) days 100, 450, or 750. Expression of C3, IBA1, and Ccl- and Cxcl- chemokines was assessed by qPCR, and in situ hybridization. Thickness of the ONL was assessed in retinal sections as a measure of photoreceptor loss, and counts were made of C3-expressing monocytes. RESULTS C3 expression increased significantly at P750, and correlated with thinning of the ONL, at P750, and up-regulation of GFAP. In situ hybridization showed that C3 was expressed by microglia/monocytes, mainly from within the retinal vasculature, and occasionally the ONL. The number of C3-expressing microglia increased significantly by P750, and coincided spatiotemporally with thinning of the ONL, and up-regulation of Ccl- and Cxcl- chemokines. CONCLUSIONS Our data suggest that recruited microglia/monocytes contribute to activation of complement in the aging retina, through local expression of C3 mRNA. C3 expression coincides with age-related thinning of the ONL at P750, although it is unclear whether the C3-expressing monocytes are a cause or consequence. These findings provide evidence of activation of complement during natural aging, and may have relevance to cellular events underling the pathogenesis of age-related retinal diseases.Funding provided by Australian Research Council Centres of Excellence Program Grant (CE0561903)

Two novel human cytomegalovirus NK cell evasion functions target MICA for lysosomal degradation

NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αβ and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1–6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12–US21; a genetic arrangement, which is suggestive of an ‘accordion’ expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family

A European multi-language initiative to make the general population aware of independent clinical research: the European Communication on Research Awareness Need project

BACKGROUND: The ECRAN (European Communication on Research Awareness Needs) project was initiated in 2012, with support from the European Commission, to improve public knowledge about the importance of independent, multinational, clinical trials in Europe. METHODS: Participants in the ECRAN consortium included clinicians and methodologists directly involved in clinical trials; researchers working in partnership with the public and patients; representatives of patients; and experts in science communication. We searched for, and evaluated, relevant existing materials and developed additional materials and tools, making them freely available under a Creative Commons licence. RESULTS: The principal communication materials developed were: 1. A website ( http://ecranproject.eu ) in six languages, including a Media centre section to help journalists to disseminate information about the ECRAN project 2. An animated film about clinical trials, dubbed in the 23 official languages of the European Community, and an interactive tutorial 3. An inventory of resources, available in 23 languages, searchable by topic, author, and media type 4. Two educational games for young people, developed in six languages 5. Testing Treatments interactive in a dozen languages, including five official European Community languages 6. An interactive tutorial slide presentation testing viewers' knowledge about clinical trials CONCLUSIONS: Over a 2-year project, our multidisciplinary and multinational consortium was able to produce, and make freely available in many languages, new materials to promote public knowledge about the importance of independent and international clinical trials. Sustained funding for the ECRAN information platform could help to promote successful recruitment to independent clinical trials supported through the European Clinical Research Infrastructure Network

CXCR7 Protein Expression in Human Adult Brain and Differentiated Neurons

Background: CXCR7 and CXCR4 are receptors for the chemokine CXCL12, which is involved in essential functions of the immune and nervous systems. Although CXCR7 transcripts are widely expressed throughout the central nervous system, little is known about its protein distribution and function in the adult brain. To evaluate its potential involvement in CXCL12/CXCR4 signaling in differentiated neurons, we studied CXCR7 protein expression in human brain and cultured neurons. Methodology/Principal Findings: Immunohistochemistry and RT-PCR analyses of cortex and hippocampus from control and HIV-positive subjects provided the first evidence of CXCR7 protein expression in human adult neurons, under normal and pathological conditions. Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane. Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons. Neuronal CXCR7 co-localizes to some extent with the endoplasmic reticulum marker ERp29, but not with early/late endosome markers. Additionally, large areas of overlap are detected in the intracellular pattern of CXCR7 and CXCR4 expression. Conclusions/Significance: Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface o

Entry of Herpes Simplex Virus Type 1 (HSV-1) into the Distal Axons of Trigeminal Neurons Favors the Onset of Nonproductive, Silent Infection

Following productive, lytic infection in epithelia, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons that is interrupted by episodes of reactivation. In order to better understand what triggers this lytic/latent decision in neurons, we set up an organotypic model based on chicken embryonic trigeminal ganglia explants (TGEs) in a double chamber system. Adding HSV-1 to the ganglion compartment (GC) resulted in a productive infection in the explants. By contrast, selective application of the virus to distal axons led to a largely nonproductive infection that was characterized by the poor expression of lytic genes and the presence of high levels of the 2.0-kb major latency-associated transcript (LAT) RNA. Treatment of the explants with the immediate-early (IE) gene transcriptional inducer hexamethylene bisacetamide, and simultaneous co-infection of the GC with HSV-1, herpes simplex virus type 2 (HSV-2) or pseudorabies virus (PrV) helper virus significantly enhanced the ability of HSV-1 to productively infect sensory neurons upon axonal entry. Helper-virus-induced transactivation of HSV-1 IE gene expression in axonally-infected TGEs in the absence of de novo protein synthesis was dependent on the presence of functional tegument protein VP16 in HSV-1 helper virus particles. After the establishment of a LAT-positive silent infection in TGEs, HSV-1 was refractory to transactivation by superinfection of the GC with HSV-1 but not with HSV-2 and PrV helper virus. In conclusion, the site of entry appears to be a critical determinant in the lytic/latent decision in sensory neurons. HSV-1 entry into distal axons results in an insufficient transactivation of IE gene expression and favors the establishment of a nonproductive, silent infection in trigeminal neurons

Herpes Simplex Virus Dances with Amyloid Precursor Protein while Exiting the Cell

Herpes simplex type 1 (HSV1) replicates in epithelial cells and secondarily enters local sensory neuronal processes, traveling retrograde to the neuronal nucleus to enter latency. Upon reawakening newly synthesized viral particles travel anterograde back to the epithelial cells of the lip, causing the recurrent cold sore. HSV1 co-purifies with amyloid precursor protein (APP), a cellular transmembrane glycoprotein and receptor for anterograde transport machinery that when proteolyzed produces A-beta, the major component of senile plaques. Here we focus on transport inside epithelial cells of newly synthesized virus during its transit to the cell surface. We hypothesize that HSV1 recruits cellular APP during transport. We explore this with quantitative immuno-fluorescence, immuno-gold electron-microscopy and live cell confocal imaging. After synchronous infection most nascent VP26-GFP-labeled viral particles in the cytoplasm co-localize with APP (72.8+/−6.7%) and travel together with APP inside living cells (81.1+/−28.9%). This interaction has functional consequences: HSV1 infection decreases the average velocity of APP particles (from 1.1+/−0.2 to 0.3+/−0.1 µm/s) and results in APP mal-distribution in infected cells, while interplay with APP-particles increases the frequency (from 10% to 81% motile) and velocity (from 0.3+/−0.1 to 0.4+/−0.1 µm/s) of VP26-GFP transport. In cells infected with HSV1 lacking the viral Fc receptor, gE, an envelope glycoprotein also involved in viral axonal transport, APP-capsid interactions are preserved while the distribution and dynamics of dual-label particles differ from wild-type by both immuno-fluorescence and live imaging. Knock-down of APP with siRNA eliminates APP staining, confirming specificity. Our results indicate that most intracellular HSV1 particles undergo frequent dynamic interplay with APP in a manner that facilitates viral transport and interferes with normal APP transport and distribution. Such dynamic interactions between APP and HSV1 suggest a mechanistic basis for the observed clinical relationship between HSV1 seropositivity and risk of Alzheimer's disease

Potential therapeutic applications of microbial surface-activecompounds

Numerous investigations of microbial surface-active compounds or biosurfactants over the past two decades have led to the discovery of many interesting physicochemical and biological properties including antimicrobial, anti-biofilm and therapeutic among many other pharmaceutical and medical applications. Microbial control and inhibition strategies involving the use of antibiotics are becoming continually challenged due to the emergence of resistant strains mostly embedded within biofilm formations that are difficult to eradicate. Different aspects of antimicrobial and anti-biofilm control are becoming issues of increasing importance in clinical, hygiene, therapeutic and other applications. Biosurfactants research has resulted in increasing interest into their ability to inhibit microbial activity and disperse microbial biofilms in addition to being mostly nontoxic and stable at extremes conditions. Some biosurfactants are now in use in clinical, food and environmental fields, whilst others remain under investigation and development. The dispersal properties of biosurfactants have been shown to rival that of conventional inhibitory agents against bacterial, fungal and yeast biofilms as well as viral membrane structures. This presents them as potential candidates for future uses in new generations of antimicrobial agents or as adjuvants to other antibiotics and use as preservatives for microbial suppression and eradication strategies