Respiratory syncytial virus interaction with human airway epithelium

Although respiratory syncytial virus (RSV) is a major human respiratory pathogen, our knowledge of how it causes disease in humans is limited. Airway epithelial cells are the primary targets of RSV infection in vivo, so the generation and exploitation of RSV infection models based on morphologically and physiologically authentic well-differentiated primary human airway epithelial cells cultured at an air-liquid interface (WD-PAECs) provide timely developments that will help to bridge this gap. Here we review the interaction of RSV with WD-PAEC cultures, the authenticity of the RSV-WD-PAEC models relative to RSV infection of human airway epithelium in vivo, and future directions for their exploitation in our quest to understand RSV pathogenesis in humans.

Effects of modified low-density lipoproteins on human retinal pericyte survival

According to a current paradigm cardiovascular diseases can be initiated by exposure of vascular cells to qualitatively modified low-density lipoproteins (LDL). Capillary leakage, an early feature of diabetic retinopathy, results in the exposure of retinal pericytes to modified LDL, including glycated (G-LDL) and heavily oxidized glycated LDL (HOG-LDL). We demonstrate here that modified LDL inhibits the proliferation and survival of cultured human retinal pericytes. Modified LDL also induced DNA fragmentation in bovine retinal pericytes. Overall, HOG-LDL produced a significantly higher extent of cytotoxicity and apoptosis in retinal pericytes. These results indicate that exposure of pericytes to HOG-LDL could be implicated in the development of diabetic retinopathy.

Effect of interleukin-6 polymorphisms on human longevity:a systematic review and meta-analysis

Several studies have assessed changes in frequency of -174 interleukin (IL)-6 single nucleotide polymorphism (SNP) with age. If IL-6 tracks with disability and age-related diseases, then there should be reduction, in the oldest old, of the frequency of homozygous GG subjects, who produce higher IL-6 levels. However, discordant results have been obtained. To explore the relationship between this polymorphism and longevity, we analyzed individual data on long-living subjects and controls from eight case-control studies conducted in Europeans, using meta-analysis. There was no significant difference in the IL-6 genotype between the oldest old and controls (Odds Ratio [OR]=0.96; 95% C.I.: 0.77-1.20; p=0.71), but there was significant between-study heterogeneity (I2=55.5%). In a subgroup analyses when male centenarians from the three Italian studies were included, the frequency of the IL-6 -174 GG genotype was significantly lower than the other genotypes (OR=0.49; 95% C.I.: 0.31-0.80; p=0.004), with no evidence of heterogeneity (I2=0%). Our data supports a negative association between the GG genotype of IL-6 SNP and longevity in Italian centenarians, with males who carry the genotype being two times less likely to reach extreme old age compared with subjects carrying CC or CG genotypes. These findings were not replicated in other European groups suggesting a possible interaction between genetics, sex and environment in reaching longevity.

Molecular detection of CF lung pathogens:current status and future potential

Molecular diagnostic tests, based on the detection and identification of nucleic acids in human biological samples, are increasingly employed in the diagnosis of infectious diseases and may be of future benefit to CF microbiology services. Our growing understanding of the complex polymicrobial nature of CF airway infection has highlighted current and likely future shortcomings in standard diagnostic practices. Failure to detect fastidious or slow growing microbes and misidentification of newly emerging pathogens could potentially be addressed using culture-independent molecular technologies with high target specificity. This review considers existing molecular diagnostic tests in the context of the key requirements for an envisaged CF microbiology focussed assay. The issues of assay speed, throughput, detection of multiple pathogens, data interpretation and antimicrobial susceptibility testing are discussed.

Chronology of the Grotte du Renne (France) and implications for the context of ornaments and human remains within the Châtelperronian

There is extensive debate concerning the cognitive and behavioral adaptation of Neanderthals, especially in the period when the earliest anatomically modern humans dispersed into Western Europe, around 35,000–40,000 B.P. The site of the Grotte du Renne (at Arcy-sur-Cure) is of great importance because it provides the most persuasive evidence for behavioral complexity among Neanderthals. A range of ornaments and tools usually associated with modern human industries, such as the Aurignacian, were excavated from three of the Châtelperronian levels at the site, along with Neanderthal fossil remains (mainly teeth). This extremely rare occurrence has been taken to suggest that Neanderthals were the creators of these items. Whether Neanderthals independently achieved this level of behavioral complexity and whether this was culturally transmitted or mimicked via incoming modern humans has been contentious. At the heart of this discussion lies an assumption regarding the integrity of the excavated remains. One means of testing this is by radiocarbon dating; however, until recently, our ability to generate both accurate and precise results for this period has been compromised. A series of 31 accelerator mass spectrometry ultra?ltered dates on bones, antlers, artifacts, and teeth from six key archaeological levels shows an unexpected degree of variation. This suggests that some mixing of material may have occurred, which implies a more complex depositional history at the site and makes it dif?cult to be con?dent about the association of artifacts with human remains in the Châtelperronian levels.

A PCR-based method to distinguish fungi of the rice sheath-blight complex, Rhizoctonia solani, R. oryzae and R. oryzae-sativae

Identification of Rhizoctonia solani, R. oryzae and R. oryzae-sativae, components of the rice sheath disease complex, is extremely difficult and often inaccurate and as a result may hinder the success of extensive breeding programmes throughout Asia. In this study, primers designed from unique regions within the rDNA internal transcribed spacers have been used to develop a rapid PCR-based diagnostic test to provide an accurate identification of the species on rice. Tests on the specificity of the primers concerned showed that they provide the means for accurate identification of the Rhizoctonia species responsible for sheath diseases in rice.

The Opportunistic Pathogen Propionibacterium acnes: Insights into Typing, Human Disease, Clonal Diversification and CAMP Factor Evolution

We previously described a Multilocus Sequence Typing (MLST) scheme based on eight genes that facilitates population genetic and evolutionary analysis of P. acnes. While MLST is a portable method for unambiguous typing of bacteria, it is expensive and labour intensive. Against this background, we now describe a refined version of this scheme based on two housekeeping (aroE; guaA) and two putative virulence (tly; camp2) genes (MLST) that correctly predicted the phylogroup (IA, IA, IB, IC, II, III), clonal complex (CC) and sequence type (ST) (novel or described) status for 91% isolates (n = 372) via cross-referencing of the four gene allelic profiles to the full eight gene versions available in the MLST database (http://pubmlst.org/pacnes/). Even in the small number of cases where specific STs were not completely resolved, the MLST method still correctly determined phylogroup and CC membership. Examination of nucleotide changes within all the MLST loci provides evidence that point mutations generate new alleles approximately 1.5 times as frequently as recombination; although the latter still plays an important role in the bacterium's evolution. The secreted/cell-associated 'virulence' factors tly and camp2 show no clear evidence of episodic or pervasive positive selection and have diversified at a rate similar to housekeeping loci. The co-evolution of these genes with the core genome might also indicate a role in commensal/normal existence constraining their diversity and preventing their loss from the P. acnes population. The possibility that members of the expanded CAMP factor protein family, including camp2, may have been lost from other propionibacteria, but not P. acnes, would further argue for a possible role in niche/host adaption leading to their retention within the genome. These evolutionary insights may prove important for discussions surrounding camp2 as an immunotherapy target for acne, and the effect such treatments may have on commensal lineages. © 2013 McDowell et al.

Growth of human B cell colonies from peripheral blood of patients with systemic lupus erythematosus

Human B cell colonies were grown from peripheral blood of 12 patients with systemic lupus erythematosus (SLE) and from 12 healthy control subjects. The SLE group showed a large increase (p less than 0.001) in the number of colony forming cells (CFC) present in peripheral blood as compared with controls. The CFC were of the pre-B cell type. There was also a loss of OKT8+ cell inhibition of B cell colony growth in the SLE group compared with control subjects.

The co-occurrence of mtDNA mutations on different oxidative phosphorylation subunits, not detected by haplogroup analysis, affects human longevity and is population specific.

To re-examine the correlation between mtDNA variability and longevity, we examined mtDNAs from samples obtained from over 2200 ultranonagenarians (and an equal number of controls) collected within the framework of the GEHA EU project. The samples were categorized by high-resolution classification, while about 1300 mtDNA molecules (650 ultranonagenarians and an equal number of controls) were completely sequenced. Sequences, unlike standard haplogroup analysis, made possible to evaluate for the first time the cumulative effects of specific, concomitant mtDNA mutations, including those that per se have a low, or very low, impact. In particular, the analysis of the mutations occurring in different OXPHOS complex showed a complex scenario with a different mutation burden in 90+ subjects with respect to controls. These findings suggested that mutations in subunits of the OXPHOS complex I had a beneficial effect on longevity, while the simultaneous presence of mutations in complex I and III (which also occurs in J subhaplogroups involved in LHON) and in complex I and V seemed to be detrimental, likely explaining previous contradictory results. On the whole, our study, which goes beyond haplogroup analysis, suggests that mitochondrial DNA variation does affect human longevity, but its effect is heavily influenced by the interaction between mutations concomitantly occurring on different mtDNA genes

Complex Interactions between Dioxin-Like and Non-Dioxin-Like Compounds for in Vitro Cellular Responses: Implications for the Identification of Dioxin Exposure Biomarkers

Despite considerable advances in reducing the production of dioxin-like toxicants in recent years, contamination of the food chain still occasionally occurs resulting in huge losses to the agri-food sector and risk to human health through exposure. Dioxin-like toxicity is exhibited by a range of stable and bioaccumulative compounds including polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), produced by certain types of combustion, and man-made coplanar polychlorinated biphenyls (PCBs), as found in electrical transformer oils. While dioxinergic compounds act by a common mode of action making exposure detection biomarker based techniques a potentially useful tool, the influence of co-contaminating toxicants on such approaches needs to be considered. To assess the impact of possible interactions, the biological responses of H4IIE cells to challenge by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in combination with PCB-52 and benzo-a-pyrene (BaP) were evaluated by a number of methods in this study. Ethoxyresorufin-O-deethylase (EROD) induction in TCDD exposed cells was suppressed by increasing concentrations of PCB-52, PCB-153, or BaP up to 10 mu M. BaP levels below 1 mu M suppressed TCDD stimulated EROD induction, but at higher concentrations, EROD induction was greater than the maximum observed when cells were treated with TCDD alone. A similar biphasic interaction of BaP with TCDD co-exposure was noted in the AlamarBlue assay and to a lesser extent with PCB-52. Surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF) profiling of peptidomic responses of cells exposed to compound combinations was compared. Cells co-exposed to TCDD in the presence of BaP or PCB-52 produced the most differentiated spectra with a substantial number of non-additive interactions observed. These findings suggest that interactions between dioxin and other toxicants create novel, additive, and non-additive effects, which may be more indicative of the types of responses seen in exposed animals than those of single exposures to the individual compounds.

Rare and common variants in extracellular matrix gene Fibrillin 2 (FBN2) are associated with macular degeneration

Neurodegenerative diseases affecting the macula constitute a major cause of incurable vision loss and exhibit considerable clinical and genetic heterogeneity, from early-onset monogenic disease to multifactorial late-onset age-related macular degeneration (AMD). As part of our continued efforts to define genetic causes of macular degeneration, we performed whole exome sequencing in four individuals of a two-generation family with autosomal dominant maculopathy and identified a rare variant p.Glu1144Lys in Fibrillin 2 (FBN2), a glycoprotein of the elastin-rich extracellular matrix (ECM). Sanger sequencing validated the segregation of this variant in the complete pedigree, including two additional affected and one unaffected individual. Sequencing of 192 maculopathy patients revealed additional rare variants, predicted to disrupt FBN2 function. We then undertook additional studies to explore the relationship of FBN2 to macular disease. We show that FBN2 localizes to Bruch's membrane and its expression appears to be reduced in aging and AMD eyes, prompting us to examine its relationship with AMD. We detect suggestive association of a common FBN2 non-synonymous variant, rs154001 (p.Val965Ile) with AMD in 10,337 cases and 11,174 controls (OR=1.10; p-value=3.79×10(-5)). Thus, it appears that rare and common variants in a single gene - FBN2 - can contribute to Mendelian and complex forms of macular degeneration. Our studies provide genetic evidence for a key role of elastin microfibers and Bruch's membrane in maintaining blood-retina homeostasis and establish the importance of studying orphan diseases for understanding more common clinical phenotypes.

V3 versican isoform alters the behavior of human melanoma cells by interfering with CD44/ErbB-dependent signaling

Versican is a hyaluronan-binding, extracellular chondroitin sulfate proteoglycan produced by several tumor types, including malignant melanoma, which exists as four different splice variants. The short V3 isoform contains the G1 and G3 terminal domains of versican that may potentially interact directly or indirectly with the hyaluronan receptor CD44 and the EGFR, respectively. We have previously described that overexpression of V3 in MeWo human melanoma cells markedly reduces tumor cell growth in vitro and in vivo. In this study we have investigated the signaling mechanism of V3 by silencing the expression of CD44 in control and V3-expressing melanoma cells. Suppression of CD44 had the same effects on cell proliferation and cell migration than those provoked by V3 expression, suggesting that V3 acts through a CD44-mediated mechanism. Furthermore, CD44-dependent hyaluronan internalization was blocked by V3 expression and CD44 silencing, leading to an accumulation of this glycosaminoglycan in the pericellular matrix and to changes in cell migration on hyaluronan. Furthermore, ERK1/2 and p38 activation after EGF treatment were decreased in V3-expressing cells suggesting that V3 may also interact with the EGFR through its G3 domain. The existence of a EGFR/ErbB2 receptor complex able to interact with CD44 was identified in MeWo melanoma cells. V3 overexpression resulted in a reduced interaction between EGFR/ErbB2 and CD44 in response to EGF treatment. Our results indicate that the V3 isoform of versican interferes with CD44 and the CD44-EGFR/ErbB2 interaction, altering the signaling pathways, such as ERK1/2 and p38 MAPK, that regulate cell proliferation and migration.

Role of versican V0/V1 and CD44 in the regulation of human melanoma cell behaviour

Versican is a hyaluronan-binding, large extracellular matrix chondroitin sulfate proteoglycan whose expression is increased in malignant melanoma. Binding to hyaluronan allows versican to indirectly interact with the hyaluronan cell surface receptor CD44. The aim of this work was to study the effect of silencing the large versican isoforms (V0 and V1) and CD44 in the SK-mel-131 human melanoma cell line. Versican V0/V1 or CD44 silencing caused a decrease in cell proliferation and migration, both in wound healing assays and in Transwell chambers. Versican V0/V1 silencing also caused an increased adhesion to type I collagen, laminin and fibronectin. These results support the proposed role of versican as a proliferative, anti-adhesive and pro-migratory molecule. On the other hand, CD44 silencing caused a decrease in cell adhesion to vitronectin, fibronectin and hyaluronan. CD44 silencing inhibited the binding of a FITC-hyaluronan complex to the cell surface and its internalization into the cytoplasm. Our results indicate that both versican and CD44 play an important role regulating the behavior of malignant melanoma cells.

Genome-wide characterization reveals complex interplay between TP53 and TP63 in response to genotoxic stress

In response to genotoxic stress the TP53 tumour suppressor activates target gene expression to induce cell cycle arrest or apoptosis depending on the extent of DNA damage. These canonical activities can be repressed by TP63 in normal stratifying epithelia to maintain proliferative capacity or drive proliferation of squamous cell carcinomas, where TP63 is frequently overexpressed/amplified. Here we use ChIP-sequencing, integrated with microarray analysis, to define the genome-wide interplay between TP53 and TP63 in response to genotoxic stress in normal cells. We reveal that TP53 and TP63 bind to overlapping, but distinct cistromes of sites through utilization of distinctive consensus motifs and that TP53 is constitutively bound to a number of sites. We demonstrate that cisplatin and adriamycin elicit distinct effects on TP53 and TP63 binding events, through which TP53 can induce or repress transcription of an extensive network of genes by direct binding and/or modulation of TP63 activity. Collectively, this results in a global TP53-dependent repression of cell cycle progression, mitosis and DNA damage repair concomitant with activation of anti-proliferative and pro-apoptotic canonical target genes. Further analyses reveal that in the absence of genotoxic stress TP63 plays an important role in maintaining expression of DNA repair genes, loss of which results in defective repair.

Clonal complex Pseudomonas aeruginosa in horses

Pseudomonas aeruginosa is associated with infectious endometritis in horses. Although infectious endometritis is often considered a venereal infection, there is relatively limited genotypic-based evidence to support this mode of transmission. The study sought to determine the relatedness between genital P. aeruginosa isolates collected from a limited geographical region using molecular strain typing. Enterobacterial repetitive intergenic consensus PCR typing was performed on 93 isolates collected between 2005 and 2009 from 2058 thoroughbred horses (including 18 stallions) at 66 studs. While P. aeruginosa was not detected in the stallions, 53/93 (57%) mares harbouring P. aeruginosa had clonally related strains, which included a single dominant genotype detected in 42 (45%) mares from 13 different studs. These novel findings suggest that most equine genital P. aeruginosa infections in this region may have been acquired from mechanisms other than direct horse to horse transmission. Instead, other potential acquisition pathways, as well as strain specific adaptation to the equine genital tract, should be investigated.