CD200 ectodomain shedding into the tumor microenvironment leads to NK cell dysfunction and apoptosis.

The basis of immune evasion, a hallmark of cancer, can differ even when cancers arise from one cell type such as in the human skin keratinocyte carcinomas: basal and squamous cell carcinoma. Here we showed that the basal cell carcinoma tumor-initiating cell surface protein CD200, through ectodomain shedding, was responsible for the near absence of NK cells within the basal cell carcinoma tumor microenvironment. In situ, CD200 underwent ectodomain shedding by metalloproteinases MMP3 and MMP11, which released biologically active soluble CD200 into the basal cell carcinoma microenvironment. CD200 bound its cognate receptor on NK cells to suppress MAPK pathway signaling that in turn blocked indirect (IFN-γ release) and direct cell killing. In addition, reduced ERK phosphorylation relinquished negative regulation of PPARγ-regulated gene transcription and led to membrane accumulation of the Fas/FADD death receptor and its ligand, FasL, which resulted in activation-induced apoptosis. Blocking CD200 inhibition of MAPK or PPARγ signaling restored NK cell survival and tumor cell killing, with relevance to many cancer types. Our results thus uncover a paradigm for CD200 as a potentially novel and targetable NK cell-specific immune checkpoint, which is responsible for NK cell-associated poor outcomes in many cancers.

Targeted disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate oligosaccharides.

Acquisition of a mucoid phenotype by Pseudomonas sp. in the lungs of cystic fibrosis (CF) patients, with subsequent over-production of extracellular polymeric substance (EPS), plays an important role in mediating the persistence of multi-drug resistant (MDR) infections. The ability of a low molecular weight (Mn = 3200 g mol-1) alginate oligomer (OligoG CF-5/20) to modify biofilm structure of mucoid Pseudomonas aeruginosa (NH57388A) was studied in vitro using scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) with Texas Red (TxRd®)-labelled OligoG and EPS histochemical staining. Structural changes in treated biofilms were quantified using COMSTAT image-analysis software of CLSM z-stack images, and nanoparticle diffusion. Interactions between the oligomers, Ca2+ and DNA were studied using molecular dynamics (MD) simulations, Fourier transform infrared spectroscopy (FTIR) and isothermal titration calorimetry (ITC). Imaging demonstrated that OligoG treatment (≥0.5%) inhibited biofilm formation, revealing a significant reduction in both biomass and biofilm height (P < 0.05). TxRd®-labelled oligomers readily diffused into established (24 h) biofilms. OligoG treatment (≥2%) induced alterations in the EPS of established biofilms; significantly reducing the structural quantities of EPS polysaccharides, and extracellular (e)DNA (P < 0.05) with a corresponding increase in nanoparticle diffusion (P < 0.05) and antibiotic efficacy against established biofilms. ITC demonstrated an absence of rapid complex formation between DNA and OligoG and confirmed the interactions of OligoG with Ca2+ evident in FTIR and MD modelling. The ability of OligoG to diffuse into biofilms, potentiate antibiotic activity, disrupt DNA-Ca2+-DNA bridges and biofilm EPS matrix highlights its potential for the treatment of biofilm-related infections.

The interaction of wood nanocellulose dressings and the wound pathogen P. aeruginosa.

Chronic wounds pose an increasingly significant worldwide economic burden (over £1 billion per annum in the UK alone). With the escalation in global obesity and diabetes, chronic wounds will increasingly be a significant cause of morbidity and mortality. Cellulose nanofibrils (CNF) are highly versatile and can be tailored with specific physical properties to produce an assortment of three-dimensional structures (hydrogels, aerogels or films), for subsequent utilization as wound dressing materials. Growth curves using CNF (diameter <20nm) in suspension demonstrated an interesting dose-dependent inhibition of bacterial growth. In addition, analysis of biofilm formation (Pseudomonas aeruginosa PAO1) on nanocellulose aerogels (20g/m2) revealed significantly less biofilm biomass with decreasing aerogel porosity and surface roughness. Importantly, virulence factor production by P. aeruginosa in the presence of nanocellulose materials, quantified for the first time, was unaffected (p>0.05) over 24h. These data demonstrate the potential of nanocellulose materials in the development of novel dressings that may afford significant clinical potential.

Non-invasive detection of early retinal neuronal degeneration by ultrahigh resolution optical coherence tomography.

Optical coherence tomography (OCT) has revolutionises the diagnosis of retinal disease based on the detection of microscopic rather than subcellular changes in retinal anatomy. However, currently the technique is limited to the detection of microscopic rather than subcellular changes in retinal anatomy. However, coherence based imaging is extremely sensitive to both changes in optical contrast and cellular events at the micrometer scale, and can generate subtle changes in the spectral content of the OCT image. Here we test the hypothesis that OCT image speckle (image texture) contains information regarding otherwise unresolvable features such as organelle changes arising in the early stages of neuronal degeneration. Using ultrahigh resolution (UHR) OCT imaging at 800 nm (spectral width 140 nm) we developed a robust method of OCT image analyses, based on spatial wavelet and texture-based parameterisation of the image speckle pattern. For the first time we show that this approach allows the non-invasive detection and quantification of early apoptotic changes in neurons within 30 min of neuronal trauma sufficient to result in apoptosis. We show a positive correlation between immunofluorescent labelling of mitochondria (a potential source of changes in cellular optical contrast) with changes in the texture of the OCT images of cultured neurons. Moreover, similar changes in optical contrast were also seen in the retinal ganglion cell- inner plexiform layer in retinal explants following optic nerve transection. The optical clarity of the explants was maintained throughout in the absence of histologically detectable change. Our data suggest that UHR OCT can be used for the non-invasive quantitative assessment of neuronal health, with a particular application to the assessment of early retinal disease.

Mitochondrial localization and ocular expression of mutant Opa3 in a mouse model of 3-methylglutaconicaciduria type III.

PURPOSE: To investigate the developmental and ocular expression of Opa3 in a mouse model of 3-methylglutaconicaciduria type III and the effect of mutation on protein localization and mitochondrial morphology. METHODS: The B6 C3-Opa3(L122P) mouse carrying a missense mutation in exon 2 (c.365T>C; p.L122P) of Opa3, which displays features of recessive 3-methylglutaconic aciduria type III was studied. The expression of Opa3 was determined with RT-PCR, quantitative PCR, and Western blot, in embryos (embryonic day [E]8 to postnatal day [P]0) and adult tissues, and by ocular immunohistochemistry. Mitochondria were stained using a mitochondrion-selective probe in mouse embryonic fibroblasts from Opa3(-/-) mutants and imaged by electron microscopy of the retinas. RESULTS: The splice variants Opa3a and Opa3b were expressed in the lenses and the retinas in the Opa3(-/-) mice, with the expression of the Opa3a isoform predominant. Opa3 was expressed throughout embryonic development, with high levels of expression in the developing brain, retina, optic nerve, and lens. Opa3 localized to the mitochondria, and the L122P mutant protein did not mislocalize. Neither protein localized to the peroxisome. Opa3(-/-) mice displayed disrupted mitochondrial morphology in the retina. Wild-type Opa3 protein increased as the lenses aged, despite the reduction in Opa3 mRNA occurring as a part of lens differentiation. However, mutant Opa3 mRNA was upregulated in homozygous mutant lenses, suggesting a compensatory increase in expression, which may further increase Opa3 protein levels. CONCLUSIONS: Mutant Opa3 protein retains its mitochondrial localization and induces disrupted mitochondrial morphology. Opa3 accumulates in the lens. The results may reflect a slow turnover of Opa3 protein in vivo and may be important in normal lens physiology.

A missense mutation in the murine Opa3 gene models human Costeff syndrome.

Opa3 mRNA is expressed in all tissues examined to date, but currently the function of the OPA3 protein is unknown. Intriguingly, various mutations in the OPA3 gene lead to two similar diseases in humans: autosomal dominant inherited optic atrophy and cataract (ADOAC) and a metabolic condition; type 3-methylglutaconic aciduria (MGA). Early onset bilateral optic atrophy is a common characteristic of both disorders; retinal ganglion cells are lost and visual acuity is impaired from an early age. In order to investigate the function of the OPA3 protein, we have generated a novel ENU-induced mutant mouse carrying a missense mutation in the OPA3 gene. The heterozygous mutation in exon 2, causes an amino acid change p.L122P (c.365T>C), which is predicted to alter tertiary protein structure. In the heterozygous state, the mice appear uncompromised however; in the homozygous state mice display some of the features of MGA. Visual function is severely reduced, consistent with significant loss of retinal ganglion cells and degeneration of axons in the optic nerve. In the homozygous optic nerve, there was evidence of increased mitochondrial activity, as demonstrated by the increased presence of mitochondrial marker Cytochrome C Oxidase (COX) histochemistry. Mice homozygous for the opa3(L122P) mutation also display a severe multi-systemic disease characterized by reduced lifespan (majority dying before 4 months), decreased weight, dilated cardiomyopathy, extrapyramidal dysfunction and gross neuro-muscular defects. All of these defects are synonymous with the phenotypic characteristics of Type III MGA found in humans. This model will be of major importance for future studies of the specific function of the OPA3 gene.

Differential incorporation of docosahexaenoic and arachidonic acids by the yolk sac membrane of the avian embryo.

During avian development, lipoproteins derived from yolk lipid are assembled in the yolk sac membrane (YSM) for secretion into the embryonic circulation. To investigate how yolk polyunsaturated fatty acids, essential for the development of certain tissues, are distributed among the lipid classes of the lipoproteins, pieces of YSM were incubated in vitro with [14C]arachidonic and [14C]docosahexaenoic acids (DHA). There was a marked difference in the partitioning of these two precursors among the lipid classes of the tissue. Of the radioactivity incorporated into total lipid from [14C]-arachidonic acid during 1 h of incubation, 67.3% was esterified as phospholipid and 29.5% as triacylglycerol. In contrast, only 14.6% of the label incorporated from [14C]-DHA was esterified as phospholipid, whereas 73.2% was recovered in triacylglycerol. This pattern of differential partitioning was observed at all time points and across a 20-fold range of fatty acid concentrations. There was no evidence for conversion of the radioactive arachidonic and DHAs to other fatty acids prior to incorporation into tissue lipids. It is suggested that the selective incorporation of yolk-derived DHA into the triacylglycerol of secreted lipoproteins represents part of a mechanism for directing this polyunsaturate to particular embryonic tissues.