A disease-associated gene desert directs macrophage inflammation through ETS2.

Increasing rates of autoimmune and inflammatory disease present a burgeoning threat to human health1. This is compounded by the limited efficacy of available treatments1 and high failure rates during drug development2, highlighting an urgent need to better understand disease mechanisms. Here we show how functional genomics could address this challenge. By investigating an intergenic haplotype on chr21q22-which has been independently linked to inflammatory bowel disease, ankylosing spondylitis, primary sclerosing cholangitis and Takayasu's arteritis3-6-we identify that the causal gene, ETS2, is a central regulator of human inflammatory macrophages and delineate the shared disease mechanism that amplifies ETS2 expression. Genes regulated by ETS2 were prominently expressed in diseased tissues and more enriched for inflammatory bowel disease GWAS hits than most previously described pathways. Overexpressing ETS2 in resting macrophages reproduced the inflammatory state observed in chr21q22-associated diseases, with upregulation of multiple drug targets, including TNF and IL-23. Using a database of cellular signatures7, we identified drugs that might modulate this pathway and validated the potent anti-inflammatory activity of one class of small molecules in vitro and ex vivo. Together, this illustrates the power of functional genomics, applied directly in primary human cells, to identify immune-mediated disease mechanisms and potential therapeutic opportunities.

Advances in image enhancement for sarcoma surgery.

The recurrence rate of soft tissue and bone sarcomas strongly correlates to the status of the surgical margin after excision, yet excessive removal of tissue may lead to distinct, otherwise avoidable morbidity. Therefore, adequate margination of sarcomas both pre- and intra-operatively is a clinical necessity that has not yet fully been met. Current guidance for soft-tissue sarcomas recommends an ultrasound scan followed by magnetic resonance imaging (MRI). For bone sarcomas, two plane radiographs are required, followed similarly by an MRI scan. The introduction of more precise imaging modalities may reduce the morbidity associated with sarcoma surgery; the PET-CT and PET-MRI approaches in particular demonstrating high clinical efficacy. Despite advancements in the accuracy in pre-operative imaging, translation of an image to surgical margins is difficult, regularly resulting in wider resection margins than required. For soft tissue sarcomas there is currently no standard technique for image guided resections, while for bone sarcomas fluoroscopy may be used, however margins are not easily discernible during the surgical procedure. Near infra-red (NIR) fluorescence guided surgery offers an intra-operative modality through which complete tumour resection with adequate tumour-free margins may be achieved, while simultaneously minimising surgical morbidity. NIR imaging presents a potentially valuable adjunct to sarcoma surgery. Early reports indicate that it may be able to provide the surgeon with helpful information on anatomy, perfusion, lymphatic drainage, tumour margins and metastases. The use of NIR fluorochromes have also been demonstrated to be well tolerated by patients. However, prior to widespread implementation, studies related to cost-effectiveness and the development of protocols are essential. Nevertheless, NIR imaging may become ubiquitous in the future, carrying the potential to transform the surgical management of sarcoma.

High-resolution air-clad imaging fibers.

We present a coherent fiber bundle comprising over 11,000 doped silica cores separated by an air-filled cladding. The fiber is characterized, and its imaging quality is shown to be a substantial improvement over the commercial state of the art, with comparable resolution over an unparalleled spectral range.

Unique transcriptome signatures and GM-CSF expression in lymphocytes from patients with spondyloarthritis.

Spondyloarthritis encompasses a group of common inflammatory diseases thought to be driven by IL-17A-secreting type-17 lymphocytes. Here we show increased numbers of GM-CSF-producing CD4 and CD8 lymphocytes in the blood and joints of patients with spondyloarthritis, and increased numbers of IL-17A+GM-CSF+ double-producing CD4, CD8, γδ and NK cells. GM-CSF production in CD4 T cells occurs both independently and in combination with classical Th1 and Th17 cytokines. Type 3 innate lymphoid cells producing predominantly GM-CSF are expanded in synovial tissues from patients with spondyloarthritis. GM-CSF+CD4+ cells, isolated using a triple cytokine capture approach, have a specific transcriptional signature. Both GM-CSF+ and IL-17A+GM-CSF+ double-producing CD4 T cells express increased levels of GPR65, a proton-sensing receptor associated with spondyloarthritis in genome-wide association studies and pathogenicity in murine inflammatory disease models. Silencing GPR65 in primary CD4 T cells reduces GM-CSF production. GM-CSF and GPR65 may thus serve as targets for therapeutic intervention of spondyloarthritis.

Investigation of a possible extended risk haplotype in the IL23R region associated with ankylosing spondylitis.

The IL23R region on chromosome 1 exhibits complex associations with ankylosing spondylitis (AS). We used publicly available epigenomic information and historical genetic association data to identify a putative regulatory element (PRE) in the intergenic region between IL23R and IL12RB2, which includes two single-nucleotide polymorphisms (SNPs) independently associated with AS-rs924080 (P=2 × 10-3) and rs11578380 (P=2 × 10-4). In luciferase reporter assays, this PRE showed silencer activity (P<0.001). Haplotype and conditional analysis of 4230 historical AS cases and 9700 controls revealed a possible AS-associated extended haplotype, including the PRE and risk variants at three SNPs (rs11209026, rs11209032 and rs924080), but excluding the rs11578380 risk variant. However, the rs924080 association was absent after conditioning on the primary association with rs11209032, which, in contrast, was robust to conditioning on all other AS-associated SNPs in this region (P<2 × 10-8). The role of this putative silencer on some IL23R extended haplotypes therefore remains unclear.

miR-10b-5p is a novel Th17 regulator present in Th17 cells from ankylosing spondylitis.

OBJECTIVE: To determine the microRNA (miR) signature in ankylosing spondylitis (AS) T helper (Th)17 cells. METHODS: Interleukin (IL)-17A-producing CD4+ T cells from patients with AS and healthy controls were FACS-sorted for miR sequencing and qPCR validation. miR-10b function was determined by miR mimic expression followed by cytokine measurement, transcriptome analysis, qPCR and luciferase assays. RESULTS: AS Th17 cells exhibited a miR signature characterised by upregulation of miR-155-5p, miR-210-3p and miR-10b. miR-10b has not been described previously in Th17 cells and was selected for further characterisation. miR-10b is transiently induced in in vitro differentiated Th17 cells. Transcriptome, qPCR and luciferase assays suggest that MAP3K7 is targeted by miR-10b. Both miR-10b overexpression and MAP3K7 silencing inhibited production of IL-17A by both total CD4 and differentiating Th17 cells. CONCLUSIONS: AS Th17 cells have a specific miR signature and upregulate miR-10b in vitro. Our data suggest that miR-10b is upregulated by proinflammatory cytokines and may act as a feedback loop to suppress IL-17A by targeting MAP3K7. miR-10b is a potential therapeutic candidate to suppress pathogenic Th17 cell function in patients with AS.

Anterior uterocervical angle measurement improves prediction of cerclage failure.

OBJECTIVE: To compare the anterior uterocervical angle and cervical length as predictors of spontaneous preterm delivery in patients with transvaginal cerclage. STUDY DESIGN: We retrospectively evaluated a cohort of 142 pregnant women with transvaginal cerclage placed over a 5-year period (2010 to 2015) were evaluated. Cervical morphology characteristics were measured from endovaginal imaging, including cervical length, cerclage height, funnel volume and anterior uterocervical angle prior to cerclage placement (UCA 1), shortly after cerclage placement (UCA 2) and the last image prior to delivery (UCA 3). Cerclage failure was defined as delivery prior to 36 weeks. Univariate analysis, receiver operator characteristic curves and binary logistic regression were used for statistical analysis. Statistical significance was defined as a P<0.05. RESULTS: Among the 142 women with a transvaginal cerclage, 38% had cerclage failure. The mean gestational age at birth was 29.3±5.2 weeks in the failure group compared with 37.9±2.8 weeks in those that did not fail (P<0.001). Univariate analysis identified cervical length (P=0.034) and UCA 3 (P<0.001) as significantly associated with gestational age at birth. Receiver operator characteristic curves demonstrated improved prediction of delivery prior to 34 weeks at UCA 3=108o (97% sensitivity, 65% specificity) compared to a cervical length of 25 mm. At <28 weeks, optimal performance of UCA 3 was found at 112o (100% sensitivity, 62% specificity) compared with cervical length of 25 mm (29% sensitivity, 39% specificity). Binary logistic regression revealed UCA 3>108o conferred an OR 35.1 (95% CI 7.7 to 160.3) for delivery prior to 34 weeks, and UCA 3>112o an OR 42.0 (95% CI 5.3 to 332.1) for delivery prior to 28 weeks. In comparison, CL<25 mm had an OR 4.7 (95% CI 1.8 to 12.2) for delivery prior to 34 weeks and OR 6.0 (95% CI 1.9 to 19.3) prior to 28 weeks. CONCLUSIONS: In patients with transvaginal cerclage, an increasingly obtuse anterior uterocervical angle reflects an increased risk of cerclage failure in the mid-trimester. Utilization of UCA measurement as a surveillance tool may improve identification of patients at risk for cerclage failure.

Chronic mucocutaneous candidiasis: characterization of a family with STAT-1 gain-of-function and development of an ex-vivo assay for Th17 deficiency of diagnostic utility.

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent and persistent superficial infections, with Candida albicans affecting the mucous membranes, skin and nails. It can be acquired or caused by primary immune deficiencies, particularly those that impair interleukin (IL)-17 and IL-22 immunity. We describe a single kindred with CMC and the identification of a STAT1 GOF mutation by whole exome sequencing (WES). We show how detailed clinical and immunological phenotyping of this family in the context of WES has enabled revision of disease status and clinical management. Together with analysis of other CMC cases within our cohort of patients, we used knowledge arising from the characterization of this family to develop a rapid ex-vivo screening assay for the detection of T helper type 17 (Th17) deficiency better suited to the routine diagnostic setting than established in-vitro techniques, such as intracellular cytokine staining and enzyme-linked immunosorbent assay (ELISA) using cell culture supernatants. We demonstrate that cell surface staining of unstimulated whole blood for CCR6⁺ CXCR3⁻ CCR4⁺ CD161⁺ T helper cells generates results that correlate with intracellular cytokine staining for IL-17A, and is able to discriminate between patients with molecularly defined CMC and healthy controls with 100% sensitivity and specificity within the cohort tested. Furthermore, removal of CCR4 and CD161 from the antibody staining panel did not affect assay performance, suggesting that the enumeration of CCR6⁺ CXCR3⁻ CD4⁺ T cells is sufficient for screening for Th17 deficiency in patients with CMC and could be used to guide further investigation aimed at identifying the underlying molecular cause.

A functional AT/G polymorphism in the 5'-untranslated region of SETDB2 in the IgE locus on human chromosome 13q14.

The immunoglobulin E (IgE)-associated locus on human chromosome 13q14 influencing asthma-related traits contains the genes PHF11 and SETDB2. SETDB2 is located in the same linkage disequilibrium region as PHF11 and polymorphisms within SETDB2 have been shown to associate with total serum IgE levels. In this report, we sequenced the 15 exons of SETDB2 and identified a single previously ungenotyped mutation (AT/G, rs386770867) in the 5'-untranslated region of the gene. The polymorphism was found to be significantly associated with serum IgE levels in our asthma cohort (P=0.0012). Electrophoretic mobility shift assays revealed that the transcription factor Ying Yang 1 binds to the AT allele, whereas SRY (Sex determining Region Y) binds to the G allele. Allele-specific transcription analysis (allelotyping) was performed in 35 individuals heterozygous for rs386770867 from a panel of 200 British families ascertained through probands with severe stage 3 asthma. The AT allele was found to be significantly overexpressed in these individuals (P=1.26×10(-21)). A dual-luciferase assay with the pGL3 luciferase reporter gene showed that the AT allele significantly affects transcriptional activities. Our results indicate that the IgE-associated AT/G polymorphism (rs386770867) regulates transcription of SETDB2.

Development of an enzymatic pretargeting strategy for dual-modality imaging.

A pretargeted imaging strategy based on the HaloTag dehalogenase enzyme is described. Here, a HaloTag-Trastuzumab conjugate has been used as the primary agent targeting HER2 expression, and three new radiolabelled HaloTag ligands have been used as secondary agents, two of which offer dual-modality (SPECT/optical) imaging capability.

Highly birefringent 98-core fiber.

We report on the fabrication and characterization of a polarization-maintaining multicore fiber. The fiber has 98 aligned cores, each with a birefringence of ≈2.3×10-4. The beat length, polarization extinction ratio, and polarization orientation are characterized.

1064 nm laser-induced defects in pure SiO2 fibers.

We investigate evidence of the formation of nonbridging oxygen hole centers in pure silica photonic crystal fibers from 5 ps 1064 nm pulses. The formation of the defects is attributed to the breaking of stressed silicon-oxygen bonds in the glass matrix through a many-photon process. We compare the photodarkening induced by the 1064 nm pump with photodarkening induced by short wavelength light in a 1064 nm pumped supercontinuum extending to 400 nm. It is shown that the higher peak power at the pump wavelength makes it a more significant cause of photodarkening when compared to the shorter wavelength light generated in the fiber.

Femtosecond pulses at 20 GHz repetition rate through spectral masking of a phase modulated signal and nonlinear pulse compression.

We present a laser system capable of producing 190 femtosecond pulses at a repetition rate of 20 GHz. The spectral masking of a phase modulated diode laser is used to produce a train of picosecond pulses which are compressed using a fibre-grating compressor followed by subsequent adiabatic soliton compression to the femtosecond regime using a tapered photonic crystal fiber.

Flexible delivery of Er:YAG radiation at 2.94 µm with negative curvature silica glass fibers: a new solution for minimally invasive surgical procedures.

We present the delivery of high energy microsecond pulses through a hollow-core negative-curvature fiber at 2.94 µm. The energy densities delivered far exceed those required for biological tissue manipulation and are of the order of 2300 J/cm(2). Tissue ablation was demonstrated on hard and soft tissue in dry and aqueous conditions with no detrimental effects to the fiber or catastrophic damage to the end facets. The energy is guided in a well confined single mode allowing for a small and controllable focused spot delivered flexibly to the point of operation. Hence, a mechanically and chemically robust alternative to the existing Er:YAG delivery systems is proposed which paves the way for new routes for minimally invasive surgical laser procedures.

Resolving the variable genome and epigenome in human disease.

The individual human genome and epigenome are being defined at unprecedented resolution by current advances in sequencing technologies with important implications for human disease. This review uses examples relevant to clinical practice to illustrate the functional consequences of genetic and epigenetic variation. The insights gained from genome-wide association studies are described together with current efforts to understand the role of rare variants in common disease, set in the context of recent successes in Mendelian traits through the application of whole exome sequencing. The application of functional genomics to interrogate the genome and epigenome, build up an integrated picture of the regulatory genomic landscape and inform disease association studies is discussed, together with the role of expression quantitative trait mapping and analysis of allele-specific gene expression.

Delivery of high energy Er:YAG pulsed laser light at 2.94 µm through a silica hollow core photonic crystal fibre.

In this paper the delivery of high power Er:YAG laser pulses through a silica hollow core photonic crystal fibre is demonstrated. The Er:YAG wavelength of 2.94 µm is well beyond the normal transmittance of bulk silica but the unique hollow core guidance allows silica to guide in this regime. We have demonstrated for the first time the ability to deliver high energy pulses through an all-silica fibre at 2.94 µm. These silica fibres are mechanically and chemically robust, biocompatible and have low sensitivity to bending. A maximum pulse energy of 14 mJ at 2.94 µm was delivered through the fibre. This, to our knowledge, is the first time a silica hollow core photonic crystal fibre has been shown to transmit 2.94 µm laser light at a fluence exceeding the thresholds required for modification (e.g. cutting and drilling) of hard biological tissue. Consequently, laser delivery systems based on these fibres have the potential for the realization of novel, minimally-invasive surgical procedures.

Modulational instability in a silicon-on-insulator directional coupler: role of the coupling-induced group velocity dispersion.

We report frequency conversion experiments in silicon-on-insulator (SOI) directional couplers. We demonstrate that the evanescent coupling between two subwavelength SOI waveguides is strongly dispersive and significantly modifies modulational instability (MI) spectra through the coupling induced group velocity dispersion (GVD). As the separation between two 380-nm-wide silicon photonic wires decreases, the increasing dispersion of the coupling makes the GVD in the symmetric supermode more normal and suppresses the bandwidth of the MI gain observed for larger separations.

Coherent supercontinuum generation in photonic crystal fiber with all-normal group velocity dispersion.

We demonstrate supercontinuum generation in a photonic crystal fiber with all-normal group velocity dispersion. Pumping a short section of this fiber with compressed pulses from a compact amplified fiber laser generates a 200 nm bandwidth continuum with typical self-phase-modulation characteristics. We demonstrate that the supercontinuum is compressible to a duration of 26 fs. It therefore has a high degree of coherence between all the frequency components, and is a single pulse in the time domain. A smooth, flat spectrum spanning 800 nm is achieved using a longer piece of fiber.