Validation of a fornix depth measurer: a putative tool for the assessment of progressive cicatrising conjunctivitis

Background/aims Documentation of conjunctival forniceal foreshortening in cases of progressive cicatrising conjunctivitis (PCC) is important in ascertaining disease stage and progression. Lower fornix shortening is often documented subjectively or semi-objectively, whereas upper forniceal obliteration is seldom quantified. Although tools such as fornix depth measurers (FDMs) have been described, their designs limit upper fornix measurement. The purpose of this study was to custom-design a FDM to evaluate the upper fornix and to assess variability in gauging fornix depth. \ud \ud Methods A polymethylmethacrylate FDM was constructed using industry-standard jewellery computer software and machinery. Two observers undertook a prospective independent evaluation of central lower fornix depth in a heterogeneous cohort of patients with clinically normal and abnormal conjunctival fornices both subjectively and by using the FDM (in mm). Upper central fornix depth was also measured. Agreement was assessed using Bland–Altman plots. \ud \ud Results Fifty-one eyes were evaluated. There was 100% intraobserver agreement to within 1 mm for each observer for lower fornix measurement. The mean difference in fornix depth loss using the FDM between observer 1 and 2 was 1.19%, with 95% confidence of agreement (±2SD) of −15% to +20%. In total, 86% (44/51) of measurements taken by the two observers agreed to within 10% of total lower fornix depth (ie, ±1 mm) versus only 63% (32/51) of the subjective measurements. Mean upper fornix difference was 0.57 mm, with 95% confidence of agreement of between −2 and + 3 mm. \ud \ud Conclusions This custom-designed FDM is well tolerated by patients and shows low intraobserver and interobserver variability. This enables repeatable and reproducible measurement of upper and lower fornix depths, facilitating improved rates of detection and better monitoring of progression of conjunctival scarring

Distinct Types of Fibrocyte Can Differentiate from Mononuclear Cells in the Presence and Absence of Serum

Background: Ageing, immunity and stresstolerance are inherent characteristics of all organisms. In animals, these traits are regulated, at least in part, by forkhead transcription factors in response to upstream signals from the Insulin/Insulin–like growth factor signalling (IIS) pathway. In the nematode Caenorhabditis elegans, these phenotypes are molecularly linked such that activation of the forkhead transcription factor DAF-16 both extends lifespan and simultaneously increases immunity and stress resistance. It is known that lifespan varies significantly among the Caenorhabditis species but, although DAF-16 signalling is highly conserved, it is unclear whether this phenotypic linkage occurs in other species. Here we investigate this phenotypic covariance by comparing longevity, stress resistance and immunity in four Caenorhabditis species. Methodology/Principal Findings: We show using phenotypic analysis of DAF-16 influenced phenotypes that among four closely related Caenorhabditis nematodes, the gonochoristic species (Caenorhabditis remanei and Caenorhabditis brenneri) have diverged significantly with a longer lifespan, improved stress resistance and higher immunity than the hermaphroditic species (C. elegans and Caenorhabditis briggsae). Interestingly, we also observe significant differences in expression levels between the daf-16 homologues in these species using Real-Time PCR, which positively correlate with the observed phenotypes. Finally, we provide additional evidence in support of a role for DAF-16 in regulating phenotypic coupling by using a combination of wildtype isolates, constitutively active daf-16 mutants and bioinformatic analysis. Conclusions: The gonochoristic species display a significantly longer lifespan (p<0.0001) and more robust immune and stress response (p<0.0001, thermal stress; p<0.01, heavy metal stress; p<0.0001, pathogenic stress) than the hermaphroditic species. Our data suggests that divergence in DAF-16 mediated phenotypes may underlie many of the differences observed between these four species of Caenorhabditis nematodes. These findings are further supported by the correlative higher daf-16 expression levels among the gonochoristic species and significantly higher lifespan, immunity and stress tolerance in the constitutively active daf-16 hermaphroditic mutants

Cerebrospinal fluid immunoglobulin light chain ratios predict disease progression in multiple sclerosis

Objective To determine whether the ratio of cerebrospinal fluid (CSF) immunoglobulin kappa to lambda light chains at time of multiple sclerosis (MS) diagnosis predicts disease progression and whether this was intrinsic to CSF plasmablasts. Methods CSF and peripheral blood were obtained from patients undergoing elective diagnostic lumbar puncture and included clinically isolated syndrome (CIS) (n=43), relapsing remitting MS (RRMS; n=50), primary progressive MS (PPMS; n=20) and other neurological disease controls, both inflammatory (ONID; n=23) and non-inflammatory (OND; n=114). CSF samples were assayed for free and immunoglobulin-associated light chains and on B cells and plasmablasts. Clinical follow-up data were collected during a 5-year follow-up period where available. Results There was an increased median CSF κ:λ free light chain (FLC) in all MS groups (CIS: 18.2, 95% CI 6.8 to 30.3; RRMS: 4.4, 95% CI 2.7 to 11.4; PPMS: 12.0, 95% CI 3.6 to 37.1) but not controls (OND: 1.61, 95% CI 1.4 to 1.9; ONID: 1.7, 95% CI 1.3 to 2.2; p10) CSF κ:λ FLC (0.0, 95% CI 0 to 2.5 vs 2.5, 95% CI 0 to 4, high vs low; p=0.049). CSF κ:λ FLC correlated with CSF IgG1 κ:λ (r=0.776; p<0.0001) and was intrinsic to CSF plasmablasts (r=0.65; p=0.026). Conclusions These data demonstrate that CSF immunoglobulin κ:λ ratios, determined at the time of diagnostic lumbar puncture, predict MS disease progression and may therefore be useful prognostic markers for early therapeutic stratification

Results from the project ‘Acceptance of CO2 capture and storage: economics, policy and technology (ACCSEPT)’

AbstractACCSEPT was a two-year research project (2005–2007) funded under the 6th research framework programme of the European Commission. The project leader was Det Norske Veritas (DNV), and the partners were Baker and McKenzie, the Energy Research Centre of the Netherlands (ECN), the Institute for European Environmental Policy (IEEP), Tyndall Centre for Climate Change Research, and Judge Business School of the University of Cambridge.There were three main focuses of the project: a Europe-wide survey of stakeholders and their opinions on CCS; stakeholder consultation through two workshops; and research into the economics, regulation, legal and social aspects of CCS. The project website is www.accsept.org, where all the outputs and related material can be found.This paper summarizes the conclusions of the work

Interaction between integrin α9β1 and vascular cell adhesion molecule-1 (VCAM-1) inhibits neutrophil apoptosis

According to the prevailing paradigm, neutrophils are short-lived cells that undergo spontaneous apoptosis within 24 hours of their release from the bone marrow. However, neutrophil survival can be significantly prolonged within inflamed tissue by cytokines, inflammatory mediators, and hypoxia. During screening experiments aimed at identifying the effect of the adhesive microenvironment on neutrophil survival, we found that VCAM-1 (CD106) was able to delay both spontaneous and Fas-induced apoptosis. VCAM-1-mediated survival was as efficient as that induced by the cytokine IFN-β and provided an additive, increased delay in apoptosis when given in combination with IFN-β. VCAM-1 delivered its antiapoptotic effect through binding the integrin α9β1. The α9β 1 signaling pathway shares significant features with the IFN-β survival signaling pathway, requiring PI3 kinase, NF-κB activation, as well as de novo protein synthesis, but the kinetics of NF-κB activation by VCAM-1 were slower and more sustained compared with IFN-β. This study demonstrates a novel functional role for α9β1 in neutrophil biology and suggests that adhesive signaling pathways provide an important extrinsic checkpoint for the resolution of inflammatory responses in tissues

Synovial fluid leukocyte apoptosis is inhibited in patients with very early rheumatoid arthritis

Synovial leukocyte apoptosis is inhibited in established rheumatoid arthritis (RA). In contrast, high levels of leukocyte apoptosis are seen in self-limiting crystal arthritis. The phase in the development of RA at which the inhibition of leukocyte apoptosis is first apparent, and the relationship between leukocyte apoptosis in early RA and other early arthritides, has not been defined. We measured synovial fluid leukocyte apoptosis in very early arthritis and related this to clinical outcome. Synovial fluid was obtained at presentation from 81 patients with synovitis of ≤ 3 months duration. The percentages of apoptotic neutrophils and lymphocytes were assessed on cytospin preparations. Patients were assigned to diagnostic groups after 18 months follow-up. The relationship between leukocyte apoptosis and patient outcome was assessed. Patients with early RA had significantly lower levels of neutrophil apoptosis than patients who developed non-RA persistent arthritis and those with a resolving disease course. Similarly, lymphocyte apoptosis was absent in patients with early RA whereas it was seen in patients with other early arthritides. The inhibition of synovial fluid leukocyte apoptosis in the earliest clinically apparent phase of RA distinguishes this from other early arthritides. The mechanisms for this inhibition may relate to the high levels of anti-apoptotic cytokines found in the early rheumatoid joint (e.g. IL-2, IL-4, IL-15 GMCSF, GCSF). It is likely that this process contributes to an accumulation of leukocytes in the early rheumatoid lesion and is involved in the development of the microenvironment required for persistent RA

Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin

Pathological processes involved in the initiation of rheumatoid synovitis remain unclear. We undertook the present study to identify immune and stromal processes that are present soon after the clinical onset of rheumatoid arthritis ( RA) by assessing a panel of T cell, macrophage, and stromal cell related cytokines and chemokines in the synovial fluid of patients with early synovitis. Synovial fluid was aspirated from inflamed joints of patients with inflammatory arthritis of duration 3 months or less, whose outcomes were subsequently determined by follow up. For comparison, synovial fluid was aspirated from patients with acute crystal arthritis, established RA and osteoarthritis. Rheumatoid factor activity was blocked in the synovial fluid samples, and a panel of 23 cytokines and chemokines measured using a multiplex based system. Patients with early inflammatory arthritis who subsequently developed RA had a distinct but transient synovial fluid cytokine profile. The levels of a range of T cell, macrophage and stromal cell related cytokines ( e. g. IL-2, IL-4, IL-13, IL-17, IL-15, basic fibroblast growth factor and epidermal growth factor) were significantly elevated in these patients within 3 months after symptom onset, as compared with early arthritis patients who did not develop RA. In addition, this profile was no longer present in established RA. In contrast, patients with non-rheumatoid persistent synovitis exhibited elevated levels of interferon-gamma at initiation. Early synovitis destined to develop into RA is thus characterized by a distinct and transient synovial fluid cytokine profile. The cytokines present in the early rheumatoid lesion suggest that this response is likely to influence the microenvironment required for persistent RA

Differential regulation of nuclear and mitochondrial Bcl-2 in T cell apoptosis

Activated T cells require anti-apoptotic cytokines for their survival. The anti-apoptotic effects of these factors are mediated by their influence on the balance of expression and localisation of pro- and anti-apoptotic members of the Bcl-2 family. Among the anti-apoptotic Bcl-2 family members, the expression level of Bcl-2 itself and its interaction with the pro-apoptotic protein Bim are now regarded as crucial for the regulation of survival in activated T cells. We studied the changes in Bcl-2 levels and its subcellular distribution in relation to mitochondrial depolarisation and caspase activation in survival factor deprived T cells. Intriguingly, the total Bcl-2 level appeared to remain stable, even after caspase 3 activation indicated entry into the execution phase of apoptosis. However, cell fractionation experiments showed that while the dominant nuclear pool of Bcl-2 remained stable during apoptosis, the level of the smaller mitochondrial pool was rapidly downregulated. Signals induced by anti-apoptotic cytokines continuously replenish the mitochondrial pool, but nuclear Bcl-2 is independent of such signals. Mitochondrial Bcl-2 is lost rapidly by a caspase independent mechanism in the absence of survival factors, in contrast only a small proportion of the nuclear pool of Bcl-2 is lost during the execution phase and this loss is a caspase dependent process. We conclude that these two intracellular pools of Bcl-2 are regulated through different mechanisms and only the cytokine-mediated regulation of the mitochondrial pool is relevant to the control of the initiation of apoptosis

SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population