Evaluation of variable new antigen receptors (vNARs) as a novel cathepsin S (CTSS) targeting strategy.

Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously "undruggable" targets for biologic-based therapy.

Quantitative proteomic analysis of HER2 expression in the selection of gastric cancer patients for trastuzumab treatment.

Background: A wide range of response rates have been reported in HER2-positive gastric cancer (GC) patients treated with trastuzumab. Other HER2-targeted therapies for GC have yet to show efficacy in clinical trials. These findings raise question about the ability of standard HER2 diagnostics to accurately distinguish between GC patients who would and would not benefit from anti-HER2 therapies. Patients and methods: GC patients (n = 237), including a subset from the Trastuzumab in GC (ToGA) trial were divided into three groups based on HER2 status and history of treatment with standard chemotherapy or chemotherapy plus trastuzumab. We applied mass spectrometry-based proteomic analysis to quantify HER2 protein expression in formalin-fixed tumor samples. Using HER2 expression as a continuous variable, we defined a predictive protein level cutoff to identify which patients would benefit from trastuzumab. We compared quantitated protein level with clinical outcome and HER2 status as determined by conventional HER2 diagnostics. Results: Quantitative proteomics detected a 115-fold range of HER2 protein expression among patients diagnosed as HER2 positive by standard methods. A protein level of 1825 amol/µg was predicted to determine benefit from the addition of trastuzumab to chemotherapy. Trastuzumab treated patients with HER2 protein levels above this cutoff had twice the median overall survival (OS) of their counterparts below the cutoff (35.0 versus 17.5 months, P = 0.011). Conversely, trastuzumab-treated patients with HER2 levels below the cutoff had outcomes similar to HER2-positive patients treated with chemotherapy. (Progression-free survival = 7.0 versus 6.5 months: P = 0.504; OS = 17.5 versus 12.6 months: P = 0.520). HER2 levels were not prognostic for response to chemotherapy. Conclusions: Proteomic analysis of HER2 expression demonstrated a quantitative cutoff that improves selection of GC patients for trastuzumab as compared with current diagnostic methods.

UTLS water vapour from SCIAMACHY limb measurementsV3.01 (2002-2012).

The SCanning Imaging Absorption spectroMeter for Atmospheric CHartographY (SCIAMACHY) aboard the Envisat satellite provided measurements from August 2002 until April 2012. SCIAMACHY measured the scattered or direct sunlight using different observation geometries. The limb viewing geometry allows the retrieval of water vapour at about 10-25 km height from the near-infrared spectral range (1353-1410 nm). These data cover the upper troposphere and lower stratosphere (UTLS), a region in the atmosphere which is of special interest for a variety of dynamical and chemical processes as well as for the radiative forcing. Here, the latest data version of water vapour (V3.01) from SCIAMACHY limb measurements is presented and validated by comparisons with data sets from other satellite and in situ measurements. Considering retrieval tests and the results of these comparisons, the V3.01 data are reliable from about 11 to 23 km and the best results are found in the middle of the profiles between about 14 and 20 km. Above 20 km in the extra tropics V3.01 is drier than all other data sets. Additionally, for altitudes above about 19 km, the vertical resolution of the retrieved profile is not sufficient to resolve signals with a short vertical structure like the tape recorder. Below 14 km, SCIAMACHY water vapour V3.01 is wetter than most collocated data sets, but the high variability of water vapour in the troposphere complicates the comparison. For 14-20 km height, the expected errors from the retrieval and simulations and the mean differences to collocated data sets are usually smaller than 10 % when the resolution of the SCIAMACHY data is taken into account. In general, the temporal changes agree well with collocated data sets except for the Northern Hemisphere extratropical stratosphere, where larger differences are observed. This indicates a possible drift in V3.01 most probably caused by the incomplete treatment of volcanic aerosols in the retrieval. In all other regions a good temporal stability is shown. In the tropical stratosphere an increase in water vapour is found between 2002 and 2012, which is in agreement with other satellite data sets for overlapping time periods.

EBV & HHV6 reactivation is infrequent and not associated with MS clinical course.

BACKGROUND: Among the environmental factors associated with multiple sclerosis (MS) causation, some of the strongest associations are with Epstein-Barr virus (EBV), and to a lesser extent human herpesvirus 6 (HHV6). Associations with clinical course are less conclusive, however. METHODS: We evaluated serum anti-EBV-EA-R IgG and anti-HHV6 IgM, and EBV and HHV6 viral load (VL) for their associations with relapse, disability, and progression in disability in a prospective cohort of 198 participants with clinically definite MS. RESULTS: Anti-EBV-EA-R IgG was detected in 81.8% of cases at study entry, and titers remained essentially unchanged during the study. Anti-HHV6 IgM was detected in only one participant, and EBV-VL (29%) and HHV6-VL (1.8%) were detected in a minority of samples, and where detected levels were low. Our previously demonstrated association between anti-HHV6 IgG and relapse hazard was not affected by adjustment for parameters of reactivation. We found no evidence that any of the viral markers were associated with disability or progression in disability. In relation to relapse, only EBV-VL was positively associated, although this was strongly influenced by a single individual. CONCLUSION: Using a prospective cohort design, we found no convincing evidence that reactivation parameters of EBV or HHV6 were associated with subsequent MS relapse hazard or progression in disability, confirming previous findings, and indicating that herpesvirus reactivation is not an important driver of relapse or disability in this established MS population.

Identification of RBCK1 as a novel regulator of FKBPL: implications for tumor growth and response to tamoxifen.

FKBPL has been implicated in processes associated with cancer, including regulation of tumor growth and angiogenesis with high levels of FKBPL prognosticating for improved patient survival. Understanding how FKBPL levels are controlled within the cell is therefore critical. We have identified a novel role for RBCK1 as an FKBPL-interacting protein, which regulates FKBPL stability at the post-translational level via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-ß-estradiol and interact within heat shock protein 90 chaperone complexes, together with estrogen receptor-α (ERα). Furthermore, FKBPL and RBCK1 associate with ERα at the promoter of the estrogen responsive gene, pS2, and regulate pS2 levels. MCF-7 clones stably overexpressing RBCK1 were shown to have reduced proliferation and increased levels of FKBPL and p21. Furthermore, these clones were resistant to tamoxifen therapy, suggesting that RBCK1 could be a predictive marker of response to endocrine therapy. RBCK1 knockdown using targeted small interfering RNA resulted in increased proliferation and increased sensitivity to tamoxifen treatment. Moreover, in support of our in vitro data, analysis of mRNA microarray data sets demonstrated that high levels of FKBPL and RBCK1 correlated with increased patient survival, whereas high RBCK1 predicted for a poor response to tamoxifen. Our findings support a role for RBCK1 in the regulation of FKBPL with important implications for estrogen receptor signaling, cell proliferation and response to endocrine therapy.

Simulated air quality and pollutant budgets over Europe in 2008.

Major gaseous and particulate pollutant levels over Europe in 2008 have been simulated using the offline-coupled WRFCMAQ chemistry and transport modeling system. The simulations are compared with surface observations from the EMEP stations, ozone (O3) soundings, ship-borne O3 and nitrogen dioxide (NO2) observations in the western Mediterranean, tropospheric NO2 vertical column densities from the SCIAMACHY instrument, and aerosol optical depths (AOD) from the AERONET. The results show that on average, surface O3 levels are underestimated by 4 to 7% over the northern European EMEP stations while they are overestimated by 7-10% over the southern European EMEP stations and underestimated in the tropospheric column (by 10-20%). Particulate matter (PM) mass concentrations are underestimated by up to 60%, particularly in southern and eastern Europe, suggesting underestimated PM sources. Larger differences are calculated for individual aerosol components, particularly for organic and elemental carbon than for the total PM mass, indicating uncertainty in the combustion sources. Better agreement has been obtained for aerosol species over urban areas of the eastern Mediterranean, particularly for nss-SO4(2), attributed to the implementation of higher quality emission inventories for that area. Simulated AOD levels are lower than the AERONET observations by 10% on average, with average underestimations of 3% north of 40°N, attributed to the low anthropogenic emissions in the model and 22% south of 40°N, suggesting underestimated natural and resuspended dust emissions. Overall, the results reveal differences in the model performance between northern and southern Europe, suggesting significant differences in the representation of both anthropogenic and natural emissions in these regions. Budget analyses indicate that O3 and peroxyacetyl nitrate (PAN) are transported from the free troposphere (FT) to the planetary boundary layer over Europe, while other species follow the reverse path and are then advected away from the source region.

High frequency of herpesvirus-specific clonotypes in the human T cell repertoire can remain stable over decades with minimal turnover.

High-throughput T cell receptor sequencing on sequentially banked blood samples from healthy individuals has shown that high-frequency clonotypes can remain relatively stable for up to 18 years, with minimal inflation, deflation, or turnover. These populations included T cell expansions specific for Epstein-Barr virus. Thus, in spite of exposure to a barrage of microorganisms over the course of life, the dominant clonotypes in the mature peripheral T cell repertoire can alter surprisingly little.

Citizens advice in primary care: a qualitative study of the views and experiences of service users and staff.

OBJECTIVES: To examine the views and experiences of staff and users of Citizens Advice Bureau (CAB) services located in general practice, and to identify key factors perceived as contributing to the intervention's effectiveness. STUDY DESIGN: A qualitative study in an urban and rural primary care setting in the UK. METHODS: Semi-structured, face-to-face interviews (n = 22) with primary care and practice staff, CAB advisors and 12 service users. RESULTS: Key positive service features reported by all groups were: the confidential, non-stigmatizing and familiar environment of a general practitioner's (GP) surgery; the ability to make appointments and experienced advisor availability and continuity. Outcomes for service users were described as financial gain, managed debt, and beneficial social and mental health impacts. Perceived staff benefits were appropriate referral and better use of GP consultation time. CONCLUSION: Welfare advice in primary care has financial benefits and was perceived by participants to offer health and other benefits to patients and staff. However, while perceptions of gain from the intervention were evident, demonstration of measurable health improvement and well-being presents challenges. Further empirical work is needed in order to explore these complex cause-effect links and the cost-effectiveness of the intervention.

Current and past Epstein-Barr virus infection in risk of initial CNS demyelination.

OBJECTIVES: To assess risk of a first clinical diagnosis of CNS demyelination (FCD) in relation to measures of Epstein-Barr virus (EBV) infection within the context of other known risk factors. METHODS: This was a multicenter incident case-control study. FCD cases (n = 282) aged 18-59 years and controls (n = 558, matched on age, sex, and region) were recruited from 4 Australian centers between November 1, 2003, and December 31, 2006. A nested study (n = 215 cases, n = 216 controls) included measurement of whole blood quantitative EBV DNA load and serum EBV-specific antibodies. Conditional logistic regression was used to analyze case-control differences. RESULTS: There were no significant case-control differences in the proportion with detectable EBV DNA (55.8% vs 50.5%, respectively, p = 0.28), or in quantitative EBV DNA load (p = 0.33). Consistent with previous work, higher anti-EBV-specific immunoglobulin G (IgG) titers and a history of infectious mononucleosis were associated with increased FCD risk and there was an additive interaction with HLA-DRB1*1501 status. We found additional interactions between high anti-EBNA IgG titer and SNPs in HLA-A (adjusted odds ratios [AOR] = 19.84 [95% confidence interval (CI) 5.95 to 66.21] for both factors compared to neither) and CTLA-4 genes (AOR = 0.31 [95% CI 0.13 to 0.76] for neither factor compared to both). EBV DNA load was lower at higher serum 25-hydroxyvitamin D concentrations in controls (r = -0.17, p = 0.01). An adverse effect of higher EBV DNA load on FCD risk was increased with higher 25-hydroxyvitamin D concentration (p[interaction] = 0.02). CONCLUSION: Past infection with EBV, but not current EBV DNA load in whole blood, is significantly associated with increased FCD risk. These associations appear to be modified by immune-related gene variants.

Adverse drug effects following oseltamivir mass treatment and prophylaxis in a school outbreak of 2009 pandemic influenza A(H1N1) in June 2009, Sheffield, United Kingdom.

During the containment phase of the 2009 influenza A(H1N1) pandemic, mass treatment and prophylaxis with oseltamivir was used to control an outbreak of pandemic influenza in a primary school in Sheffield, United Kingdom, where ten cases of pandemic influenza had been laboratory confirmed over a three day period in June 2009. A subsequent cross-sectional survey showed that 51 of 297 (17%) pupils and 10 of 58 (17%) reported an influenza-like illness. The most common symptoms were headache, cough, fever, tiredness, sore throat and nausea. Fifty-three staff and 273 pupils took oseltamivir for treatment or prophylaxis. Of this group, 41% (113/273) of pupils and 47% (25/53) of staff reported adverse effects. Overall, 14% (37/273) of pupils and 20% (11/53) of staff did not complete the course of oseltamivir, primarily due to adverse effects. Nausea, vomiting and rash were statistically significantly associated with failing to complete the course of oseltamivir. Given the potential for side effects from oseltamivir, particularly among those without influenza who receive the drug for prophylaxis, our findings have two important implications. Firstly, the benefits of mass treatment in an outbreak setting must clearly be greater than the benefits of targeted treatment. Secondly, any large scale regional or state level system for distribution of antiviral drugs for treatment should ideally include a robust quantification of an individual s probability of infection with influenza virus in order to avoid unnecessary treatment.

Strains of Epstein-Barr virus infecting multiple sclerosis patients.

Both epidemiological and experimental studies have indicated that the ubiquitous herpesvirus Epstein-Barr virus (EBV) plays a role in the pathogenesis of multiple sclerosis (MS). Some features of MS epidemiology, such as the decline in risk among migrants from high to low MS prevalence areas, suggest the presence of variant EBV strains that increase MS risk. The objective of this study was to investigate whether genetic variability in EBV is associated with MS. Genes encoding for two EBV antigens (EBNA1 and BRRF2) were sequenced in EBV isolates from 40 MS patients and a similar number of control subjects. These viral antigens were chosen for analysis because they are known to stimulate atypical immune responses in MS. Extensive sequence polymorphism was observed within the EBNA1 and BRRF2 genes in isolates from both MS patients and controls. Interestingly, several single nucleotide polymorphisms within the EBNA1 gene, and one within the BRRF2 gene, were found to occur at marginally different frequencies in EBV strains infecting MS patients versus controls. Although this study does not find a simple causal relationship between EBV strains and the occurrence of MS, the existence of haplotypes that occur at different frequencies in MS patients versus controls may provide an area for future study of the role of EBV strain variation in multiple sclerosis.

Inequalities and healthcare reform in Chile: equity of what?

Chile has achieved great success in terms of growth and development. However, growing inequalities exist in relation to income and health status. The previous Chilean government began to reform the healthcare system with the aim of reducing health inequities. What is meant by "equity" in this context? What is the extent of the equity aimed for? A normative framework is required for public policy-makers to consider ideas about fairness in their decisions about healthcare reform. This paper aims to discuss the main features of the Chilean healthcare reform and their implications for such a normative framework.

De-ubiquitinating enzymes: intracellular signalling and disease.

Ubiquitination is now accepted as an important process for regulating intracellular signalling and the realization that many known signalling molecules exhibit E3 ligase activity has led to great strides in our understanding of how these pathways are regulated. However, as most of the de-ubiquitinating enzymes have as yet no identified substrate, little is known about their potential role in the regulation of intracellular signalling. Here, we examine what is known about de-ubiquitinating enzymes and signalling, with particular emphasis on their role in the regulation of immune signalling and the initiation of DNA repair. In addition, we look at the evidence implicating these enzymes in the pathogenesis of diseases such as cancer and neurodegenerative diseases.

Retrieval and monitoring of atmospheric trace gas concentrations in nadir and limb geometry using the space-borne SCIAMACHY instrument.

The Scanning Imaging Absorption Spectrometer for Atmospheric Chartography (SCIAMACHY) onboard the European Envisat spacecraft performs continuous spectral observations of reflected, scattered and transmitted sunlight in various observation geometries. A unique feature of SCIAMACHY is the capability of probing the atmosphere in three different observation geometries:The nadir, limb, and occultation measurement modes. In nadir mode, column densities of trace gases are retrieved with a spatial resolution of typically 30 x 60 km using the Differential Optical Absorption Spectroscopy (DOAS) technique (Platt and Perner, 1983). Alternating with the nadir measurement, vertical profiles of absorber concentration in the stratosphere are derived in limb and occultation. In this paper we present an overview over some applications of SCIAMACHY data in space-based monitoring of atmospheric pollution. The DOAS algorithms for the retrieval of total column amounts from nadir spectra are briefly described and case studies of pollution events are presented. We also illustrate the technique used to derive stratospheric concentration profiles from limb observations and show comparisons with other remote sensing systems. Special emphasis will be given to techniques, which take advantage of SCIAMACHY's different viewing geometries. In particular, we will discuss the potential and limits of strategies to infer tropospheric abundances of O3 and NO2.

Prognostic significance of Subjective Global Assessment (SGA) in advanced colorectal cancer.

OBJECTIVE: To evaluate the prognostic significance of Subjective Global Assessment (SGA) in advanced colorectal cancer and create statistically distinct prognostic groups of colorectal cancer patients based on clinical and nutritional variables. DESIGN: A retrospective clinical epidemiologic study. SETTING: A private tertiary care American Cancer Center. SUBJECTS: In total, 234 colorectal cancer patients aged 29-82 y treated at Cancer Treatment Centers of America at Midwestern Regional Medical Center between January 1995 and March 2001. INTERVENTION: SGA Questionnaire. SGA A-well nourished; SGA B-moderately malnourished; and SGA C-severely malnourished. Malnutrition was defined as either SGA B or SGA C. RESULTS: The prevalence of malnutrition in this patient population, as determined by SGA, was 52% (113/217). The median survival of patients with SGA A was 12.8 months (95% CI; 9.1-16.5), those with SGA B was 8.8 months (95% CI; 6.7-10.9) and those with SGA C was 6 months (95% CI; 3.9-8.1); the difference being statistically significant at P=0.0013. Regression tree analysis identified prior treatment history, lactate dehydrogenase (LDH) and SGA to be important predictors of survival for our patient cohort. Patients with no prior treatment history (newly diagnosed disease), low LDH scores, and SGA A had the best overall survival of 40.4 months (95% CI; 30.45-50.4), whereas patients with prior treatment history (progressive disease), high LDH scores, and SGA B/C had the worst overall survival of 4.5 months (95% CI; 2.22-6.76). CONCLUSION: The SGA provides useful prognostic information in patients with advanced colorectal cancer.

Measuring the impact of forensic science in detecting burglary and autocrime offences.

As UK investment in forensic science has increased, the government has taken a fresh interest in how far this has led to dividends in terms of the detection of crime and its reduction. The Home Office funded 'Pathfinder Project' sought to monitor and document the complex relationships between the collection and use of forensic material (looking at a range of forensic science techniques) and its impact on crime detection. The project specifically targeted the 'volume' crimes of burglary and vehicle crime. Detailed data was gathered on all stages of the process between the collection and use of forensic material and crime detection. The model falls into two conceptual phases--scene attendance to suspect identification and identification to detection. From the analysis it was found that approximately one third of burglary and autocrime scenes are visited by SOCOs. While scientific identifications are only made in a minority of burglary and autocrime offences overall, it belies their importance. About one in ten of burglary and autocrime cases are cleared up by the police and it is estimated that fingerprints and SGMPlus were a contributory factor in achieving one third of these clear ups.

Mitochondrial cytochrome c release precedes transmembrane depolarisation and caspase-3 activation during ceramide-induced apoptosis of Jurkat T cells.

Whilst the role of ceramide, a second messenger of the sphingolipid family, in the initiation of receptor-mediated apoptosis is controversial, a growing body of evidence is emerging for a role of ceramide in the amplification of apoptosis via mitochondrial perturbations that culminate in the activation of execution caspases. Treatment of Jurkat T cells with the cell-permeable analog, C(2)-ceramide, resulted in the rapid onset of apoptosis as evidenced by Annexin V-FITC staining of externalised phosphatidylserine residues. Cells bearing this early apoptotic marker had a reduced mitochondrial transmembrane potential (Delta(Psi)m) that was preceded by the release of cytochrome c from mitochondria. Subsequent activation of caspase-3 provides the link between these ceramide-induced mitochondrial changes and execution caspases that ultimately result in the physical destruction of the cell. Collectively these results demonstrate that ceramide signalling results in caspase-mediated apoptosis via mitochondrial cytochrome c release and are further supportive of the role of ceramide in the amplification of apoptosis.