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.

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.

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.

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.

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.

An alloresponse in humans is dominated by cytotoxic T lymphocytes (CTL) cross-reactive with a single Epstein-Barr virus CTL epitope: implications for graft-versus-host disease.

The phenomenon of T cell allorecognition is difficult to accommodate within the framework of a T cell repertoire positively selected in the thymus, unless allorecognition results from the cross-reactions of self-major histocompatibility complex restricted T cells. Herein, we demonstrate the dual specificity of cytotoxic T lymphocyte (CTL) clones for the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, presented on HLA-B8, and the alloantigen HLA-B*4402. CTL which recognized peptide FLRGRAYGL in association with HLA-B8 could be reactivated in vitro from healthy individuals who had been exposed previously to EBV, using stimulator cells expressing the cross-reacting alloantigen HLA-B*4402. Limiting dilution analysis of the alloresponse to HLA-B*4402 in eight healthy individuals revealed that HLA-B8+, EBV-sero+ donors had higher CTL precursor frequencies for alloantigen HLA-B*4402 than EBV-sero- control donors. It is surprising that the majority (65-100%) of anti-HLA-B*4402 CTL, generated in limiting dilution mixed lymphocyte reactions between responder cells from HLA-B8+, EBV-sero+ individuals and HLA-B*4402+ stimulators, also recognized the EBV CTL epitope FLRGRAYGL/HLA-B8. In contrast to previous studies showing extensive diversity in the T cell repertoire against individual alloantigens, these data demonstrate that the response to an alloantigen can be dominated by CTL cross-reactive with a single viral epitope, thus illustrating a possible mechanism for the frequent clinical association between herpesvirus exposure and graft-versus-host disease after bone marrow transplants.

Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis.

The importance of cytotoxic T lymphocytes (CTLs) in the immunosurveillance of Epstein-Barr virus (EBV)-infected B cells is firmly established, and the viral antigens of CTL recognition in latent infection are well defined. The epitopes targeted by CTLs during primary infection have not been identified, however, and there is only limited information about T cell receptor (TCR) selection. In the present report, we have monitored the development of memory TCR-beta clonotypes selected in response to natural EBV infection in a longitudinal study of an HLA-B8+ individual with acute infectious mononucleosis (IM). By stimulating peripheral blood lymphocytes with HLA-B8+ EBV-transformed B lymphoblastoid cells, the primary virus-specific CTL response was shown to include specificities for two HLA-B8-restricted antigenic determinants, FLRGRAYGL and QAKWRLQTL, which are encoded within the latent EBV nuclear antigen EBNA-3. TCR-beta sequence analysis of CTL clones specific for each epitope showed polyclonal TCR-beta repertoire selection, with structural restrictions on recognition that indicated antigen-driven selection. Furthermore, longitudinal repertoire analysis revealed long-term preservation of a multiclonal effector response throughout convalescence, with the reemergence of distinct memory T cell clonotypes sharing similar structural restrictions. Tracking the progression of specific TCR-beta clonotypes and antigen-specific TCR-V beta family gene expression in the peripheral repertoire ex vivo using semiquantitative PCR strongly suggested that selective TCR-beta expansions were present at the clonotype level, but not at the TCR-V beta family level. Overall, in this first analysis of antigen-specific TCR development in IM, a picture of polyclonal TCR stimulation is apparent. This diversity may be especially important in the establishment of an effective CTL control during acute EBV infection and in recovery from disease.

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.

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.

Energetic Charged-Particle Phenomena in the Jovian Magnetosphere: First Results from the Ulysses COSPIN Collaboration.

The Ulysses spacecraft made the first exploration of the region of Jupiter's magnetosphere at high Jovigraphic latitudes ( approximately 37 degrees south) on the dusk side and reached higher magnetic latitudes ( approximately 49 degrees north) on the day side than any previous mission to Jupiter. The cosmic and solar particle investigations (COSPIN) instrumentation achieved a remarkably well integrated set of observations of energetic charged particles in the energy ranges of approximately 1 to 170 megaelectron volts for electrons and 0.3 to 20 megaelectron volts for protons and heavier nuclei. The new findings include (i) an apparent polar cap region in the northern hemisphere in which energetic charged particles following Jovian magnetic field lines may have direct access to the interplanetary medium, (ii) high-energy electron bursts (rise times approximately 17 megaelectron volts) on the dusk side that are apparently associated with field-aligned currents and radio burst emissions, (iii) persistence of the global 10-hour relativistic electron "clock" phenomenon throughout Jupiter's magnetosphere, (iv) on the basis of charged-particle measurements, apparent dragging of magnetic field lines at large radii in the dusk sector toward the tail, and (v) consistent outflow of megaelectron volt electrons and large-scale departures from corotation for nucleons.

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.

Isolation and mapping of a human septin gene to a region on chromosome 17q, commonly deleted in sporadic epithelial ovarian tumors.

Allele losses from chromosome 17 are common in sporadic ovarian tumors. Previously, we reported high rates of LOH (up to 70%) from 17q25 at the marker THH59 in a bank of malignant ovarian tumors. We have extended this study to 70 tumors with 17 markers from the long arm of chromosome 17. In most cases, the data are consistent with whole chromosome loss, but we have identified a minimal region of deletion that is centered around 4 microsatellites with zero recombination at map position 106.9 cM. A P1/BAC contig across the region (approximately 200 kb) was constructed and used to determine the precise position and order of the microsatellites. The contig was shown to hybridize to 17q25 by fluorescence in situ hybridization analysis. The DNA sequence of the entire contig was determined and analyzed by BLAST searches. A 4-kb cDNA was subsequently identified with homology to the yeast, Drosophila and mammalian septin family of genes. We have designated this gene Ovarian/Breast (Ov/Br) septin. Two splice variants were demonstrated within the 200-kb contig, which differ only at exon 1. Within the contig, approximately 45% of the septin alpha transcript was identified and 38% of the septin beta transcript. The septins are a family of genes involved in cytokinesis and cell cycle control. Their known functions are consistent with the hypothesis that the human 17q25 septin gene is a candidate for the ovarian tumor suppressor gene.

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.

Genomic organization, complex splicing pattern and expression of a human septin gene on chromosome 17q25.3.

The Ov/Br septin gene, which is also a fusion partner of MLL in acute myeloid leukaemia, is a member of a family of novel GTP binding proteins that have been implicated in cytokinesis and exocytosis. In this study, we describe the genomic and transcriptional organization of this gene, detailing seventeen exons distributed over 240 kb of sequence. Extensive database analyses identified orthologous rodent cDNAs that corresponded to new, unidentified 5' splice variants of the Ov/Br septin gene, increasing the total number of such variants to six. We report that splicing events, occurring at non-canonical sites within the body of the 3' terminal exon, remove either 1801 bp or 1849 bp of non-coding sequence and facilitate access to a secondary open reading frame of 44 amino acids maintained near the end of the 3' UTR. These events constitute a novel coding arrangement and represent the first report of such a design being implemented by a eukaryotic gene. The various Ov/Br proteins either differ minimally at their amino and carboxy termini or are equivalent to truncated versions of larger isoforms. Northern analysis with an Ov/Br septin 3' UTR probe reveals three transcripts of 4.4, 4 and 3 kb, the latter being restricted to a sub-set of the tissues tested. Investigation of the identified Ov/Br septin isoforms by RT-PCR confirms a complex transcriptional pattern, with several isoforms showing tissue-specific distribution. To date, none of the other human septins have demonstrated such transcriptional complexity.

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.

Morphine tolerance offers protection from radiogenic performance deficits.

When rats are exposed to a sufficiently large dose of ionizing radiation they exhibit lethargy, hypokinesia, and deficits in performance. These and other behavioral changes parallel those often observed in this species after a large dose of morphine. Since the release of endogenous opiates has been implicated in some stress reactions, we sought to determine if they might play a part in radiogenic behavioral deficits. Rats were trained to criterion on a signaled avoidance task. Some subjects were then implanted with a pellet containing 75 mg of morphine. Other animals received placebo implants. Over a number of days, morphine tolerance was evaluated by measurement of body temperature changes. Prior to 2500 rad 60Co exposure or sham irradiation, morphine (or placebo) pellets were removed. Twenty-four hours later rats were retested to assess their performance on the avoidance task. Morphine-tolerant subjects performed significantly better than the irradiated placebo-implanted group and no differently than morphine-tolerant/sham-irradiated animals. Morphine tolerance seems to provide a degree of behavioral radiation resistance. These data are consistent with the hypothesis that endogenous opiate hyperexcretion may play some part in the behavioral deficits often observed after irradiation.

Evaluation of calibration routines in pharmaceutical and bioanalytical separations, with reference to Monte Carlo simulations.

A Monte Carlo method for the estimation of the bias and inter-assay component of precision attributable to given combinations of assay and calibration routine is described. Simulations are performed by a computer program which uses the actual concentration versus response and concentration versus intra-assay precision characteristics of the assay under investigation as its database. In operation, a calibration routine is selected, the calibrators defined and the analytical process of calibration followed by the analysis of unknowns is reproduced. In this way the mean bias and interassay component of precision at a number of pre-defined concentrations covering the analytical range of the assay is estimated. The program is intended for use by analysts as a practical aid in the selection and optimization of appropriate calibration routines in an experimental environment.

Development and application of a calibration regression routine in conjunction with linear and non-linear chromatographic detector responses.

Linear regression is widely used in the calibration of chromatographic assays even in conjunction with some chromatographic detectors which show significant non-linearity in their response characteristics. A calibration routine, based upon the curve y = axlnx+bx+c is presented which describes the non-linear behaviour of some chromatographic systems, including electron capture, nitrogen-phosphorus and UV photometric detectors, and gives comparable results to weighted linear regression with assays showing linear concentration versus response relationships. The ratio of the coefficients a and b in the equation allows quantification of the deviation from linearity and provides a more sensitive indicator of linearity than the correlation coefficient often quoted with linear regression.

A pre-embedding reaction method for localizing NADH reductase and succinic dehydrogenase in skeletal muscle.

Paraffin wax embedding methods suitable for demonstrating the distribution of enzyme activity in tissues sections are uncommon; most procedures rely on the use of frozen section techniques. This paper describes a system for demonstrating certain enzymes which involves incubation of the tissue with appropriate substrates before a Paramat wax embedding procedure. While it has distinct merits of its own, the procedure is eminently suitable for use where a cryostat is not available; it can also be readily applied to other enzymes and tissues.