Severe pulmonary arterial hypertension is characterized by increased neutrophil elastase and relative elafin deficiency

BACKGROUND: Preclinical evidence implicates neutrophil elastase (NE) in PAH pathogenesis, and the NE inhibitor elafin is under early therapeutic investigation. RESEARCH QUESTION: Are circulating NE and elafin levels abnormal in PAH and associated with clinical severity? STUDY DESIGN/METHODS: . In an observational Stanford University PAH cohort (N=249), plasma NE and elafin were measured in comparison to healthy controls (N=106) then related to clinical features and relevant ancillary biomarkers. Cox regression models were fitted with cubic spline functions to associate NE and elafin with survival. To validate prognostic relationships, we analyzed two United Kingdom cohorts (N=75, N=357). Mixed effects models evaluated NE and elafin changes during disease progression. Finally, we studied effects of NE/elafin balance on pulmonary artery endothelial cells (PAECs) from PAH patients. RESULTS: Relative to controls, patients had increased NE (205.1 [123.6-387.3] vs. 97.6 [74.4-126.6] ng/mL, P168.5 ng/mL portended increased mortality risk after adjustment for known clinical predictors (HR 2.52, CI 1.36-4.65, P=0.003) or prognostic cytokines (HR 2.63, CI 1.42-4.87, P=0.001), and NE added incremental value to established PAH risk scores. Similar prognostic thresholds were identified in validation cohorts. Longitudinal NE changes tracked with clinical trends and outcomes. PAH-PAECs exhibited increased apoptosis and attenuated angiogenesis when exposed to NE at the level observed in patients' blood. Elafin rescued PAEC homeostasis, yet the required dose exceeded levels found in patients. INTERPRETATION: NE is increased and elafin deficient across PAH subtypes. NE associates with disease severity and outcomes, and this target-specific biomarker could facilitate therapeutic development of elafin

Analytical Validation of Multiplex Biomarker Assay to Stratify Colorectal Cancer into Molecular Subtypes.

Previously, we classified colorectal cancers (CRCs) into five CRCAssigner (CRCA) subtypes with different prognoses and potential treatment responses, later consolidated into four consensus molecular subtypes (CMS). Here we demonstrate the analytical development and validation of a custom NanoString nCounter platform-based biomarker assay (NanoCRCA) to stratify CRCs into subtypes. To reduce costs, we switched from the standard nCounter protocol to a custom modified protocol. The assay included a reduced 38-gene panel that was selected using an in-house machine-learning pipeline. We applied NanoCRCA to 413 samples from 355 CRC patients. From the fresh frozen samples (n = 237), a subset had matched microarray/RNAseq profiles (n = 47) or formalin-fixed paraffin-embedded (FFPE) samples (n = 58). We also analyzed a further 118 FFPE samples. We compared the assay results with the CMS classifier, different platforms (microarrays/RNAseq) and gene-set classifiers (38 and the original 786 genes). The standard and modified protocols showed high correlation (> 0.88) for gene expression. Technical replicates were highly correlated (> 0.96). NanoCRCA classified fresh frozen and FFPE samples into all five CRCA subtypes with consistent classification of selected matched fresh frozen/FFPE samples. We demonstrate high and significant subtype concordance across protocols (100%), gene sets (95%), platforms (87%) and with CMS subtypes (75%) when evaluated across multiple datasets. Overall, our NanoCRCA assay with further validation may facilitate prospective validation of CRC subtypes in clinical trials and beyond

Options for early breast cancer follow-up in primary and secondary care : a systematic review

Background Both incidence of breast cancer and survival have increased in recent years and there is a need to review follow up strategies. This study aims to assess the evidence for benefits of follow-up in different settings for women who have had treatment for early breast cancer. Method A systematic review to identify key criteria for follow up and then address research questions. Key criteria were: 1) Risk of second breast cancer over time - incidence compared to general population. 2) Incidence and method of detection of local recurrence and second ipsi and contra-lateral breast cancer. 3) Level 1–4 evidence of the benefits of hospital or alternative setting follow-up for survival and well-being. Data sources to identify criteria were MEDLINE, EMBASE, AMED, CINAHL, PSYCHINFO, ZETOC, Health Management Information Consortium, Science Direct. For the systematic review to address research questions searches were performed using MEDLINE (2011). Studies included were population studies using cancer registry data for incidence of new cancers, cohort studies with long term follow up for recurrence and detection of new primaries and RCTs not restricted to special populations for trials of alternative follow up and lifestyle interventions. Results Women who have had breast cancer have an increased risk of a second primary breast cancer for at least 20 years compared to the general population. Mammographically detected local recurrences or those detected by women themselves gave better survival than those detected by clinical examination. Follow up in alternative settings to the specialist clinic is acceptable to women but trials are underpowered for survival. Conclusions Long term support, surveillance mammography and fast access to medical treatment at point of need may be better than hospital based surveillance limited to five years but further large, randomised controlled trials are needed

Microvascular function is preserved in newly diagnosed rheumatoid arthritis and low systemic inflammatory activity

Rheumatoid arthritis (RA) is associated with increased cardiovascular morbidity and mortality. Microvascular function has been linked to several risk factors for cardiovascular disease and may be affected in RA. It is, however, presently unknown at what point in the disease course the abnormalities in microvascular function occur. We determined whether microvascular function is already disturbed in early disease-modifying antirheumatic drugs (DMARD)-naive RA patients with low systemic inflammation. Fifteen consecutive RA patients with a median symptom duration of 5 months, a C-reactive protein level of ≤20 mg/l and without a history of cardiovascular disease, and age 15 and sex-matched healthy controls were recruited. Endothelium-dependent and endothelium-independent vasodilatation in skin was evaluated with laser Doppler fluxmetry after iontophoresis of acetylcholine and sodium nitroprusside, respectively. Videomicroscopy was used to measure recruitment of skin capillaries after arterial occlusion. CRP and ESR levels were mildly, but significantly elevated in patients compared to controls. No differences in both endothelium-dependent vasodilatation and capillary recruitment were observed between groups [709% (95% CI, 457–961%) vs 797% (95% CI, 556–1,037%), P = 0.59 and 37% (95% CI, 26–47%) vs 41% (95% CI, 31–50%), P = 0.59, respectively]. Skin microvascular function is preserved in early, DMARD-naive RA patients with moderately active RA but low systemic inflammatory activity. Both the extent of the systemic inflammation and disease duration, therefore, may be important determinants of microvascular dysfunction and subsequent increased risk for cardiovascular disease

Agronomic Management of Indigenous Mycorrhizas

Many of the advantages conferred to plants by arbuscular mycorrhiza (AM) are associated to the ability of AM plants to explore a greater volume of soil through the extraradical mycelium. Sieverding (1991) estimates that for each centimetre of colonized root there is an increase of 15 cm3 on the volume of soil explored, this value can increase to 200 cm3 depending on the circumstances. Due to the enhancement of the volume of soil explored and the ability of the extraradical mycelium to absorb and translocate nutrients to the plant, one of the most obvious and important advantages resulting from mycorrhization is the uptake of nutrients. Among of which the ones that have immobilized forms in soil, such as P, assume particular significance. Besides this, many other benefits are recognized for AM plants (Gupta et al, 2000): water stress alleviation (Augé, 2004; Cho et al, 2006), protection from root pathogens (Graham, 2001), tolerance to toxic heavy metals and phytoremediation (Audet and Charest, 2006; Göhre and Paszkowski, 2006), tolerance to adverse conditions such as very high or low temperature, high salinity (Sannazzaro et al, 2006), high or low pH (Yano and Takaki, 2005) or better performance during transplantation shock (Subhan et al, 1998). The extraradical hyphae also stabilize soil aggregates by both enmeshing soil particles (Miller e Jastrow, 1992) and producing a glycoprotein, golmalin, which may act as a glue-like substance to adhere soil particles together (Wright and Upadhyaya, 1998). Despite the ubiquous distribution of mycorrhizal fungi (Smith and Read, 2000) and only a relative specificity between host plants and fungal isolates (McGonigle and Fitter, 1990), the obligate nature of the symbiosis implies the establishment of a plant propagation system, either under greenhouse conditions or in vitro laboratory propagation. These techniques result in high inoculum production costs, which still remains a serious problem since they are not competitive with production costs of phosphorus fertilizer. Even if farmers understand the significance of sustainable agricultural systems, the reduction of phosphorus inputs by using AM fungal inocula alone cannot be justified except, perhaps, in the case of high value crops (Saioto and Marumoto, 2002). Nurseries, high income horticulture farmers and no-agricultural application such as rehabilitation of degraded or devegetated landscapes are examples of areas where the use of commercial inoculum is current. Another serious problem is quality of commercial available products concerning guarantee of phatogene free content, storage conditions, most effective application methods and what types to use. Besides the information provided by suppliers about its inoculum can be deceiving, as from the usually referred total counts, only a fraction may be effective for a particular plant or in specific soil conditions. Gianinazzi and Vosátka (2004) assume that progress should be made towards registration procedures that stimulate the development of the mycorrhizal industry. Some on-farm inoculum production and application methods have been studied, allowing farmers to produce locally adapted isolates and generate a taxonomically diverse inoculum (Mohandas et al, 2004; Douds et al, 2005). However the inocula produced this way are not readily processed for mechanical application to the fields, being an obstacle to the utilization in large scale agriculture, especially row crops, moreover it would represent an additional mechanical operation with the corresponding economic and soil compaction costs. It is well recognized that inoculation of AM fungi has a potential significance in not only sustainable crop production, but also environmental conservation. However, the status quo of inoculation is far from practical technology that can be widely used in the field. Together a further basic understanding of the biology and diversity of AM fungi is needed (Abbott at al, 1995; Saito and Marumoto, 2002). Advances in ecology during the past decade have led to a much more detailed understanding of the potential negative consequences of species introductions and the potential for negative ecological consequences of invasions by mycorrhizal fungi is poorly understood. Schwartz et al, (2006) recommend that a careful assessment documenting the need for inoculation, and the likelihood of success, should be conducted prior to inoculation because inoculations are not universally beneficial. Agricultural practices such as crop rotation, tillage, weed control and fertilizer apllication all produce changes in the chemical, physical and biological soil variables and affect the ecological niches available for occupancy by the soil biota, influencing in different ways the symbiosis performance and consequently the inoculum development, shaping changes and upset balance of native populations. The molecular biology tools developed in the latest years have been very important for our perception of these changes, ensuing awareness of management choice implications in AM development. In this context, for extensive farming systems and regarding environmental and economic costs, the identification of agronomic management practices that allow controlled manipulation of the fungal community and capitalization of AM mutualistic effect making use of local inoculum, seem to be a wise option for mycorrhiza promotion and development of sustainable crop production

Chemo-hormone therapy of non-well-differentiated endocrine tumours from different anatomic sites with cisplatinum, etoposide and slow release lanreotide formulation

We report the results of a phase II trial in patients with metastatic endocrine tumours from different sites, which aimed to evaluate the anti-tumour activity and toxicity of a cisplatinum and etoposide regimen administered in combination with the somatostatin agonist lanreotide given in slow release formulation. Between January 1999 and November 2003, 27 patients with histological diagnoses of endocrine tumours with different degrees of differentiation, excluding well differentiated carcinoid neoplasms, received intravenous (i.v.) administration of cisplatinum (30 mg m−2) and etoposide (100 mg m−2) on days 1–3 and intramuscular administration of 60 mg lanreotide on day 1, in a 21-day cycle. All of the patients were evaluable for toxicity and response. The treatment was very well tolerated as no grade 4 toxicity was observed. Four patients achieved a complete response, six a partial response, 12 experienced disease stabilisation and five disease progression. The average time to progression and to survival were 9 and 24 months respectively. These results suggest that this chemo-hormone therapy regimen is well tolerated and active in patients with non-well differentiated endocrine tumours

Women's attitudes towards mechanisms of action of family planning methods: survey in primary health centres in Pamplona, Spain

Irala J de, Lopez del Burgo C, Lopez de Fez CM, Arredondo J, Mikolajczyk RT, Stanford JB. Women's attitudes towards mechanisms of action of family planning methods: survey in primary health centres in Pamplona, Spain. BMC Women's Health. 2007;7(1): 10.Background: Informed consent in family planning includes knowledge of mechanism of action. Some methods of family planning occasionally work after fertilization. Knowing about postfertilization effects may be important to some women before choosing a certain family planning method. The objective of this survey is to explore women's attitudes towards postfertilization effects of family planning methods, and beliefs and characteristics possibly associated with those attitudes. Methods: Cross-sectional survey in a sample of 755 potentially fertile women, aged 18–49, from Primary Care Health Centres in Pamplona, Spain. Participants were given a 30-item, self-administered, anonymous questionnaire about family planning methods and medical and surgical abortion. Logistic regression was used to identify variables associated with women's attitudes towards postfertilization effects. Results: The response rate was 80%. The majority of women were married, held an academic degree and had no children. Forty percent of women would not consider using a method that may work after fertilization but before implantation and 57% would not consider using one that may work after implantation. While 35.3% of the sample would stop using a method if they learned that it sometimes works after fertilization, this percentage increased to 56.3% when referring to a method that sometimes works after implantation. Women who believe that human life begins at fertilization and those who consider it is important to distinguish between natural and induced embryo loss were less likely to consider the use of a method with postfertilization effects. Conclusion: Information about potential postfertilization effects of family planning methods may influence women's acceptance and choice of a particular family planning method. Additional studies in other populations are necessary to evaluate whether these beliefs are important to those populations

Embodying limb absence in the negotiation of sexual intimacy

Some people with physical disabilities experience difficulties in forming and maintaining intimate and sexual relationships (Taleporos and McCabe, 2001). Individuals with physical impairments may variously be seen as inferior, ‘not up to scratch’ and can be less valued than those thought to embody the cultural ideal of ‘normality’ (Edwards and Imrie, 2003). The loss of an anatomical part such as an arm or a leg can therefore set up a complex series of perceptual, emotional and psychological responses that can limit or prevent individuals from fully experiencing the kinds of intimate relationships that many of their ‘non-disabled’ counterparts can more freely enjoy (Oliver, 1990). Drawing on a series of semi-structured interviews and a group visual workshop with five men and two women with varying acquired and congenital limb absences, this study explores the variety of ways in which sexual relationships and intimacies are negotiated and managed. Certain participants reported a great deal of anxiety regarding their perceptions of sexual attractiveness, and experienced feelings of loss of desirability as a sexual partner. Others reported engaging in an ‘active management of visual information’ about the body, through methods of concealment and exposure. Compensatory strategies were also reported as a way to effectively maintain existing intimate relationships. Finally, hyper-masculine or hyper-feminine enactments of sexuality were described within a range of social and intimate contexts as a means to ‘normalise’ the disabled body. The findings of this study provide a richer and more contextualised understanding of the highly complex adjustment process faced by individuals with limb absence, who actively engage in sexual and intimate relationships

Combining a prioritization strategy and functional studies nominates 5’UTR variants underlying inherited retinal disease

BACKGROUND: 5’ untranslated regions (5’UTRs) are essential modulators of protein translation. Predicting the impact of 5’UTR variants is challenging and rarely performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and functional assays to evaluate 5’UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs). METHODS: We performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of their 5’UTRs by different whole exome sequencing (WES) kits. The selected 5’UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2397 WGS) and an in-house database (n = 1682 WES), respectively. Identified variants were annotated for 5’UTR-relevant features and classified into seven categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse approaches. RESULTS: Isoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5’UTR compared to that of their canonical isoform. Depending on the probe design, 3–20% of IRD genes have 5’UTRs fully captured by WES. After analyzing these regions in both cohorts, we prioritized 11 (likely) pathogenic variants in 10 genes (ARL3, MERTK, NDP, NMNAT1, NPHP4, PAX6, PRPF31, PRPF4, RDH12, RD3), of which 7 were novel. Functional analyses further supported the pathogenicity of three variants. Mis-splicing was demonstrated for the PRPF31:c.-9+1G>T variant. The MERTK:c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. The RDH12:c.-123C>T variant was found in cis with the hypomorphic RDH12:c.701G>A (p.Arg234His) variant in 11 patients. This 5’UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels. CONCLUSIONS: This study demonstrates the importance of 5’UTR variants implicated in IRDs and provides a systematic approach for 5’UTR annotation and validation that is applicable to other inherited diseases