Peroxisome Proliferator-Activated Receptor alpha (PPAR alpha) down-regulation in cystic fibrosis lymphocytes

Background: PPARs exhibit anti-inflammatory capacities and are potential modulators of the inflammatory response. We hypothesized that their expression and/or function may be altered in cystic fibrosis (CF), a disorder characterized by an excessive host inflammatory response. Methods: PPARα, β and γ mRNA levels were measured in peripheral blood cells of CF patients and healthy subjects via RT-PCR. PPARα protein expression and subcellular localization was determined via western blot and immunofluorescence, respectively. The activity of PPARα was analyzed by gel shift assay. Results: In lymphocytes, the expression of PPARα mRNA, but not of PPARβ, was reduced (-37%; p < 0.002) in CF patients compared with healthy persons and was therefore further analyzed. A similar reduction of PPARα was observed at protein level (-26%; p < 0.05). The transcription factor was mainly expressed in the cytosol of lymphocytes, with low expression in the nucleus. Moreover, DNA binding activity of the transcription factor was 36% less in lymphocytes of patients (p < 0.01). For PPARα and PPARβ mRNA expression in monocytes and neutrophils, no significant differences were observed between CF patients and healthy persons. In all cells, PPARγ mRNA levels were below the detection limit. Conclusion: Lymphocytes are important regulators of the inflammatory response by releasing cytokines and antibodies. The diminished lymphocytic expression and activity of PPARα may therefore contribute to the inflammatory processes that are observed in CF

γδ T lymphocytes from cystic fibrosis patients and healthy donors are high TNF-α and IFN-γ-producers in response to Pseudomonas aeruginosa

BACKGROUND: γδ T cells have an important immunoregulatory and effector function through cytokine release. They are involved in the responses to Gram-negative bacterium and in protection of lung epithelium integrity. On the other hand, they have been implicated in airway inflammation. METHODS: The aim of the present work was to study intracytoplasmic IL-2, IL-4, IFN-γ and TNF-α production by γδ and αβ T lymphocytes from cystic fibrosis patients and healthy donors in response to Pseudomonas aeruginosa (PA). Flow cytometric detection was performed after peripheral blood mononuclear cells (PBMC) culture with a cytosolic extract from PA and restimulation with phorbol ester plus ionomycine. Proliferative responses, activation markers and receptor usage of γδ T cells were also evaluated. RESULTS: The highest production of cytokine was of TNF-α and IFN-γ, γδ being better producers than αβ. No differences were found between patients and controls. The Vγ9δ2 subset of γδ T cells was preferentially expanded. CD25 and CD45RO expression by the αβ T subset and PBMC proliferative response to PA were defective in cystic fibrosis lymphocytes. CONCLUSION: Our results support the hypothesis that γδ T lymphocytes play an important role in the immune response to PA and in the chronic inflammatory lung reaction in cystic fibrosis patients. They do not confirm the involvement of a supressed Th1 cytokine response in the pathogenesis of this disease

A gene signature for post-infectious chronic fatigue syndrome

Background: At present, there are no clinically reliable disease markers for chronic fatigue syndrome. DNA chip microarray technology provides a method for examining the differential expression of mRNA from a large number of genes. Our hypothesis was that a gene expression signature, generated by microarray assays, could help identify genes which are dysregulated in patients with post-infectious CFS and so help identify biomarkers for the condition. Methods: Human genome-wide Affymetrix GeneChip arrays (39,000 transcripts derived from 33,000 gene sequences) were used to compare the levels of gene expression in the peripheral blood mononuclear cells of male patients with post-infectious chronic fatigue (n = 8) and male healthy control subjects (n = 7). Results: Patients and healthy subjects differed significantly in the level of expression of 366 genes. Analysis of the differentially expressed genes indicated functional implications in immune modulation, oxidative stress and apoptosis. Prototype biomarkers were identified on the basis of differential levels of gene expression and possible biological significance Conclusion: Differential expression of key genes identified in this study offer an insight into the possible mechanism of chronic fatigue following infection. The representative biomarkers identified in this research appear promising as potential biomarkers for diagnosis and treatment

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer

INTRODUCTION Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years

Effective nebulization of interferon-γ using a novel vibrating mesh.

BACKGROUND: Interferon gamma (IFN-γ) is a clinically relevant immunomodulatory cytokine that has demonstrated significant potential in the treatment and management of respiratory diseases such as tuberculosis and pulmonary fibrosis. As with all large biomolecules, clinical translation is dependent on effective delivery to the disease site and delivery of IFN-γ as an aerosol offers a logical means of drug targeting. Effective localization is often hampered by instability and a lack of safe and efficient delivery systems. The present study sought to determine how effectively IFN-γ can be nebulized using two types of vibrating mesh nebulizer, each with differing mesh architectures, and to investigate the comparative efficiency of delivery of therapeutically active IFN-γ to the lungs. METHODS: Nebulization of IFN-γ was carried out using two different Aerogen vibrating mesh technologies with differing mesh architectures. These technologies represent both a standard commercially available mesh type (Aerogen Solo®) and a new iteration mesh (Photo-defined aperture plate (PDAP®). Extensive aerosol studies (aerosol output and droplet analysis, non-invasive and invasive aerosol therapy) were conducted in line with regulatory requirements and characterization of the stability and bioactivity of the IFN-γ post-nebulization was confirmed using SDS-PAGE and stimulation of Human C-X-C motif chemokine 10 (CXCL 10) also known as IFN-γ-induced protein 10KDa (IP 10) expression from THP-1 derived macrophages (THP-1 cells). RESULTS: Aerosol characterization studies indicated that a significant and reproducible dose of aerosolized IFN-γ can be delivered using both vibrating mesh technologies. Nebulization using both devices resulted in an emitted dose of at least 93% (100% dose minus residual volume) for IFN-γ. Characterization of aerosolized IFN-γ indicated that the PDAP was capable of generating droplets with a significantly lower mass median aerodynamic diameter (MMAD) with values of 2.79 ± 0.29 μm and 4.39 ± 0.25 μm for the PDAP and Solo respectively. The volume median diameters (VMD) of aerosolized IFN-γ corroborated this with VMDs of 2.33 ± 0.02 μm for the PDAP and 4.30 ± 0.02 μm for the Solo. SDS-PAGE gels indicated that IFN-γ remains stable after nebulization by both devices and this was confirmed by bioactivity studies using a THP-1 cell model in which an alveolar macrophage response to IFN-γ was determined. IFN-γ nebulized by the PDAP and Solo devices had no significant effect on the key inflammatory biomarker cytokine IP-10 release from this model in comparison to non-nebulized controls. Here we demonstrate that it is possible to combine IFN-γ with vibrating mesh nebulizer devices and facilitate effective aerosolisation with minimal impact on IFN-γ structure or bioactivity. CONCLUSIONS: It is possible to nebulize IFN-γ effectively with vibrating mesh nebulizer devices without compromising its stability. The PDAP allows for generation of IFN-γ aerosols with improved aerodynamic properties thereby increasing its potential efficiency for lower respiratory tract deposition over current technology, whilst maintaining the integrity and bioactivity of IFN-γ. This delivery modality therefore offers a rational means of facilitating the clinical translation of inhaled IFN-γ

Quantitative and Qualitative Responses to Topical Cold in Healthy Caucasians Show Variance between Individuals but High Test-Retest Reliability.

Increased sensitivity to cold may be a predictor of persistent pain, but cold pain threshold is often viewed as unreliable. This study aimed to determine the within-subject reliability and between-subject variance of cold response, measured comprehensively as cold pain threshold plus pain intensity and sensation quality at threshold. A test-retest design was used over three sessions, one day apart. Response to cold was assessed at four sites (thenar eminence, volar forearm, tibialis anterior, plantar foot). Cold pain threshold was measured using a Medoc thermode and standard method of limits. Intensity of pain at threshold was rated using a 10cm visual analogue scale. Quality of sensation at threshold was quantified with indices calculated from subjects' selection of descriptors from a standard McGill Pain Questionnaire. Within-subject reliability for each measure was calculated with intra-class correlation coefficients and between-subject variance was evaluated as group coefficient of variation percentage (CV%). Gender and site comparisons were also made. Forty-five healthy adults participated: 20 male, 25 female; mean age 29 (range 18-56) years. All measures at all four test sites showed high within-subject reliability: cold pain thresholds r = 0.92-0.95; pain rating r = 0.93-0.97; McGill pain quality indices r = 0.87-0.85. In contrast, all measures showed wide between-subject variance (CV% between 51.4% and 92.5%). Upper limb sites were consistently more sensitive than lower limb sites, but equally reliable. Females showed elevated cold pain thresholds, although similar pain intensity and quality to males. Females were also more reliable and showed lower variance for all measures. Thus, although there was clear population variation, response to cold for healthy individuals was found to be highly reliable, whether measured as pain threshold, pain intensity or sensation quality. A comprehensive approach to cold response testing therefore may add validity and improve acceptance of this potentially important pain measure.Thus, although there was clear population variation, response to cold for healthy individuals was found to be highly reliable, whether measured as pain threshold, pain intensity or sensation quality. A comprehensive approach to cold response testing therefore may add validity and improve acceptance of this potentially important pain measure

A Mathematical Framework for Estimating Pathogen Transmission Fitness and Inoculum Size Using Data from a Competitive Mixtures Animal Model

We present a method to measure the relative transmissibility (“transmission fitness”) of one strain of a pathogen compared to another. The model is applied to data from “competitive mixtures” experiments in which animals are co-infected with a mixture of two strains. We observe the mixture in each animal over time and over multiple generations of transmission. We use data from influenza experiments in ferrets to demonstrate the approach. Assessment of the relative transmissibility between two strains of influenza is important in at least three contexts: 1) Within the human population antigenically novel strains of influenza arise and compete for susceptible hosts. 2) During a pandemic event, a novel sub-type of influenza competes with the existing seasonal strain(s). The unfolding epidemiological dynamics are dependent upon both the population's susceptibility profile and the inherent transmissibility of the novel strain compared to the existing strain(s). 3) Neuraminidase inhibitors (NAIs), while providing significant potential to reduce transmission of influenza, exert selective pressure on the virus and so promote the emergence of drug-resistant strains. Any adverse outcome due to selection and subsequent spread of an NAI-resistant strain is exquisitely dependent upon the transmission fitness of that strain. Measurement of the transmission fitness of two competing strains of influenza is thus of critical importance in determining the likely time-course and epidemiology of an influenza outbreak, or the potential impact of an intervention measure such as NAI distribution. The mathematical framework introduced here also provides an estimate for the size of the transmitted inoculum. We demonstrate the framework's behaviour using data from ferret transmission studies, and through simulation suggest how to optimise experimental design for assessment of transmissibility. The method introduced here for assessment of mixed transmission events has applicability beyond influenza, to other viral and bacterial pathogens

Use of dietary supplements by female seniors in a large Northern California health plan

BACKGROUND: Women aged ≥ 65 years are high utilizers of prescription and over-the-counter medications, and many of these women are also taking dietary supplements. Dietary supplement use by older women is a concern because of possible side effects and drug-supplement interactions. The primary aim of this study was to provide a comprehensive picture of dietary supplement use among older women in a large health plan in Northern California, USA, to raise awareness among health care providers and pharmacists about the need for implementing structural and educational interventions to minimize adverse consequences of self-directed supplement use. A secondary aim was to raise awareness about how the focus on use of herbals and megavitamins that has occurred in most surveys of complementary and alternative therapy use results in a significant underestimate of the proportion of older women who are using all types of dietary supplements for the same purposes. METHODS: We used data about use of different vitamin/mineral (VM) supplements and nonvitamin, nonmineral (NVNM) supplements, including herbals, from a 1999 general health survey mailed to a random sample of adult members of a large Northern California health plan to estimate prevalence of and characteristics associated with supplement use among women aged 65–84 (n = 3,109). RESULTS: Based on weighted data, 84% had in the past 12 months used >1 dietary supplement, 82% a VM, 59% a supplement other than just multivitamin or calcium, 32% an NVNM, and 25% an herbal. Compared to white, nonHispanic women, African-Americans and Latinas were significantly less likely to use VM and NVNM supplements and Asian/Pacific Islanders were less likely to use NVNM supplements. Higher education was strongly associated with use of an NVNM supplement. Prevalence did not differ by number of prescription medications taken. Among white, nonHispanic women, multiple logistic regression models showed that college education, good health, belief that health practices have at least a moderate effect on health, and having arthritis or depression significantly increased likelihood of NVNM use, while having diabetes decreased likelihood. CONCLUSIONS: An extremely high proportion of older women are using dietary supplements other than multivitamins and calcium, many in combination with multiple prescription medications. Increased resources should be devoted to helping clinicians, pharmacists, supplement vendors, and consumers become more aware of the safety, effectiveness, and potential side effects of dietary supplements

MICE: The muon ionization cooling experiment. Step I: First measurement of emittance with particle physics detectors

Copyright @ 2011 APSThe Muon Ionization Cooling Experiment (MICE) is a strategic R&D project intended to demonstrate the only practical solution to providing high brilliance beams necessary for a neutrino factory or muon collider. MICE is under development at the Rutherford Appleton Laboratory (RAL) in the United Kingdom. It comprises a dedicated beamline to generate a range of input muon emittances and momenta, with time-of-flight and Cherenkov detectors to ensure a pure muon beam. The emittance of the incoming beam will be measured in the upstream magnetic spectrometer with a scintillating fiber tracker. A cooling cell will then follow, alternating energy loss in Liquid Hydrogen (LH2) absorbers to RF cavity acceleration. A second spectrometer, identical to the first, and a second muon identification system will measure the outgoing emittance. In the 2010 run at RAL the muon beamline and most detectors were fully commissioned and a first measurement of the emittance of the muon beam with particle physics (time-of-flight) detectors was performed. The analysis of these data was recently completed and is discussed in this paper. Future steps for MICE, where beam emittance and emittance reduction (cooling) are to be measured with greater accuracy, are also presented.This work was supported by NSF grant PHY-0842798

Proinflammatory Phenotype and Increased Caveolin-1 in Alveolar Macrophages with Silenced CFTR mRNA

The inflammatory milieu in the respiratory tract in cystic fibrosis (CF) has been linked to the defective expression of the cystic transmembrane regulator (CFTR) in epithelial cells. Alveolar macrophages (AM), important contibutors to inflammatory responses in the lung, also express CFTR. The present study analyzes the phenotype of human AM with silenced CFTR. Expression of CFTR mRNA and the immature form of the CFTR protein decreased 100-fold and 5.2-fold, respectively, in AM transfected with a CFTR specific siRNA (CFTR-siRNA) compared to controls. Reduction of CFTR expression in AM resulted in increased secretion of IL-8, increased phosphorylation of NF-κB, a positive regulator of IL-8 expression, and decreased expression of IκB-α, the inhibitory protein of NF-κB activation. AM with silenced CFTR expression also showed increased apoptosis. We hypothesized that caveolin-1 (Cav1), a membrane protein that is co-localized with CFTR in lipid rafts and that is related to inflammation and apoptosis in macrophages, may be affected by decreased CFTR expression. Messenger RNA and protein levels of Cav1 were increased in AM with silenced CFTR. Expression and transcriptional activity of sterol regulatory element binding protein (SREBP), a negative transcriptional regulator of Cav1, was decreased in AM with silenced CFTR, but total and free cholesterol mass did not change. These findings indicate that silencing of CFTR in human AM results in an inflammatory phenotype and apoptosis, which is associated to SREBP-mediated regulation of Cav1