Chlamydia muridarum lung infection in infants alters hematopoietic cells to promote allergic airway disease in mice

Background Viral and bacterial respiratory tract infections in early-life are linked to the development of allergic airway inflammation and asthma. However, the mechanisms involved are not well understood. We have previously shown that neonatal and infant, but not adult, chlamydial lung infections in mice permanently alter inflammatory phenotype and physiology to increase the severity of allergic airway disease by increasing lung interleukin (IL)-13 expression, mucus hyper-secretion and airway hyper-responsiveness. This occurred through different mechanisms with infection at different ages. Neonatal infection suppressed inflammatory responses but enhanced systemic dendritic cell:T-cell IL-13 release and induced permanent alterations in lung structure (i.e., increased the size of alveoli). Infant infection enhanced inflammatory responses but had no effect on lung structure. Here we investigated the role of hematopoietic cells in these processes using bone marrow chimera studies. Methodology/Principal Findings Neonatal (<24-hours-old), infant (3-weeks-old) and adult (6-weeks-old) mice were infected with C. muridarum. Nine weeks after infection bone marrow was collected and transferred into recipient age-matched irradiated naïve mice. Allergic airway disease was induced (8 weeks after adoptive transfer) by sensitization and challenge with ovalbumin. Reconstitution of irradiated naïve mice with bone marrow from mice infected as neonates resulted in the suppression of the hallmark features of allergic airway disease including mucus hyper-secretion and airway hyper-responsiveness, which was associated with decreased IL-13 levels in the lung. In stark contrast, reconstitution with bone marrow from mice infected as infants increased the severity of allergic airway disease by increasing T helper type-2 cell cytokine release (IL-5 and IL-13), mucus hyper-secretion, airway hyper-responsiveness and IL-13 levels in the lung. Reconstitution with bone marrow from infected adult mice had no effects. Conclusions These results suggest that an infant chlamydial lung infection results in long lasting alterations in hematopoietic cells that increases the severity of allergic airway disease in later-life.

Gene expression profiling in human breast cancer – toward personalised therapeutics?

The most integrated approach toward understanding the multiple molecular events and mechanisms by which cancer may develop is the application of gene expression profiling using microarray technologies. As molecular alterations in breast cancer are complex and involve cross-talk between multiple cellular signalling pathways, microarray technology provides a means of capturing and comparing the expression patterns of the entire genome across multiple samples in a high throughput manner. Since the development of microarray technologies, together with the advances in RNA extraction methodologies, gene expression studies have revolutionised the means by which genes suitable as targets for drug development and individualised cancer treatment can be identified. As of the mid-1990s, expression microarrays have been extensively applied to the study of cancer and no cancer type has seen as much genomic attention as breast cancer. The most abundant area of breast cancer genomics has been the clarification and interpretation of gene expression patterns that unite both biological and clinical aspects of tumours. It is hoped that one day molecular profiling will transform diagnosis and therapeutic selection in human breast cancer toward more individualised regimes. Here, we review a number of prominent microarray profiling studies focussed on human breast cancer and examine their strengths, their limitations, clinical implications including prognostic relevance and gene signature significance along with potential improvements for the next generation of microarray studies.

Hemi-field and full-field form-deprivation induce timing changes in multifocal ERG responses in chick

Purpose: In animal models hemi-field deprivation results in localised, graded vitreous chamber elongation and presumably deprivation induced localised changes in retinal processing. The aim of this research was to determine if there are variations in ERG responses across the retina in normal chick eyes and to examine the effect of hemi-field and full-field deprivation on ERG responses across the retina and at earlier times than have previously been examined electrophysiologically. Methods: Chicks were either untreated, wore monocular full-diffusers or half-diffusers (depriving nasal retina) (n = 6-8 each group) from day 8. mfERG responses were measured using the VERIS mfERG system across the central 18.2º× 16.7º (H × V) field. The stimulus consisted of 61 unscaled hexagons with each hexagon modulated between black and white according to a pseudorandom binary m-sequence. The mfERG was measured on day 12 in untreated chicks, following 4 days of hemi-field diffuser wear, and 2, 48 and 96 h after application of full-field diffusers. Results: The ERG response of untreated chick eyes did not vary across the measured field; there was no effect of retinal location on the N1-P1 amplitude (p = 0.108) or on P1 implicit time (p > 0.05). This finding is consistent with retinal ganglion cell density of the chick varying by only a factor of two across the entire retina. Half-diffusers produced a ramped retina and a graded effect of negative lens correction (p < 0.0001); changes in retinal processing were localized. The untreated retina showed increasing complexity of the ERG waveform with development; form-deprivation prevented the increasing complexity of the response at the 2, 48 and 96 h measurement times and produced alterations in response timing. Conclusions: Form-deprivation and its concomitant loss of image contrast and high spatial frequency images prevented development of the ERG responses, consistent with a disruption of development of retinal feedback systems. The characterisation of ERG responses in normal and deprived chick eyes across the retina allows the assessment of concurrent visual and retinal manipulations in this model. (Ophthalmic & Physiological Optics © 2013 The College of Optometrists.)

EUELC project : a multi-centre, multipurpose study to investigate early stage NSCLC, and to establish a biobank for ongoing collaboration

The European Early Lung Cancer (EUELC) project aims to determine if specific genetic alterations occurring in lung carcinogenesis are detectable in the respiratory epithelium. In order to pursue this objective, nonsmall cell lung cancer (NSCLC) patients with a very high risk of developing progressive lung cancer were recruited from 12 centres in eight European countries: France, Germany, southern Ireland, Italy, the Netherlands, Poland, Spain and the UK. In addition, NSCLC patients were followed up every 6 months for 36 months. A European Bronchial Tissue Bank was set up at the University of Liverpool (Liverpool, UK) to optimise the use of biological specimens. The molecular - pathological investigations were subdivided into specific work packages that were delivered by EUELC Partners. The work packages encompassed mutational analysis, genetic instability, methylation profiling, expression profiling utilising immunohistochemistry and chip-based technologies, as well as in-depth analysis of FHIT and RARβ genes, the telomerase catalytic subunit hTERT and genotyping of susceptibility genes in specific pathways. The EUELC project engendered a tremendous collaborative effort, and it enabled the EUELC Partners to establish protocols for assessing molecular biomarkers in early lung cancer with the view to using such biomarkers for early diagnosis and as intermediate end-points in future chemopreventive programmes. Copyright©ERS Journals Ltd 2009.

Gait parameters associated with hallux valgus : a systematic review

Background Hallux valgus (HV) has been linked to functional disability and increased falls risk in older adults. However, specific gait alterations in individuals with HV are unclear. This systematic review investigated gait parameters associated with HV in otherwise healthy adults. Methods Electronic databases (Medline, Embase, CINAHL) were searched to October 2011, including cross-sectional studies with clearly defined HV and non-HV comparison groups. Two investigators independently rated studies for methodological quality. Effect sizes (95% confidence intervals (CI)) were calculated as standardized mean differences (SMD) for continuous data and risk ratios (RR) for dichotomous data. Results Nine studies included a total of 589 participants. Three plantar pressure studies reported increased hallux loading (SMD 0.56 to 1.78) and medial forefoot loading (SMD 0.62 to 1.21), while one study found reduced first metatarsal loading (SMD −0.61, CI −1.19 to −0.03) in HV participants. HV participants demonstrated less ankle and rearfoot motion during terminal stance (SMD −0.81 to −0.63) and increased intrinsic muscle activity (RR 1.6, 1.1 to 2.2). Most studies reported no differences in spatio-temporal parameters; however, one study found reduced speed (SMD −0.73, -1.25 to −0.20), step length (SMD −0.66 to −0.59) and less stable gait patterns (SMD −0.86 to −0.78) in older adults with HV. Conclusions HV impacts on particular gait parameters, and further understanding of potentially modifiable factors is important for prevention and management of HV. Cause and effect relationships cannot be inferred from cross-sectional studies, thus prospective studies are warranted to elucidate the relationship between HV and functional disability.

Gemcitabine reactivates epigenetically silenced genes and functions as a DNA methyltransferase inhibitor

Gemcitabine is indicated in combination with cisplatin as first-line therapy for solid tumours including non-small cell lung cancer (NSCLC), bladder cancer and mesothelioma. Gemcitabine is an analogue of pyrimidine cytosine and functions as an anti-metabolite. Structurally, however, gemcitabine has similarities to 5-aza-2-deoxycytidine (decitabine/Dacogen®), a DNA methyltransferase inhibitor (DNMTi). NSCLC, mesothelioma and prostate cancer cell lines were treated with decitabine and gemcitabine. Reactivation of epigenetically silenced genes was examined by RT-PCR/qPCR. DNA methyltransferase activity in nuclear extracts and recombinant proteins was measured using a DNA methyltransferase assay, and alterations in DNA methylation status were examined using methylation-specific PCR (MS-PCR) and pyrosequencing. We observe a reactivation of several epigenetically silenced genes including GSTP1, IGFBP3 and RASSF1A. Gemcitabine functionally inhibited DNA methyltransferase activity in both nuclear extracts and recombinant proteins. Gemcitabine dramatically destabilised DNMT1 protein. However, DNA CpG methylation was for the most part unaffected by gemcitabine. In conclusion, gemcitabine both inhibits and destabilises DNA methyltransferases and reactivates epigenetically silenced genes having activity equivalent to decitabine at concentrations significantly lower than those achieved in the treatment of patients with solid tumours. This property may contribute to the anticancer activity of gemcitabine.

In vivo confocal microscopy of the ocular surface : from bench to bedside

In vivo confocal microscopy (IVCM) is an emerging technology that provides minimally invasive, high resolution, steady-state assessment of the ocular surface at the cellular level. Several challenges still remain but, at present, IVCM may be considered a promising technique for clinical diagnosis and management. This mini-review summarizes some key findings in IVCM of the ocular surface, focusing on recent and promising attempts to move “from bench to bedside”. IVCM allows prompt diagnosis, disease course follow-up, and management of potentially blinding atypical forms of infectious processes, such as acanthamoeba and fungal keratitis. This technology has improved our knowledge of corneal alterations and some of the processes that affect the visual outcome after lamellar keratoplasty and excimer keratorefractive surgery. In dry eye disease, IVCM has provided new information on the whole-ocular surface morphofunctional unit. It has also improved understanding of pathophysiologic mechanisms and helped in the assessment of prognosis and treatment. IVCM is particularly useful in the study of corneal nerves, enabling description of the morphology, density, and disease- or surgically induced alterations of nerves, particularly the subbasal nerve plexus. In glaucoma, IVCM constitutes an important aid to evaluate filtering blebs, to better understand the conjunctival wound healing process, and to assess corneal changes induced by topical antiglaucoma medications and their preservatives. IVCM has significantly enhanced our understanding of the ocular response to contact lens wear. It has provided new perspectives at a cellular level on a wide range of contact lens complications, revealing findings that were not previously possible to image in the living human eye. The final section of this mini-review provides a focus on advances in confocal microscopy imaging. These include 2D wide-field mapping, 3D reconstruction of the cornea and automated image analysis.

ATF3 suppresses metastasis of bladder cancer by regulating gelsolin-mediated remodeling of the actin cytoskeleton

Bladder cancer is associated with high recurrence and mortality rates due to metastasis. The elucidation of metastasis suppressors may offer therapeutic opportunities if their mechanisms of action can be elucidated and tractably exploited. In this study, we investigated the clinical and functional significance of the transcription factor activating transcription factor 3 (ATF3) in bladder cancer metastasis. Gene expression analysis revealed that decreased ATF3 was associated with bladder cancer progression and reduced survival of patients with bladder cancer. Correspondingly, ATF3 overexpression in highly metastatic bladder cancer cells decreased migration in vitro and experimental metastasis in vivo. Conversely, ATF3 silencing increased the migration of bladder cancer cells with limited metastatic capability in the absence of any effect on proliferation. In keeping with their increased motility, metastatic bladder cancer cells had increased numbers of actin filaments. Moreover, ATF3 expression correlated with expression of the actin filament severing protein gelsolin (GSN). Mechanistic studies revealed that ATF3 upregulated GSN, whereas ATF3 silencing reduced GSN levels, concomitant with alterations in the actin cytoskeleton. We identified six ATF3 regulatory elements in the first intron of the GSN gene confirmed by chromatin immunoprecipitation analysis. Critically, GSN expression reversed the metastatic capacity of bladder cancer cells with diminished levels of ATF3. Taken together, our results indicate that ATF3 suppresses metastasis of bladder cancer cells, at least in part through the upregulation of GSN-mediated actin remodeling. These findings suggest ATF3 coupled with GSN as prognostic markers for bladder cancer metastasis.

Epigenetic regulation of chemokine/chemokine receptor expression

Epigenetic regulation of gene expression is an important event for normal cellular homeostasis. Gene expression may be "switched" on or "turned" off via epigenetic means through adjustments in DNA architecture. These structural alterations result from changes to the DNA methylation status in addition to histone posttranslational modifications such as acetylation and methylation. Drugs which can alter the status of these epigenetic markers are currently undergoing clinical trials in a wide variety of diseases, including cancer.We illustrate the treatment of cell lines with histone deacetylase (HDi) and DNA methyltransferase inhibitors and the subsequent RNA isolation and reverse transcriptase polymerase chain reaction for several members of the CXC (ELR(+)) chemokine family. In addition we describe a chromatin immunoprecipitation assay to determine the association between chromatin transcription markers and DNA following pretreatment of cell cultures with an HDi, Trichostatin A (TSA). This assay allows us to determine whether treatment with TSA dynamically remodels the promoter region of our selected genes, as judged by the differences in the PCR product between our treated and untreated samples.

Genome stability pathways in head and neck cancers

Genomic instability underlies the transformation of host cells toward malignancy, promotes development of invasion and metastasis and shapes the response of established cancer to treatment. In this review, we discuss recent advances in our understanding of genomic stability in squamous cell carcinoma of the head and neck (HNSCC), with an emphasis on DNA repair pathways. HNSCC is characterized by distinct profiles in genome stability between similarly staged cancers that are reflected in risk, treatment response and outcomes. Defective DNA repair generates chromosomal derangement that can cause subsequent alterations in gene expression, and is a hallmark of progression toward carcinoma. Variable functionality of an increasing spectrum of repair gene polymorphisms is associated with increased cancer risk, while aetiological factors such as human papillomavirus, tobacco and alcohol induce significantly different behaviour in induced malignancy, underpinned by differences in genomic stability. Targeted inhibition of signalling receptors has proven to be a clinically-validated therapy, and protein expression of other DNA repair and signalling molecules associated with cancer behaviour could potentially provide a more refined clinical model for prognosis and treatment prediction. Development and expansion of current genomic stability models is furthering our understanding of HNSCC pathophysiology and uncovering new, promising treatment strategies. © 2013 Glenn Jenkins et al.

Posttraumatic stress disorder and traumatic stress : from bench to bedside, from war to disaster

War is a tragic event and its mental health consequences can be profound. Recent studies indicate substantial rates of posttraumatic stress disorder and other behavioral alterations because of war exposure. Understanding the psychological, behavioral, and neurobiological mechanism of mental health and behavioral changes related to war exposure is critical to helping those in need of care. Substantial work to encourage bench to bedside to community knowledge and communication is a core component of addressing this world health need.

Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence

The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.

Assessment of gene expression of intracellular calcium channels, pumps and exchangers with epidermal growth factor-induced epithelial-mesenchymal transition in a breast cancer cell line

Background Epithelial-mesenchymal transition (EMT) is a process implicated in cancer metastasis that involves the conversion of epithelial cells to a more mesenchymal and invasive cell phenotype. In breast cancer cells EMT is associated with altered store-operated calcium influx and changes in calcium signalling mediated by activation of cell surface purinergic receptors. In this study, we investigated whether MDA-MB-468 breast cancer cells induced to undergo EMT exhibit changes in mRNA levels of calcium channels, pumps and exchangers located on intracellular calcium storing organelles, including the Golgi, mitochondria and endoplasmic reticulum (ER). Methods Epidermal growth factor (EGF) was used to induce EMT in MDA-MB-468 breast cancer cells. Serum-deprived cells were treated with EGF (50 ng/mL) for 12 h and gene expression was assessed using quantitative RT-PCR. Results and conclusions These data reveal no significant alterations in mRNA levels of the Golgi calcium pump secretory pathway calcium ATPases (SPCA1 and SPCA2), or the mitochondrial calcium uniporter (MCU) or Na+/Ca2+ exchanger (NCLX). However, EGF-induced EMT was associated with significant alterations in mRNA levels of specific ER calcium channels and pumps, including (sarco)-endoplasmic reticulum calcium ATPases (SERCAs), and inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RYR) calcium channel isoforms. The most prominent change in gene expression between the epithelial and mesenchymal-like states was RYR2, which was enriched 45-fold in EGF-treated MDA-MB-468 cells. These findings indicate that EGF-induced EMT in breast cancer cells may be associated with major alterations in ER calcium homeostasis.

Dynamic changes in high and low mammographic density human breast tissues maintained in murine tissue engineering chambers during various murine peripartum states and over time

Mammographic density (MD) is a strong heritable risk factor for breast cancer, and may decrease with increasing parity. However, the biomolecular basis for MD-associated breast cancer remains unclear, and systemic hormonal effects on MD-associated risk is poorly understood. This study assessed the effect of murine peripartum states on high and low MD tissue maintained in a xenograft model of human MD. Method High and low MD human breast tissues were precisely sampled under radiographic guidance from prophylactic mastectomy specimens of women. The high and low MD tissues were maintained in separate vascularised biochambers in nulliparous or pregnant SCID mice for 4 weeks, or mice undergoing postpartum involution or lactation for three additional weeks. High and low MD biochamber material was harvested for histologic and radiographic comparisons during various murine peripartum states. High and low MD biochamber tissues in nulliparous mice were harvested at different timepoints for histologic and radiographic comparisons. Results High MD biochamber tissues had decreased stromal (p = 0.0027), increased adipose (p = 0.0003) and a trend to increased glandular tissue areas (p = 0.076) after murine postpartum involution. Stromal areas decreased (p = 0.042), while glandular (p = 0.001) and adipose areas (p = 0.009) increased in high MD biochamber tissues during lactation. A difference in radiographic density was observed in high (p = 0.0021) or low MD biochamber tissues (p = 0.004) between nulliparous, pregnant and involution groups. No differences in tissue composition were observed in high or low MD biochamber tissues maintained for different durations, although radiographic density increased over time. Conclusion High MD biochamber tissues had measurable histologic changes after postpartum involution or lactation. Alterations in radiographic density occurred in biochamber tissues between different peripartum states and over time. These findings demonstrate the dynamic nature of the human MD xenograft model, providing a platform for studying the biomolecular basis of MD-associated cancer risk. © 2013 Springer Science+Business Media New York.

The effect of exercise on the skeletal muscle phospholipidome of rats fed a high-fat diet

The aim of this study was to examine the effect of endurance training on skeletal muscle phospholipid molecular species from high-fat fed rats. Twelve female Sprague-Dawley rats were fed a high-fat diet (78.1% energy). The rats were randomly divided into two groups, a sedentary control group and a trained group (125 min of treadmill running at 8 m/min, 4 days/wk for 4 weeks). Forty-eight hours after their last training bout phospholipids were extracted from the red and white vastus lateralis and analyzed by electrospray-ionization mass spectrometry. Exercise training was associated with significant alterations in the relative abundance of a number of phospholipid molecular species. These changes were more prominent in red vastus lateralis than white vastus lateralis. The largest observed change was an increase of similar to 30% in the abundance of 1-palmitoyl-2-linoleoyl phosphatidylcholine ions in oxidative fibers. Reductions in the relative abundance of a number of phospholipids containing long-chain n-3 polyunsaturated fatty acids were also observed. These data suggest a possible reduction in phospholipid remodeling in the trained animals. This results in a decrease in the phospholipid n-3 to n-6 ratio that may in turn influence endurance capacity.