Differential Proliferative Characteristics of Alveolar Fibroblasts in Interstitial Lung Diseases: Regulative Role of IL-1 and PGE2

Fibroblasts (Fb) from patients with sarcoidosis (SA) and hypersensitivity pneumonitis (HP) exhibited a lower proliferative capacity compared with Fb obtained from control (CO) and diffuse interstitial fibrosis patients (DIF). Proliferation of Fb from SA or lip patients was suppressed by autologous LPS-stimulated alveolar macrophages (AM) supernatants but not by those from CO patients. Similarly, alveolar macrophages (AM) derived supernatant, obtained from CO, did not suppress the proliferation of SA and HP Fb. AM from SA and HP patients secreted higher amounts of IL-1α and β compared with controls and compared with Fb from SA and HP patients. Steady levels of IL-1α and βmRNA were expressed in unstimulated and stimulated cultures. Fb from SA and HP patients could be stimulated by LPS to secrete significantly higher levels of PGE2 than those detected in supernatants from LPS stimulated Fb of DIF patients. Only the proliferation of Fb from SA and HP patients was sensitive to amounts of IL-1 equivalent to those detected in the lung of these diseases. As SA and HP are two diseases where irreversible deterioration occurs in only 20% of the patients, we hypothesize that mediators in the lung may modulate Fb proliferation. IL-1 of AM origin and PGE2 of Fb origin secreted at high levels, may be candidates for this suppression because it was abrogated by anti IL-1β and indomethacin

Detection of HER2 amplification in circulating free DNA in patients with breast cancer.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in 20-25% of breast cancers. This study investigated circulating free DNA (cfDNA) for detection of HER2 gene amplification in patients with breast cancer. METHODS: Circulating free DNA was extracted from plasma of unselected patients with primary breast cancer (22 before surgery and 68 following treatment), 30 metastatic patients and 98 female controls using the QIAamp Blood DNA Mini Kit (Qiagen). The ratio of HER2 to an unamplified reference gene (contactin-associated protein 1 (CNTNAP1)) was measured in cfDNA samples by quantitative PCR (qPCR) using SK-BR-3 cell line DNA as a positive control. RESULTS: We validated the qPCR assay with DNA extracted from 23 HER2 3+ and 40 HER2-negative tumour tissue samples; the results agreed for 60 of 63 (95.2%) tumours. Amplification was detected in cfDNA for 8 of 68 patients following primary breast cancer treatment and 5 of 30 metastatic patients, but was undetected in 22 patients with primary breast cancer and 98 healthy female controls. Of the patients with amplification in cfDNA, 10 had HER2 3+ tumour status by immunohistochemistry. CONCLUSIONS: The results demonstrate for the first time the existence of amplified HER2 in cfDNA in the follow-up of breast cancer patients who are otherwise disease free. This approach could potentially provide a marker in patients with HER2-positive breast cancer

Dynamic Computational Model Suggests That Cellular Citizenship Is Fundamental for Selective Tumor Apoptosis

Computational models in the field of cancer research have focused primarily on estimates of biological events based on laboratory generated data. We introduce a novel in-silico technology that takes us to the next level of prediction models and facilitates innovative solutions through the mathematical system. The model's building blocks are cells defined phenotypically as normal or tumor, with biological processes translated into equations describing the life protocols of the cells in a quantitative and stochastic manner. The essentials of communication in a society composed of normal and tumor cells are explored to reveal “protocols” for selective tumor eradication. Results consistently identify “citizenship properties” among cells that are essential for the induction of healing processes in a healthy system invaded by cancer. These properties act via inter-cellular communication protocols that can be optimized to induce tumor eradication along with system recovery. Within the computational systems, the protocols universally succeed in removing a wide variety of tumors defined by proliferation rates, initial volumes, and apoptosis resistant phenotypes; they show high adaptability for biological details and allow incorporation of population heterogeneity. These protocols work as long as at least 32% of cells obey extra-cellular commands and at least 28% of cancer cells report their deaths. This low percentage implies that the protocols are resilient to the suboptimal situations often seen in biological systems. We conclude that our in-silico model is a powerful tool to investigate, to propose, and to exercise logical anti-cancer solutions. Functional results should be confirmed in a biological system and molecular findings should be loaded into the computational model for the next level of directed experiments

Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection

Structure of the Full-Length Major Pilin from Streptococcus pneumoniae: Implications for Isopeptide Bond Formation in Gram-Positive Bacterial Pili

The surface of the pneumococcal cell is adorned with virulence factors including pili. The major pilin RrgB, which forms the pilus shaft on pathogenic Streptococcus pneumoniae, comprises four immunoglobulin (Ig)-like domains, each with a common CnaB topology. The three C-terminal domains are each stabilized by internal Lys-Asn isopeptide bonds, formed autocatalytically with the aid of an essential Glu residue. The structure and orientation of the crucial N-terminal domain, which provides the covalent linkage to the next pilin subunit in the shaft, however, remain incompletely characterised. We report the crystal structure of full length RrgB, solved by X-ray crystallography at 2.8 Å resolution. The N-terminal (D1) domain makes few contacts with the rest of the RrgB structure, and has higher B-factors. This may explain why D1 is readily lost by proteolysis, as are the N-terminal domains of many major pilins. D1 is also found to have a triad of Lys, Asn and Glu residues in the same topological positions as in the other domains, yet mass spectrometry and the crystal structure show that no internal isopeptide bond is formed. We show that this is because β-strand G of D1, which carries the Asn residue, diverges from β-strand A, carrying the Lys residue, such that these residues are too far apart for bond formation. Strand G also carries the YPKN motif that provides the essential Lys residue for the sortase-mediated intermolecular linkages along the pilus shaft. Interaction with the sortase and formation of the intermolecular linkage could result in a change in the orientation of this strand, explaining why isopeptide bond formation in the N-terminal domains of some major pilins appears to take place only upon assembly of the pili

Reduced ventricular proliferation in the foetal cortex following maternal inflammation in the mouse

It has been well established that maternal inflammation during pregnancy alters neurological function in the offspring, but its impact on cortical development and long-term consequences on the cytoarchitecture is largely unstudied. Here we report that lipopolysaccharide-induced systemic maternal inflammation in C57Bl/6 mice at embryonic Day 13.5 of pregnancy, as early as 8 h after challenge, caused a significant reduction in cell proliferation in the ventricular zone of the developing cerebral cortex, as revealed by quantification of anti-phospho-Histone H3 immunoreactivity and bromodeoxyuridine pulse labelling. The angle of mitotic cleavage, determined from analysis of haematoxylin and eosin staining, cyclin E1 gene expression and the pattern of β-catenin immunoreactivity were also altered by the challenge, which suggests a change from symmetric to asymmetric division in the radial progenitor cells. Modifications of cortical lamination and gene expression patterns were detected at post-natal Day 8 suggesting prolonged consequences of these alterations during embryonic development. Cellular uptake of proteins from the cerebrospinal fluid was observed in brains from lipopolysaccharide-treated animals in radial progenitor cells. However, the foetal blood–brain barrier to plasma proteins remained intact. Together, these results indicate that maternal inflammation can disrupt the ventricular surface and lead to decreased cellular proliferation. Changes in cell density in Layers IV and V at post-natal Day 8 show that these initial changes have prolonged effects on cortical organization. The possible shift in the fate of progeny and the resulting alterations in the relative cell numbers in the cerebral cortex following a maternal inflammatory response shown here will require further investigation to determine the long-term consequences of inflammation on the development of neuronal circuitry and behaviour

The presence of disseminated tumour cells in the bone marrow is inversely related to circulating free DNA in plasma in breast cancer dormancy.

BACKGROUND: The aim of this study was to gain insight into breast cancer dormancy by examining different measures of minimal residual disease (MRD) over time in relation to known prognostic factors. METHODS: Sixty-four primary breast cancer patients on follow-up (a median of 8.3 years post surgery) who were disease free had sequential bone marrow aspirates and blood samples taken for the measurement of disseminated tumour cells (DTCs), circulating tumour cells (CTCs) by CellSearch and qPCR measurement of overlapping (96-bp and 291-bp) amplicons in circulating free DNA (cfDNA). RESULTS: The presence of CTCs was correlated with the presence of DTCs measured by immunocytochemistry (P=0.01) but both were infrequently detected. Increasing cfDNA concentration correlated with ER, HER2 and triple-negative tumours and high tumour grade, and the 291-bp amplicon was inversely correlated with DTCs measured by CK19 qRT-PCR (P=0.047). CONCLUSION: Our results show that breast cancer patients have evidence of MRD for many years after diagnosis despite there being no overt evidence of disease. The inverse relationship between bone marrow CK19 mRNA and the 291-bp amplicon in cfDNA suggests that an inverse relationship between a measure of cell viability in the bone marrow (DTCs) and cell death in the plasma occurs during the dormancy phase of breast cancer

Hypoxia and Prostaglandin E Receptor 4 Signalling Pathways Synergise to Promote Endometrial Adenocarcinoma Cell Proliferation and Tumour Growth

The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E2. PTGS2 expression and PGE2 biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE2 regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1–4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE2 and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE2 and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells

Estrogen Receptor Silencing Induces Epithelial to Mesenchymal Transition in Human Breast Cancer Cells

We propose the hypothesis that loss of estrogen receptor function which leads to endocrine resistance in breast cancer, also results in trans-differentiation from an epithelial to a mesenchymal phenotype that is responsible for increased aggressiveness and metastatic propensity. siRNA mediated silencing of the estrogen receptor in MCF7 breast cancer cells resulted in estrogen/tamoxifen resistant cells (pII) with altered morphology, increased motility with rearrangement and switch from a keratin/actin to a vimentin based cytoskeleton, and ability to invade simulated components of the extracellular matrix. Phenotypic profiling using an Affymetrix Human Genome U133 plus 2.0 GeneChip indicated geometric fold changes ≥3 in approximately 2500 identifiable unique sequences, with about 1270 of these being up-regulated in pII cells. Changes were associated with genes whose products are involved in cell motility, loss of cellular adhesion and interaction with the extracellular matrix. Selective analysis of the data also showed a shift from luminal to basal cell markers and increased expression of a wide spectrum of genes normally associated with mesenchymal characteristics, with consequent loss of epithelial specific markers. Over-expression of several peptide growth factors and their receptors are indicative of an increased contribution to the higher proliferative rates of pII cells as well as aiding their potential for metastatic activity. Signalling molecules that have been identified as key transcriptional drivers of epithelial to mesenchymal transition were also found to be elevated in pII cells. These data support our hypothesis that induced loss of estrogen receptor in previously estrogen/antiestrogen sensitive cells is a trigger for the concomitant loss of endocrine dependence and onset of a series of possibly parallel events that changes the cell from an epithelial to a mesenchymal type. Inhibition of this transition through targeting of specific mediators may offer a useful supplementary strategy to circumvent the effects of loss of endocrine sensitivity