Cytokine phenotype, genotype, and renal outcomes at cardiac surgery

Cardiac surgery modulates pro- and anti-inflammatory cytokine balance involving plasma tumour necrosis factor alpha (TNFa) and interleukin-10 (IL-10) together with urinary transforming growth factor beta-1 (TGFß1), interleukin-1 receptor antagonist (IL1ra) and tumour necrosis factor soluble receptor-2 (TNFsr2). Effects on post-operative renal function are unclear. We investigated if following cardiac surgery there is a relationship between cytokine (a) phenotype and renal outcome; (b) genotype and phenotype and (c) genotype and renal outcome. Since angiotensin-2 (AG2), modulates TGFß1 production, we determined whether angiotensin converting enzyme insertion/deletion (ACE I/D) genotype affects urinary TGFß1 phenotype as well as renal outcome.

Intrafamilial variation of phenotype in Stargardt macular dystrophy- fundus flavimaculatus

Purpose. To evaluate the intrafamilial phenotypic variation in Stargardt macular dystrophy-Fundus flavimaculatus (SMD-FFM). Methods. Thirty-one siblings from 15 families with SMD-FFM were examined. Age of onset, visual acuity, and clinical features on fundus examination and fundus autofluorescence images, including presence or absence of central and peripheral atrophy and distribution of flecks, were recorded. In addition, electrophysiological studies were undertaken. Results. Large differences between siblings in age of onset (median, 12 years; range, 5-23 years) were observed in six of the 15 families studied, whereas in 9 families differences in age of onset between siblings were small (median, 1 year; range, 0-3 years). Visual acuity varied two or more lines among siblings in nine families. In 10 families (67%) siblings were found to have different clinical appearance on fundus examination and fundus autofluorescence images, whereas in 5 families (33%), affected siblings had similar clinical features. Electrodiagnostic tests were performed on affected members of 12 families and disclosed similar qualitative findings among siblings. In nine families there was loss of central function only; in two, global loss of cone function; and in one, global loss of cone and rod function. Conclusions. In this series, although differences in age of onset, visual acuity, and fundus appearance were observed between siblings, electrophysiological studies demonstrated intrafamilial homogeneity in retinal function. The findings are difficult to reconcile with expression studies showing ABCR transcripts in rod photoreceptors but not in cones.

Abnormal changes in voltage-gated sodium channels NaV1.1, NaV1.2, NaV1.3, NaV1.6 and in Calmodulin/calmodulin-dependent protein kinase II, within the brains of spontaneously epileptic rats and tremor rats

Voltage-gated sodium channels (VGSCs) play a crucial role in epilepsy. The expressions of different VGSCs subtypes are varied in diverse animal models of epilepsy that may reflect their multiple phenotypes or the complexity of the mechanisms of epilepsy. In a previous study, we reported that NaV1.1 and NaV1.3 were up-regulated in the hippocampus of the spontaneously epileptic rat (SER). In this study, we further analyzed both the expression and distribution of the typical VGSC subtypes NaV1.1, NaV1.2, NaV1.3 and NaV1.6 in the hippocampus and in the cortex of the temporal lobe of two genetic epileptic animal models: the SER and the tremor rat (TRM). The expressions of calmodulin (CaM) and calmodulin-dependent protein kinase II (CaMKII) were also analyzed with the purpose of assessing the effect of the CaM/CaMKII pathway in these two models of epilepsy. Increased expression of the four VGSC subtypes and CaM, accompanied by a decrease in CaMKII was observed in the hippocampus of both the SERs and the TRM rats. However, the changes observed in the expression of VGSC subtypes and CaM were decreased with an elevated CaMKII in the cortex of their temporal lobes. Double-labeled immunofluorescence data suggested that in SERs and TRM rats, the four subtypes of the VGSC proteins were present throughout the CA1, CA3 and dentate gyrus regions of the hippocampus and temporal lobe cortex and these were co-localized in neurons with CaM. These data represent the first evidence of abnormal changes in expression of four VGSC subtypes (NaV1.1, NaV1.2, NaV1.3 and NaV1.6) and CaM/CaMKII in the hippocampus and temporal lobe cortex of SERs and TRM rats. These changes may be involved in the generation of epileptiform activity and underlie the observed seizure phenotype in these rat models of genetic epilepsy.

Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer

Secretory factors that drive cancer progression are attractive immunotherapeutic targets. We used a whole-genome data-mining approach on multiple cohorts of breast tumours annotated for clinical outcomes to discover such factors. We identified Serine protease inhibitor Kazal-type 1 (SPINK1) to be associated with poor survival in estrogen receptor-positive (ER+) cases. Immunohistochemistry showed that SPINK1 was absent in normal breast, present in early and advanced tumours, and its expression correlated with poor survival in ER+ tumours. In ER- cases, the prognostic effect did not reach statistical significance. Forced expression and/or exposure to recombinant SPINK1 induced invasiveness without affecting cell proliferation. However, down-regulation of SPINK1 resulted in cell death. Further, SPINK1 overexpressing cells were resistant to drug-induced apoptosis due to reduced caspase-3 levels and high expression of Bcl2 and phospho-Bcl2 proteins. Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain. Thus, SPINK1 affects multiple aggressive properties in breast cancer: survival, invasiveness and chemoresistance. Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.

Achaete-scute homolog-1 linked to remodeling and preneoplasia of pulmonary epithelium

The basic helix-loop-helix protein achaete-scute homolog-1 (ASH1) is involved in lung neuroendocrine (NE) differentiation and tumor promotion in SV40 transgenic mice. Constitutive expression of human ASH-1 (hASH1) in mouse lung results in hyperplasia and remodeling that mimics bronchiolization of alveoli (BOA), a potentially premalignant lesion of human lung carcinomas. We now show that this is due to sustained cellular proliferation in terminal bronchioles and resistance to apoptosis. Throughout the airway epithelium the expression of anti-apoptotic Bcl-2 and c-Myb was increased and Akt/mTOR pathway activated. Moreover, the expression of matrix metalloproteases (MMPs) including MMP7 was specifically enhanced at the bronchiolo-alveolar duct junction and BOA suggesting that MMPs play a key role in this microenvironment during remodeling. We also detected MMP7 in 70% of human BOA lesions. Knockdown of hASH1 gene in human lung cancer cells in vitro suppressed growth by increasing apoptosis. We also show that forced expression of hASH1 in immortalized human bronchial epithelial cells decreases apoptosis. We conclude that the impact of hASH1 is not limited to cells with NE phenotype. Rather, constitutive expression of hASH1 in lung epithelium promotes remodeling through multiple pathways that are commonly activated during lung carcinogenesis. The collective results suggest a novel model of BOA formation via hASH1-induced suppression of the apoptotic pathway. Our study yields a promising new preclinical tool for chemoprevention of peripheral lung carcinomas. © 2007 USCAP, Inc All rights reserved.

Pigmented paravenous chorioretinal atrophy is associated with a mutation within the crumbs homolog 1 (CRB1) gene

Pigmented paravenous chorioretinal atrophy (PPCRA) is an unusual retinal degeneration characterized by accumulation of pigmentation along retinal veins. The purpose of this study was to describe the phenotype of a family with PPCRA, determine the mode of inheritance, and identify the causal mutation.

Nestor-Guillermo Progeria Syndrome: a biochemical insight into Barrier-to-Autointegration Factor 1, alanine 12 threonine mutation

Background: Premature aging syndromes recapitulate many aspects of natural aging and provide an insight into this phenomenon at a molecular and cellular level. The progeria syndromes appear to cause rapid aging through disruption of normal nuclear structure. Recently, a coding mutation (c.34G > A [p.A12T]) in the Barrier to Autointegration Factor 1 (BANF1) gene was identified as the genetic basis of Nestor-Guillermo Progeria syndrome (NGPS). This mutation was described to cause instability in the BANF1 protein, causing a disruption of the nuclear envelope structure.

Results: Here we demonstrate that the BANF1 A12T protein is indeed correctly folded, stable and that the observed phenotype, is likely due to the disruption of the DNA binding surface of the A12T mutant. We demonstrate, using biochemical assays, that the BANF1 A12T protein is impaired in its ability to bind DNA while its interaction with nuclear envelope proteins is unperturbed. Consistent with this, we demonstrate that ectopic expression of the mutant protein induces the NGPS cellular phenotype, while the protein localizes normally to the nuclear envelope.

Conclusions: Our study clarifies the role of the A12T mutation in NGPS patients, which will be of importance for understanding the development of the disease.

Characterisation of the frequent exacerbator phenotype in COPD patients in a large UK primary care population

BACKGROUND: The 'frequent exacerbator' is recognised as an important phenotype in COPD. Current understanding about this phenotype comes from prospective longitudinal clinical trials in secondary/tertiary care with little information reported in primary care populations.

AIMS: To characterize the frequent-exacerbator phenotype and identify associated risk factors in a large UK primary care COPD population.

METHODS: Using a large database of primary care patients from 80 UK general practices, patients were categorised using GOLD 2014 criteria into high and low risk groups based on exacerbation history. A multivariate logistic regression model was used to investigate covariates associated with the frequent-exacerbator phenotype and risk of experiencing a severe exacerbation (leading to hospitalisation).

RESULTS: Of the total study population (n = 9219), 2612 (28%) fulfilled the criteria for high risk frequent-exacerbators. Independent risk factors (adjusted odds ratio [95% CI]) for ≥2 exacerbations were: most severely impaired modified Medical Research Council (mMRC) dyspnoea score (mMRC grade 4: 4.37 [2.64-7.23]), lower FEV1 percent predicted (FEV1 <30%: 2.42 [1.61-3.65]), co-morbid cardiovascular disease (1.42 [1.19-1.68]), depression (1.56 [1.22-1.99]) or osteoporosis (1.54 [1.19-2.01]), and female gender (1.20 [1.01-1.43]). Older patients (≥75 years), those with most severe lung impairment (FEV1 <30%), those with highest mMRC score and those with co-morbid osteoporosis were identified as most at risk of experiencing exacerbations requiring hospitalisation.

CONCLUSIONS: Although COPD exacerbations occur across all grades of disease severity, female patients with high dyspnoea scores, more severely impaired lung function and co-morbidities are at greatest risk. Elderly patients, with severely impaired lung function, high mMRC scores and osteoporosis are associated with experience of severe exacerbations requiring hospitalisation.

DNA methylation subgroups and the CpG island methylator phenotype in gastric cancer: a comprehensive profiling approach

BACKGROUND: Methylation-induced silencing of promoter CpG islands in tumor suppressor genes plays an important role in human carcinogenesis. In colorectal cancer, the CpG island methylator phenotype (CIMP) is defined as widespread and elevated levels of DNA methylation and CIMP+ tumors have distinctive clinicopathological and molecular features. In contrast, the existence of a comparable CIMP subtype in gastric cancer (GC) has not been clearly established. To further investigate this issue, in the present study we performed comprehensive DNA methylation profiling of a well-characterised series of primary GC.

METHODS: The methylation status of 1,421 autosomal CpG sites located within 768 cancer-related genes was investigated using the Illumina GoldenGate Methylation Panel I assay on DNA extracted from 60 gastric tumors and matched tumor-adjacent gastric tissue pairs. Methylation data was analysed using a recursively partitioned mixture model and investigated for associations with clinicopathological and molecular features including age, Helicobacter pylori status, tumor site, patient survival, microsatellite instability and BRAF and KRAS mutations.

RESULTS: A total of 147 genes were differentially methylated between tumor and matched tumor-adjacent gastric tissue, with HOXA5 and hedgehog signalling being the top-ranked gene and signalling pathway, respectively. Unsupervised clustering of methylation data revealed the existence of 6 subgroups under two main clusters, referred to as L (low methylation; 28% of cases) and H (high methylation; 72%). Female patients were over-represented in the H tumor group compared to L group (36% vs 6%; P = 0.024), however no other significant differences in clinicopathological or molecular features were apparent. CpG sites that were hypermethylated in group H were more frequently located in CpG islands and marked for polycomb occupancy.

CONCLUSIONS: High-throughput methylation analysis implicates genes involved in embryonic development and hedgehog signaling in gastric tumorigenesis. GC is comprised of two major methylation subtypes, with the highly methylated group showing some features consistent with a CpG island methylator phenotype.

Regulating macrophage phenotype and function by retinal pigment epithelial cells

Purpose: We have shown previously that macrophages/microglia accumulate in the subretinal space and express CD68 and Arginase-1 in the aging eye. Subretinal macrophages are in close contact with retinal pigment epithelial (RPE) cells. We hypothesize that RPE cells may play an important role in regulating macrophage/microglial phenotype and function. The aim of this study was to investigate the effect of RPE cells on the phenotype and function of bone marrow–derived macrophages (BM-DMs).
Methods: BM-DM from C57BL/6J mice were cultured in DMEM supplemented with 20%L929 cell supernatant for 5 days. The phenotype of BM-DMs was confirmed by flow cytometry as CD11b+F4/80+. Primary RPE cells were cultured from C57BL/6J mice and confirmed by RPE65 and cytokeratin staining. BMDMs were co-cultured with different types of RPE cells (healthy, oxidized, and apoptotic RPE) and then isolated from the co-culture system for phenotypic and functional assays.
Results: Co-culture of BM-DMs with RPE cells results in a time-dependent down-regulation of MHC-II expression and the generation of CD11b+F4/80+Ly6G+ myeloid-derived suppressor cells (MDSC). qRT-PCR analysis showed that RPE-induced MDSCs expressed high levels of IL-6, IL-1β, and Arginase-1, but lower levels of IL-12p40 and TNF-a compared to naïve BM-DMs. The expression levels of iNOS, TGF-β and Ym1 did not differ 207 between naive BMDMs and RPE-induced MDSCs. Furthermore, functional studies showed that these cells had reduced phagocytic activity and lower ability to stimulate T cell activation and proliferation. When RPE cells were pre-treated with oxidized photoreceptor outer segments before co-culturing with BMDMs, the expression of IL-1β and IL-6 in BMDMs was increased whereas the expression of Arginase-1 was decreased. 
Conclusion: Our results suggest that healthy RPE cells can convert BMDMs into myeloid-derived suppressor cells under in vitro culture conditions, RPE-induced myeloid-derived suppressor cells are CD11b+F4/80+Ly6G+MHCIIlowIL6+IL1b+Arg-1+. The ability of RPE cells is reduced when suffering from oxidative insults.

Calibration of BAS-TR image plate response to high energy (3-300 MeV) carbon ions

The paper presents the calibration of Fuji BAS-TR image plate (IP) response to high energy carbon ions of different charge states by employing an intense laser-driven ion source, which allowed access to carbon energies up to 270 MeV. The calibration method consists of employing a Thomson parabola spectrometer to separate and spectrally resolve different ion species, and a slotted CR-39 solid state detector overlayed onto an image plate for an absolute calibration of the IP signal. An empirical response function was obtained which can be reasonably extrapolated to higher ion energies. The experimental data also show that the IP response is independent of ion charge states.

Vitamin D3 suppresses morphological evolution of the cribriform cancerous phenotype

Development of cribriform morphology (CM) heralds malignant change in human colon but lack of mechanistic understanding hampers preventive therapy. This study investigated CM pathobiology in three-dimensional (3D) Caco-2 culture models of colorectal glandular architecture, assessed translational relevance and tested effects of 1,25(OH)2D3, the active form of vitamin D. CM evolution was driven by oncogenic perturbation of the apical polarity (AP) complex comprising PTEN, CDC42 and PRKCZ (phosphatase and tensin homolog, cell division cycle 42 and protein kinase C zeta). Suppression of AP genes initiated a spatiotemporal cascade of mitotic spindle misorientation, apical membrane misalignment and aberrant epithelial configuration. Collectively, these events promoted “Swiss cheese-like” cribriform morphology (CM) comprising multiple abnormal “back to back” lumens surrounded by atypical stratified epithelium, in 3D colorectal gland models. Intestinal cancer driven purely by PTEN-deficiency in transgenic mice developed CM and in human CRC, CM associated with PTEN and PRKCZ readouts. Treatment of PTEN-deficient 3D cultures with 1,25(OH)2D3 upregulated PTEN, rapidly activated CDC42 and PRKCZ, corrected mitotic spindle alignment and suppressed CM development. Conversely, mutationally-activated KRAS blocked 1,25(OH)2D3 rescue of glandular architecture. We conclude that 1,25(OH)2D3 upregulates AP signalling to reverse CM in a KRAS wild type (wt), clinically predictive CRC model system. Vitamin D could be developed as therapy to suppress inception or progression of a subset of colorectal tumors.

Phenotype-Based Discovery of 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol as a Novel Regulator of Ocular Angiogenesis

Retinal angiogenesis is tightly regulated to meet oxygenation and nutritional requirements. In diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, uncontrolled angiogenesis can lead to blindness. Our goal is to better understand the molecular processes controlling retinal angiogenesis and discover novel drugs that inhibit retinal neovascularization. Phenotype-based chemical screens were performed using the ChemBridge Diverset^TM library and inhibition of hyaloid vessel angiogenesis in Tg(fli1:EGFP) zebrafish. 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol, (quininib) robustly inhibits developmental angiogenesis at 4–10 μM in zebrafish and significantly inhibits angiogenic tubule formation in HMEC-1 cells, angiogenic sprouting in aortic ring explants, and retinal revascularization in oxygen-induced retinopathy mice. Quininib is well tolerated in zebrafish, human cell lines, and murine eyes. Profiling screens of 153 angiogenic and inflammatory targets revealed that quininib does not directly target VEGF receptors but antagonizes cysteinyl leukotriene receptors 1 and 2 (CysLT_1–2) at micromolar IC₅₀ values. In summary, quininib is a novel anti-angiogenic small-molecule CysLT receptor antagonist. Quininib inhibits angiogenesis in a range of cell and tissue systems, revealing novel physiological roles for CysLT signaling. Quininib has potential as a novel therapeutic agent to treat ocular neovascular pathologies and may complement current anti-VEGF biological agents.