Assessing venous thrombus in renal cell carcinoma: preliminary results for unenhanced 3D-SSFP MRI.

AIM: To test the potential of unenhanced cardiac- and respiratory-motion-corrected three-dimensional steady-state free precession (3D-SSFP) magnetic resonance imaging (MRI) for the assessment of inferior vena cava (IVC) thrombus in patients with clear-cell renal cell carcinoma (cRCC), compared to standard contrast-enhanced (CE)-MRI and CE-computed tomography (CT). MATERIALS AND METHODS: Eighteen patients with cRCC and IVC thrombus, who received CE-MRI and 3D-SSFP at 1.5 T between June 2015 and December 2017, were included. The diagnostic performance of 3D-SSFP in determining the level of thrombus extension, contrast-to-noise ratio (CNR), and image quality were compared with standard MRI/CT and validated against intraoperative and histopathology results. RESULTS: There was 100% agreement between 3D-SSFP, 83.3% agreement between CE-MRI, and 71.4% agreement between CE-CT and surgical findings regarding the level of IVC thrombus. In addition, 3D-SSFP showed a slightly superior estimate of pathological IVC volume. 3D-SSFP reached a significantly higher CNR in the supra- and infrarenal IVC compared to the morphological sequence T2-weighted half-Fourier axial single-shot fast spin-echo (T2-HASTE) and all phases of CE-MRI. More specifically, 3D-SSFP showed a significantly higher CNR in the infrarenal IVC (mean CNR of 10.09±5.74 versus 4.21±2.33 in the delayed phase, p≤0.001) and in the suprarenal IVC (mean CNR of 9.22±4.11 versus 4.84±5.74 in the late arterial phase, p=0.015). CE-CT also was significantly inferior to 3D-SSFP (p≤0.01) and slightly inferior to CE-MRI (p>0.05). The thrombus delineation score for 3D-SSFP (4.38±0.67) was higher compared to CE-MRI (3.76±0.56, p=0.005). CONCLUSION: This preliminary study indicates that 3D-SSFP can achieve an accurate assessment of IVC thrombus in cRCC patients without the need for contrast medium administration, being superior to standard MRI and CT.

Distinguishing globally-driven changes from regional- and local-scale impacts: The case for long-term and broad-scale studies of recovery from pollution.

Marine ecosystems are subject to anthropogenic change at global, regional and local scales. Global drivers interact with regional- and local-scale impacts of both a chronic and acute nature. Natural fluctuations and those driven by climate change need to be understood to diagnose local- and regional-scale impacts, and to inform assessments of recovery. Three case studies are used to illustrate the need for long-term studies: (i) separation of the influence of fishing pressure from climate change on bottom fish in the English Channel; (ii) recovery of rocky shore assemblages from the Torrey Canyon oil spill in the southwest of England; (iii) interaction of climate change and chronic Tributyltin pollution affecting recovery of rocky shore populations following the Torrey Canyon oil spill. We emphasize that "baselines" or "reference states" are better viewed as envelopes that are dependent on the time window of observation. Recommendations are made for adaptive management in a rapidly changing world.

Suppressed expression of tubedown-1 in retinal neovascularization of proliferative diabetic retinopathy.

PURPOSE: Retinal neovascularization occurring as a complication of diabetes mellitus can cause vision loss and blindness. The identification and study of novel genes involved in retinal angiogenesis may define new targets to suppress retinal neovascularization in diabetes and other ocular diseases. A novel acetyltransferase subunit, tubedown-1 (tbdn-1), has been isolated, the expression of which is regulated during blood vessel development. Tbdn-1 is not detected in most adult vascular beds but persists at high levels in the adult ocular vasculature. The purpose of this study was to gain insight into the possible role of tbdn-1 in retinal blood vessels by characterizing its expression patterns in adult homeostasis and in retinal neovascularization associated with diabetes. METHODS: Western blot analysis and immunohistochemistry were performed to study the expression patterns of tbdn-1 during adult homeostasis in normal human retinas, in a model of choroid-retina endothelial capillary outgrowth in vitro, and in retinas showing neovascularization in patients with proliferative diabetic retinopathy (PDR). RESULTS: In adults during homeostasis, tbdn-1 was expressed highly in normal endothelium of retinal and limbic blood vessels. Tbdn-1 was also expressed in RF/6A, a rhesus macaque choroid-retina-derived endothelial cell line. In an in vitro model system using the RF/6A cell line, tbdn-1 expression was downregulated during the outgrowth of these cells into capillary-like structures on a reconstituted basement membrane matrix. Similar to this in vitro model, tbdn-1 expression is specifically suppressed in the endothelial cells of blood vessels and capillary fronds in vivo in both the neural retinal tissue and in preretinal membranes in eyes of patients with PDR. CONCLUSIONS: High levels of expression of tbdn-1 are associated with ocular endothelial homeostasis in adults. Conversely, low levels of tbdn-1 expression are associated with endothelial capillary outgrowth in vitro and retinal neovascularization in vivo. Because the tbdn-1 acetyltransferase subunit is a member of a family of regulatory enzymes that are known to control a range of processes, including cell growth and differentiation, through posttranslational modification, the current results support a hypothesis that tbdn-1 may be involved in maintaining homeostasis and preventing retinal neovascularization.

MK/T-1, an immortalized fibroblast cell line derived using cultures of mouse corneal stroma.

PURPOSE: Immortalized cell lines representing fibroblast cells from corneal stroma would facilitate studies of corneal cell biology and injury response. METHODS: Primary cultures of cells derived from mouse corneal stroma were transfected with a human telomerase reverse transcriptase (hTERT) expression construct to maximize chances of cellular immortalization. A resulting cell line was analyzed for telomerase activity, cell growth characteristics, senescence and gene expression patterns. Specific responses to transforming growth factor beta (TGF-beta) were also analyzed. RESULTS: An immortalized cell line was derived and was named MK/T-1. MK/T-1 cells show no signs of cellular senescence or transformation at over 100 passages. Telomerase activity was significantly higher in MK/T-1 cells as compared to the parental cell cultures. However, relative telomere length (RTL) in the MK/T-1 and parental cells was not significantly different. Senescence associated beta-galactosidase (SA-beta-Gal) activity was not detected in late passage MK/T-1 cells while the parental cells had already upregulated SA-beta-Gal at high levels by passage 9. The MK/T-1 cells express vimentin, tubulin, lumican, mimecan, decorin and collagen I, but not keratocan. Exposure of the MK/T-1 cells to TGF-beta induces the expression of smooth muscle alpha-actin (ASMA), the activation of MAP Kinase (p38-MAPK) and morphological changes consistent with cytoskeletal reorganization. CONCLUSIONS: MK/T-1 cells represent an immortalized fibroblast cell line derived using cultures from corneal stroma cell preparations. Expression of hTERT may contribute to immortalization of the MK/T-1 cells by a mechanism other than increases in RTL. MK/T-1 cells may be a useful model in which to study the responses of corneal fibroblast cells to cytokines and other diverse environmental factors in vitro.

Correlating differences in larval survival and development of bollworm (Lepidoptera: Noctuidae) and fall armyworm (Lepidoptera: Noctuidae) to differential expression of Cry1A(c) delta-endotoxin in various plant parts among commercial cultivars of transgenic Bacillus thuringiensis cotton.

Differences in larval survival and development of bollworm, Helicoverpa zea (Boddie), and fall armyworm, Spodoptera frugiperda (J. E. Smith), respectively, were found to exist among commercially available Cry1A(c) transgenic Bacillus thuringiensis Berliner (Bt) varieties. Using a quantification assay (ELISA) to measure the levels of delta-endotoxin in two of these varieties ('DP 451B/RR' and 'NuCOTN 33B'), differences in the amount of delta-endotoxin present in various plant parts was correlated with larval survival of bollworms and larval development of fall armyworms throughout the growing season. Larvae that were fed on DP 451B/RR completed development faster and exhibited better survivorship than those larvae fed NuCOTN 33B, whereas lower levels of delta-endotoxin were generally detected in plant parts from DP 451B/RR compared with NuCOTN 33B. These differences may impact population dynamics of these pests which may be a critical factor in managing resistance to Bt. Furthermore, the utility of using this system for providing information to the grower concerning varietal choices may be more common in the future.

Field efficacy and seasonal expression profiles for terminal leaves of single and double Bacillus thuringiensis toxin cotton genotypes.

Examination of commercial Cry1Ac transgenic Bacillus thuringiensis Berliner (Bt) cotton varieties (Bollgard, Monsanto, St. Louis, MO) and an experimental Cry1Ac + Cry2Ab transgenic Bt cotton variety (Bollgard II, Monsanto) for lepidopteran field efficacy was conducted during the 2000 growing season. In addition, a commercially available (Envirologix, Portland, ME) quantification assay (ELISA) was used to measure and profile the expression levels of Cry proteins in two of these varieties ['DP50B, Bollgard'; 'DP50BII, Bollgard II' (Delta & Pine Land, Scott, MS)]. Populations of beet army worms, Spodoptera exigua (Hübner), and soybean loopers, Pseudoplusia includens (Walker), were significantly lower (P < 0.05) in Bollgard II plots compared with Bollgard. Population numbers for fall army worms, Spodoptera frugiperda (J. E. Smith), and salt marsh caterpillars, Estigmene acrea (Drury), were lower in Bollgard II plots compared with Bollgard but means did not differ significantly (P > 0.05). Single and dual-toxin genotypes remained superior (P < 0.05) compared with conventional cotton against the tobacco budworm, Heliothis virescens (F.). The addition of Cry2Ab had no significant (P > 0.05) impact on Cry1Ac expression in Bollgard II compared with Cry1Ac expression in Bollgard. Furthermore, throughout the season Cry2Ab was present at much higher levels in the plant compared with Cry1Ac for Bollgard II plants. Possible species-specific reasons for increased efficacy of Bollgard II over Bollgard are discussed.

Tubedown-1, a novel acetyltransferase associated with blood vessel development.

We have used an embryonic endothelial cell line (IEM cells) as an experimental system for identifying and characterizing new molecules which are regulated during blood vessel development. A novel gene isolated from IEM cells, tubedown-1 (tbdn-1), is expressed at high levels in unstimulated IEM cells and is downregulated during formation of capillary tube structures by the IEM cells induced by basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) in vitro. Tbdn-1 is also downregulated in M1 myeloid leukemia cells after differentiation in response to LIF in vitro. Tbdn-1 is homologous to the yeast NAT-1 N-terminal acetyltransferases and encodes a novel protein of approximately 69 kDa associated with an acetyltransferase activity. Levels and distribution of tbdn-1 expression are regulated in both endothelial and hematopoietic cells during development in tissues such as the yolk sac blood islands, heart, and liver blood vessels. In the adult, tbdn-1 expression is low or undetected in most organs examined with the exception of the atrial endocardium, the endothelial and myeloid compartments of bone marrow, and the remodeling vascular bed of atretic ovarian follicles. The distribution and regulation of expression of tbdn-1 suggest that this novel acetyltransferase may be involved in regulating vascular and hematopoietic development and physiologic angiogenesis.

The use of single cell gel electrophoresis and flow cytometry to identify antimutagens from commercial soybean by-products.

Single cell gel electrophoresis (alkaline Comet assay) and flow cytometric methods were combined into an assay that enables the analysis of direct DNA damage and longer-term whole cell clastogenicity in mammalian cells. We employed these techniques to analyze the antimutagenic activity of by-products of commercial soybean processing. At a concentration of 1 mg/ml, the soybean molasses by-product was found to repress 66% of the mutagenic capacity of the direct-acting mutagen 2-acetoxyacetylaminofluorene (2AAAF) in Chinese hamster lung (CHL) cells. At a concentration of 50 microg/ml, fraction PCC (an ethanol extract of soybean molasses) repressed 70% of the genotoxic potency of 500 nM 2AAAF as measured by the Comet assay. Fraction PCC was also effective in protecting CHL cells from 2AAAF-induced clastogenic damage. Using a forward mutation assay in Chinese hamster ovary cells (line AS52), PCC protected the cells against 2AAAF-induced cytotoxicity and point mutation at a specific gene target. These data indicate that agronomic crops such as soybean may yield a wealth of commercially available antimutagenic agents that may be suitable as chemoprotective food supplements.

Differentiation responses of embryonic endothelium to leukemia inhibitory factor.

The IEM cell line is a murine embryonic endothelial cell line that responds to combinations of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) by undergoing proliferation and vasculogenic differentiation in vitro and in vivo. Exposure to LIF and bFGF in vitro permits the IEM cells to specifically chimerize endothelium in vivo and recapitulate normal endothelial development after blastocyst injection. We report here that unmanipulated IEM cells form vascular neoplasias when injected into immunodeficient nude mice. Examination of IEM neoplasia following exposure in vitro to bFGF and LIF before injection into nude mice profoundly reduced or completely suppressed the neoplastic growth of IEM cells. Furthermore, this suppression was observed by treatment with LIF alone, while bFGF treatment did not significantly alter IEM neoplasia and did not modify the LIF-mediated suppression. Characterization of the IEM responses to LIF revealed that the LIF suppression of IEM neoplasia depended on how long the cells were exposed to LIF in vitro. The IEM cell response to LIF was associated with the specific activation of the transcription factor Stat3. Stat1 activation could not be detected in response to LIF, although it is expressed in IEM cells. Our results demonstrate that the LIF-induced differentiation of IEM cells involves suppression of IEM-derived neoplasia and is associated with the specific activation of Stat3.