Optical imaging modalities are emerging as digital microscopy tools for tissue examination. The investigation of these techniques for potential applications in anatomic pathology practice has focused ...primarily on surgical pathology and has not included cytopathological specimens. We evaluated the feasibility of using digital confocal microscopy (CM) to examine cytopathological specimens. Smears and cell suspensions collected in RPMI solution were prepared from tissue scrapes obtained from surgical resections of breast, lung, liver, and kidney. Air-dried smears and cell pellets obtained from centrifugation of the cell suspensions were stained with 0.6 mM acridine orange and imaged with a CM platform. After completion of imaging, the smears were stained with Diff-Quik (DQ), and cell pellets were routinely processed, embedded in paraffin wax, cut, and stained with hematoxylin and eosin (H&E). We evaluated the mean time to acquire digital CM images; quality of images based on the extent of tissue recognition (0%, grade 0; 1-19%, grade 1; 20-50%, grade 2; >50%, grade 3); comparison of images with DQ- and H&E-stained specimens; and ability to make specific diagnoses. We imaged 91 smears and 52 cell pellets and acquired digital CM images within 2-3 min, with 92% and 88% of images, respectively, from smears and cell pellets showing grade 3 quality. On the basis of CM images, 8 smears (9%) and 7 cell pellets (14%) were categorized as benign, and 83 (91%) and 45 (88%), respectively, as malignant. Specific diagnoses were made by using digital CM images of smears and cell pellets that matched accurately with corresponding DQ- and H&E-stained preparations. The results of our first feasibility study clearly indicated the utility of CM as a next-generation digital microscopy tool for evaluating cytology specimens. Prospective clinical studies are warranted for validating our findings for potential incorporation into cytopathological clinical practice.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
RET Signaling in Prostate Cancer Ban, Kechen; Feng, Shu; Shao, Longjiang ...
Clinical cancer research,
08/2017, Volume:
23, Issue:
16
Journal Article
Peer reviewed
Open access
Large diameter perineural prostate cancer is associated with poor outcomes. GDNF, with its coreceptor GFRα1, binds RET and activates downstream pro-oncogenic signaling. Because both GDNF and GFRα1 ...are secreted by nerves, we examined the role of RET signaling in prostate cancer.
Expression of RET, GDNF, and/or GFRα1 was assessed. The impact of RET signaling on proliferation, invasion and soft agar colony formation, perineural invasion, and growth
was determined. Cellular signaling downstream of RET was examined by Western blotting.
RET is expressed in all prostate cancer cell lines. GFRα1 is only expressed in 22Rv1 cells, which is the only line that responds to exogenous GDNF. In contrast, all cell lines respond to GDNF plus GFRα1. Conditioned medium from dorsal root ganglia contains secreted GFRα1 and promotes transformation-related phenotypes, which can be blocked by anti-GFRα1 antibody. Perineural invasion in the dorsal root ganglion assay is inhibited by anti-GFRα antibody and RET knockdown.
, knockdown of RET inhibits tumor growth. RET signaling activates ERK or AKT signaling depending on context, but phosphorylation of p70S6 kinase is markedly increased in all cases. Knockdown of p70S6 kinase markedly decreases RET induced transformed phenotypes. Finally, RET is expressed in 18% of adenocarcinomas and all three small-cell carcinomas examined.
RET promotes transformation associated phenotypes, including perineural invasion in prostate cancer via activation of p70S6 kinase. GFRα1, which is secreted by nerves, is a limiting factor for RET signaling, creating a perineural niche where RET signaling can occur.
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Intravenous tranexamic acid (TXA) is an effective adjunct after hemorrhagic shock (HS) because of its antifibrinolytic properties. TXA is also a serine protease inhibitor, and recent laboratory data ...demonstrated that intraluminal TXA into the small bowel inhibited digestive proteases and protected the gut. A Disintegrin And Metalloproteinase 17 (ADAM-17) and tumor necrosis factor α (TNF-α) are effective sheddases of intestinal syndecan-1, which when shed, exposes the underlying intestinal epithelium to digestive proteases and subsequent systemic insult. We therefore hypothesized that intraluminal TXA as a serine protease inhibitor would reduce intestinal sheddases and syndecan-1 shedding, mitigating gut and distant organ (lung) damage.
Mice underwent 90 minutes of HS to a mean arterial pressure of 35 ± 5 mm Hg followed by the intraluminal administration of TXA or vehicle. After 3 hours, the small intestine, lung, and blood were collected for analysis.
Intraluminal TXA significantly reduced gut and lung histopathologic injury and inflammation compared with HS alone. Gut, lung, and systemic ADAM-17 and TNF-α were significantly increased by HS but lessened by TXA. In addition, gut and lung syndecan-1 immunostaining were preserved and systemic shedding lessened after TXA. TXA reduced ADAM-17 and TNF-α, but not syndecan-1, in TXA-sham animals compared with sham vehicles.
Results of the present study demonstrate a beneficial effect of intraluminal TXA in the gut and lung after experimental HS in part because of the inhibition of the syndecan-1 shedding by ADAM-17 and TNF-α. Further studies are needed to determine if orally administered TXA could provide similar intestinal protection and thus be of potential benefit to patients with survivable hemorrhage at risk for organ injury. This is particularly relevant in patients or soldiers who may not have access to timely medical care.
Providing surgical margin information during breast cancer surgery is crucial for the success of the procedure. The margin is defined as the distance from the tumor to the cut surface of the ...resection specimen. The consensus among surgeons and radiation oncologists is that there should be no tumor left within 1 to maximum 2 mm from the surface of the surgical specimen. If a positive margin remains, there is substantial risk for tumor recurrence, which may also result in potentially reduced cosmesis and eventual need for mastectomy. In this paper we report a novel multimodal optical imaging instrument based on combined high-resolution confocal microscopy-optical coherence tomography imaging for assessing the presence of potential positive margins on surgical specimens. Since rapid specimen analysis is critical during surgery, this instrument also includes a fluorescence imaging channel to enable rapid identification of the areas of the specimen that have potential positive margins. This is possible by specimen incubation with a cancer specific agent prior to imaging. In this study we used a quenched contrast agent, which is activated by cancer specific enzymes, such as urokinase plasminogen activators (uPA). Using this agent or a similar one, one may limit the use of high-resolution optical imaging to only fluorescence-highlighted areas for visualizing tissue morphology at the sub-cellular scale and confirming or ruling out cancer presence. Preliminary evaluation of this technology was performed on 20 surgical specimens and testing of the optical imaging findings was performed against histopathology. The combination of the three imaging modes allowed for high correlation between optical image analysis and histological ground-truth. The initial results are encouraging, showing instrument capability to assess margins on clinical specimens with a positive predictive value of 1.0 and a negative predictive value of 0.83.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The role of extracellular signal-regulated protein kinase (ERK) in intestinal ischemia/reperfusion (I/R) injury has not been well investigated. The aim of the current study was to examine the effect ...of inhibition of the ERK pathway in an in vitro and in vivo model of intestinal I/R injury.
ERK1/2 activity was inhibited using the specific inhibitor, U0126, in intestinal epithelial cells under hypoxia/reoxygenation conditions and in mice subjected to 1 hour of intestinal ischemia followed by 6 hours reperfusion. In vitro, cell proliferation was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, apoptosis by DNA fragmentation, and migration using an in vitro model of intestinal wound healing. Cells were also transfected with a p70S6K plasmid and the effects of overexpression similarly analyzed. In vivo, the effects of U0126 on intestinal cell proliferation and apoptosis, intestinal permeability, lung and intestinal neutrophil infiltration and injury, and plasma cytokine levels were measured. Survival was also assessed after U0126. Activity of p70S6 kinase (p70S6K) was measured by Western blot.
In vitro, inhibition of ERK1/2 by U0126 significantly decreased cell proliferation and migration but enhanced cell apoptosis. Overexpression of p70S6K promoted cell proliferation and decreased cell apoptosis. In vivo, U0126 significantly increased cell apoptosis and decreased cell proliferation in the intestine, increased intestinal permeability, intestinal and lung neutrophil infiltration, and injury, as well as systemic pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β. Mortality was also significantly increased by U0126. Inhibition of ERK1/2 by U0126 also abolished activity of p70S6K both in vitro and in vivo models.
Pharmacologic inhibition of ERK1/2 by U0126 worsens intestinal IR injury. The detrimental effects are mediated, at least in part, by inhibition of p70S6K, the major effector of mammalian target of rapamycin pathway.
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ABSTRACTWe have shown in a rodent model of hemorrhagic shock (HS) that fresh frozen plasma (FFP) reduces lung inflammation and injury that are correlated with restitution of syndecan-1. As the gut is ...believed to contribute to distant organ injury and inflammation after shock, the current study sought to determine if the protective effects of plasma would extend to the gut and to elucidate the contribution of syndecan-1 to this protective effect. We also examined the potential role of TNFα, and a disintegrin and metalloproteinase (ADAM)-17, both intestinal sheddases of syndecan-1. Wild-type (WT) and syndecan-1 (KO) mice were subjected to HS followed by resuscitation with lactated Ringerʼs (LR) or FFP and compared with shock alone and shams. Small bowel and blood were obtained after 3 h for analysis of mucosal injury and inflammation and TNFα and ADAM-17 protein expression and activity. After HS, gut injury and inflammation were significantly increased compared with shams. Resuscitation with LR decreased both injury and inflammation that were further lessened by FFP. KO mice displayed worsened gut injury and inflammation after HS compared with WT mice, and LR and FFP equivalently inhibited injury and inflammation. Both systemic and intestinal TNFα and ADAM-17 followed similar trends, with increases after HS, reduction by LR, and a further decrease by FFP in WT but not KO mice. In conclusion, FFP decreased gut injury and inflammation after hemorrhagic shock, an effect that was abrogated in syndecan-1 mice. Plasma also decreased TNFα and ADAM-17, representing a potential mechanistic link to its protection via syndecan-1.
The mTOR signaling pathway plays a crucial role in the regulation of cell growth, proliferation, survival and in directing immune responses. As the intestinal epithelium displays rapid cell growth ...and differentiation and is an important immune regulatory organ, we hypothesized that mTOR may play an important role in the protection against intestinal ischemia reperfusion (I/R)-induced injury. To better understand the molecular mechanisms by which the mTOR pathway is altered by intestinal I/R, p70S6K, the major effector of the mTOR pathway, was investigated along with the effects of rapamycin, a specific inhibitor of mTOR and an immunosuppressant agent used clinically in transplant patients. In vitro experiments using an intestinal epithelial cell line and hypoxia/reoxygenation demonstrated that overexpression of p70S6K promoted cell growth and migration, and decreased cell apoptosis. Inhibition of p70S6K by rapamycin reversed these protective effects. In a mouse model of gut I/R, an increase of p70S6K activity was found by 5 min and remained elevated after 6 h of reperfusion. Inhibition of p70S6K by rapamycin worsened gut injury, promoted inflammation, and enhanced intestinal permeability. Importantly, rapamycin treated animals had a significantly increased mortality. These novel results demonstrate a key role of p70S6K in protection against I/R injury in the intestine and suggest a potential danger in using mTOR inhibitors in patients at risk for gut hypoperfusion.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Glutamine plays a key role in intestinal growth and maintenance of gut function, and as we have shown protects the postischemic gut (Kozar RA, Scultz SG, Bick RJ, Poindexter BJ, Desoigne R, Weisbrodt ...NW, Haber MM, Moore FA. Shock 21: 433-437, 2004). However, the precise mechanisms of the gut protective effects of glutamine have not been well elucidated. In the present study, RNA microarray was performed to obtain differentially expressed genes in intestinal epithelial IEC-6 cells following either 2 mM or 10 mM glutamine. The result demonstrated that specificity protein 3 (Sp3) mRNA expression was downregulated 3.1-fold. PCR and Western blot confirmed that Sp3 expression was decreased by glutamine in a time- and dose-dependent fashion. To investigate the role of Sp3, Sp3 gene siRNA silencing was performed and apoptosis was assessed. Silencing of Sp3 demonstrated a significant increase in Bcl-2 and decrease in Bax protein expression, as well as a decrease in caspase-3, -8, and -9 protein expression and activity. The protein expression of apoptosis-related proteins after hypoxia/reoxygenation was similar to that of normoxia and correlated with a decrease in DNA fragmentation. Importantly, the addition of glutamine to Sp3-silenced cells did not further lessen apoptosis, suggesting that Sp3 plays a major role in the inhibitory effect of glutamine on apoptosis. This novel finding may explain in part the gut-protective effects of glutamine.
The transcription factor Forkhead box M1 (FoxM1) is overexpressed in malignant glioma. However, the functional importance of this factor in human glioma is not known. In the present study, we found ...that FoxM1B was the predominant FoxM1 isoform expressed in human glioma but not in normal brain tissue. The level of FoxM1 protein expression in human glioma tissues was directly correlated with the glioma grade. The level of FoxM1 protein expression in human glioblastoma tissues was inversely correlated with patient survival. Enforced FoxM1B expression caused SW1783 and Hs683 glioma cells, which do not form tumor xenografts, to regain tumorigenicity in nude mouse model systems. Moreover, gliomas that arose from FoxM1B-transfected anaplastic astrocytoma SW1783 cells displayed glioblastoma multiforme phenotypes. Inhibition of FoxM1 expression in glioblastoma U-87MG cells suppressed their anchorage-independent growth in vitro and tumorigenicity in vivo. Furthermore, we found that FoxM1 regulates the expression of Skp2 protein, which is known to promote degradation of the cell cycle regulator p27(Kip1). These results showed that FoxM1 is overexpressed in human glioblastomas and contributes to glioma tumorigenicity. Therefore, FoxM1 might be a new potential target of therapy for human malignant gliomas.
Signaling initiated by the BCR-ABL1 kinase causes chronic myelogenous leukemia (CML). Recently, we reported that expression of Fyn, a Src kinase, is heightened in CML cells and patient specimens and ...confers in vitro and in vivo proliferative advantages. Fyn is regulated by redox, and because BCR-ABL1 raises intracellular oxidant levels, which have been implicated in CML progression, we explored the molecular regulation of Fyn. Here we identify the transcription factors that drive redox- and BCR-ABL1-dependent Fyn expression. Promoter deletion analysis in 293T, BaF3, BaF3-p210, and K562 cells identified the region essential for basal transcriptional activity. Mutation of Sp1 and Egr1 binding sites within the essential region diminished Fyn promoter activity and identified Egr1 as conferring redox sensitivity. Gel shift and chromatin immunoprecipitation assays confirmed the binding of Sp1 and Egr1 to the promoter fragments. Importantly, knockdown of Sp1 or Egr1 with small interference RNA or inhibition of Sp1 binding by mithramycin A repressed Fyn protein expression. Our work is the first to define transcription factors that are responsible for endogenous, oxidative stress-dependent and BCR-ABL1-dependent Fyn expression.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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