Dynamic expression of Schlafen 11 (SLFN11) in circulating tumour cells as a liquid biomarker in small cell lung cancer.

INTRODUCTION: Small cell lung cancer (SCLC) is an aggressive malignancy with no established biomarkers. Schlafen 11(SLFN11), a DNA/RNA helicase that sensitises cancer cells to DNA-damaging agents, has emerged as a promising predictive biomarker for several drug classes including platinum and PARP inhibitors. Detection of SLFN11 in circulating tumour cells (CTCs) may provide a valuable alternative to tissue sampling. METHODS: SLFN11 expression was evaluated in tumour samples and characterised in circulating tumour cells (CTC) longitudinally to determine its potential role as a biomarker of response. RESULTS: Among 196 SCLC tumours, 51% expressed SLFN11 by IHC. In addition, 20/29 extra-thoracic high-grade neuroendocrine tumours expressed SLFN11 expression. In 64 blood samples from 42 SCLC patients, 83% (53/64) of samples had detectable CTCs, and SLFN11-positive CTCs were detected in 55% (29/53). Patients actively receiving platinum treatment had the lowest number of CTCs and a lower percentage of SLFN11-positive CTCs (p = 0.014). Analysis from patients with longitudinal samples suggest a decrease in CTC number and in SLFN11 expression that correlates with clinical response. CONCLUSIONS: SLFN11 levels can be monitored in CTCs from SCLC patients using non-invasive liquid biopsies. The ability to detect SLFN11 in CTCs from SCLC patients adds a valuable tool for the detection and longitudinal monitoring of this promising biomarker.

Cell proliferation in human epiretinal membranes: characterization of cell types and correlation with disease condition and duration.

PURPOSE: To quantify the extent of cellular proliferation and immunohistochemically characterize the proliferating cell types in epiretinal membranes (ERMS) from four different conditions: proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy, post-retinal detachment, and idiopathic ERM. METHODS: Forty-six ERMs were removed from patients undergoing vitrectomy and immediately fixed in paraformaldehyde. The membranes were processed whole and immunolabeled with either anti-MIB-1 or anti-SP6 to detect the K(i)-67 protein in proliferating cells, in combination with anti-glial fibrillary acidic protein or anti-vimentin to identify glia, anti-ezrin to identify retinal pigment epithelial cells, Ricinus communis to identify immune cells, and Hoechst to label nuclei. Digital images were collected using a laser scanning confocal microscope. The cell types were identified, their combined proliferative indices were tabulated as the average number of anti-K(i)-67-positive cells/mm(2) of tissue, and the number of dividing cells was related to the specific ocular condition and estimated disease duration. RESULTS: ERMs of all four types were shown to be highly cellular and contained proliferating cells identified as glia, retinal pigment epithelium, and of immune origin. In general, membranes identified as PVR had many more K(i)-67-positive cells in comparison to those in the other three categories, with the average number of K(i)-67-positive cells identified per mm(2) of tissue being 20.9 for proliferative diabetic retinopathy, 138.3 for PVR, 12.2 for post-retinal detachment, and 19.3 for idiopathic ERM. While all membrane types had dividing cells, their number was a relatively small fraction of the total number of cells present. CONCLUSIONS: The four ERM types studied demonstrated different cell types actively dividing at the time of removal, confirming that proliferation is a common event and does continue over many months. The low number of dividing cells at the time of removal in comparison to the total number of cells present, however, is an indicator that proliferation alone may not be responsible for the problems observed with the ERMs. Treatment strategies may need to take into consideration the timing of drug administration, as well as the contractile and possibly the inflammatory characteristics of the membranes to prevent the ensuing effects on the retina.

The effects of transient retinal detachment on cavity size and glial and neural remodeling in a mouse model of X-linked retinoschisis.

PURPOSE: To determine the cellular consequences of retinal detachment in retinoschisin knockout (Rs1-KO) mice, a model for retinoschisin in humans. METHODS: Experimental retinal detachments (RDs) were induced in the right eyes of both Rs1-KO and wild-type (wt) control mice. Immunocytochemistry was performed on retinal tissue at 1, 7, or 28 days after RD with antibodies to anti-GFAP, -neurofilament, and -rod opsin to examine cellular changes after detachment. Images of the immunostained tissue were captured by laser scanning confocal microscopy. Quantitative analysis was performed to measure the number of Hoechst-stained photoreceptor nuclei and their density, number, and size of inner retinal cavities, as well as the number of subretinal glial scars. RESULTS: Since detachments were created with balanced salt solution, by examination, all retinas had spontaneously reattached by 1 day. Cellular responses common to many photoreceptor degenerations occurred in the nondetached retinas of Rs1-KO mice, and, of importance, RD did not appear to significantly accentuate these responses. The number of schisis cavities was not changed after detachment, but their size was reduced. CONCLUSIONS: These data indicate that large short-term RD in Rs1-KO mice, followed by a period of reattachment may cause a slight increase in photoreceptor cell death, but detachments do not accentuate the gliosis and neurite sprouting already present and may in fact reduce the size of existing retinal cavities. This finding suggests that performing subretinal injections to deliver therapeutic agents may be a viable option in the treatment of patients with retinoschisis without causing significant cellular damage to the retina.

Muller cell reactivity and photoreceptor cell death are reduced after experimental retinal detachment using an inhibitor of the Akt/mTOR pathway.

PURPOSE: To test the effect of Palomid 529, an inhibitor of the Akt/mTOR pathway, on Müller cell proliferation, subretinal glial scar formation, and photoreceptor survival after experimental retinal detachment (RD). METHODS: Palomid 529 (600 microg) in balanced salt solution or balanced salt solution alone was injected intravitreally immediately after RD into the right eyes of 12 rabbits. Ten micrograms of BrdU was injected intravitreally on day 3. Animals were killed on day 3 or 7, at which time retinal sections were labeled with anti-BrdU to detect dividing cells, with anti-vimentin to identify Müller cells, and with the isolectin B4 to identify microglia and macrophages. Outer nuclear layer (ONL) thickness was measured from fluorescence-labeled nuclear-stained sections. Labeling was imaged using confocal microscopy. Six additional animals received either drug or balanced salt solution injections into normal eyes, and paraffin sections were stained with hematoxylin and eosin. RESULTS: In the drug-treated eyes there was a significant decrease in the number of anti-BrdU-labeled Müller cells, the number and size of subretinal scars, and the number of isolectin B4-labeled cells. The ONL was also significantly thicker, and there was no evidence of toxic effects. CONCLUSIONS: Palomid 529 is an effective suppressor of Müller cell proliferation, glial scar formation, and photoreceptor cell death in a rabbit model of RD. This suggests that inhibiting the Akt/mTOR signal transduction pathway may be an effective strategy to decrease proliferation and photoreceptor cell death induced by detachment and perhaps represents a novel therapy for related human diseases such as proliferative vitreoretinopathy.

"Columnlike" structure of the cross-sectional morphology of bulk heterojunction materials.

The cross-sectional morphology of the bulk heterojunction (BHJ) films comprising regio-regular poly(3-hexylthiophene) (rrP3HT) and [6,6]-phenyl-C61 butyric acid methyl ester (PCBM) was observed with transmission electron microscopy (TEM). The cross-sectional TEM images of the BHJ film provide information on the pathways for charge transport through the film thickness. The length scale of the phase separation was obtained from spatial Fourier transform analysis of the TEM images and from calculations of the autocorrelation function.

Abnormal reactivity of muller cells after retinal detachment in mice deficient in GFAP and vimentin.

PURPOSE: To determine the roles of glial fibrillary acidic protein (GFAP) and vimentin in Müller cell reactivity. METHODS: Retinal detachments were created in mice deficient for GFAP and vimentin (GFAP(-/-)vim(-/-)) and age-matched wild-type (wt) mice. The reactivity of the retina was studied by immunofluorescence and electron microscopy. RESULTS: Müller cell morphology was different and glutamine synthetase immunoreactivity was reduced in the undisturbed GFAP(-/-)vim(-/-) retinas. After retinal detachment, Müller cells formed subretinal glial scars in the wt mice. In contrast, such scars were not observed in GFAP(-/-)vim(-/-) mice. Müller cells, which normally elongate and thicken in response to detachment, appeared compressed, thin, and "spikey" in the GFAP(-/-)vim(-/-) mice. The end foot region of Müller cells in the GFAP(-/-)vim(-/-) mice often sheared away from the rest of the retina during detachment, corroborating earlier results showing decreased resistance of this region in GFAP(-/-)vim(-/-) retinas to mechanical stress. In regions with end foot shearing, ganglion cells showed intense neurite sprouting, as revealed by anti-neurofilament labeling, a response rarely observed in wt mice. CONCLUSIONS: Müller cells are subtly different in the GFAP(-/-)vim(-/-) mouse retina before detachment. The end foot region of these cells may be structurally reinforced by the presence of the intermediate filament cytoskeleton, and our data suggest a critical role for these proteins in Müller cell reaction to retinal detachment and participation in subretinal gliosis.

Automated tool for the detection of cell nuclei in digital microscopic images: application to retinal images.

PURPOSE: To develop an automated tool that provides reliable, consistent, and accurate results for counting cell nuclei in tissue sections. METHODS: We propose a novel method based on an image processing algorithm to analyze large sets of digital micrographs. The nucleus detector design is based on a Laplacian of Gaussian filter. We use the leave-one-out cross validation method for estimating the generalization error, which is then used to choose the model and parameters of the proposed nucleus detector with both fluorescent and dye stained images. We also evaluate the performance of a nucleus detector by comparing the results with manual counts. RESULTS: When our nucleus detector is applied to previously unanalyzed images of feline retina, it correctly counts nuclei within the outer nuclear layer (ONL) with an average error of 3.67% ranging from 0 to 6.07%, and nuclei within the inner nuclear layer (INL) with an average error of 8.55% ranging from 0 to 13.76%. Our approach accurately identifies the location of cell bodies. Even though we have a relatively large error in the INL due to the large intra-observer variation, both manual counting and nucleus detector result in the same conclusion. This is the first time that cell death in the INL in response to retinal detachment is analyzed quantitatively. We also test the proposed tool with various images and show that it is applicable to a wide range of image types with nuclei varying in size and staining intensity. CONCLUSIONS: The proposed method is simple and reliable. It also has widespread applicability to a variety of sample preparation and imaging methods. Our approach will be immediately useful in quantifying cell number in large sets of digital micrographs and from high-throughput imaging. The tool is available as a plug-in for Image J.

Fibroblast growth factor-2 converts PACAP growth action on embryonic hindbrain precursors from stimulation to inhibition.

Pituitary adenylyl cyclase activating peptide (PACAP) has been shown either to stimulate or to inhibit neural cell proliferation depending on the origin of the cell population. We show here that, depending on the presence or absence of fibroblast growth factor-2 (FGF-2, also called basic FGF), PACAP may either stimulate or inhibit DNA synthesis in neural precursors isolated from embryonic day 10.5 mouse hindbrain. In the absence of FGF-2, PACAP stimulated 3H-thymidine incorporation in a dose-dependent manner. This stimulatory action was unaffected by antagonists of protein kinases A and C but was abolished in the presence of the MEK1/2 antagonist PD98059. In contrast, when FGF-2 was present, PACAP inhibited DNA synthesis. This inhibitory action was insensitive to PD98059 but was fully blocked by the protein kinase A (PKA) inhibitor H89. The differential blockades by MEK1/2 and PKA inhibitors indicate that the FGF-2-induced switch in PACAP action on DNA synthesis was accomplished by a change in PACAP signaling pathways. We hypothesize that the actions of PACAP in the specific parts of the developing nervous system are determined in part by the presence or absence of FGFs and other growth factors.