Slow degradation in phagocytic astrocytes can be enhanced by lysosomal acidification.

Inefficient lysosomal degradation is central in the development of various brain disorders, but the underlying mechanisms and the involvement of different cell types remains elusive. We have previously shown that astrocytes effectively engulf dead cells, but then store, rather than degrade the ingested material. In the present study we identify reasons for the slow digestion and ways to accelerate degradation in primary astrocytes. Our results show that actin-rings surround the phagosomes for long periods of time, which physically inhibit the phago-lysosome fusion. Furthermore, astrocytes express high levels of Rab27a, a protein known to reduce the acidity of lysosomes by Nox2 recruitment, in order to preserve antigens for presentation. We found that Nox2 colocalizes with the ingested material, indicating that it may influence antigen processing also in astrocytes, as they express MHC class II. By inducing long-time acidification of astrocytic lysosomes using acidic nanoparticles, we could increase the digestion of astrocyte-ingested, dead cells. The degradation was, however, normalized over time, indicating that inhibitory pathways are up-regulated in response to the enhanced acidification. GLIA 2015;63:1997-2009.

Telomerase antagonist imetelstat inhibits esophageal cancer cell growth and increases radiation-induced DNA breaks.

Telomerase is mainly active in human tumor cells, which provides an opportunity for a therapeutic window on telomerase targeting. We sought to evaluate the potential of the thio-phosphoramidate oligonucleotide inhibitor of telomerase, imetelstat, as a drug candidate for treatment of esophageal cancer. Our results showed that imetelstat inhibited telomerase activity in a dose-dependent manner in esophageal cancer cells. After only 1 week of imetelstat treatment, a reduction of colony formation ability of esophageal cancer cells was observed. Furthermore, long-term treatment with imetelstat decreased cell growth of esophageal cancer cells with different kinetics regarding telomere lengths. Short-term imetelstat treatment also increased γ-H2AX and 53BP1 foci staining in the esophageal cancer cell lines indicating a possible induction of DNA double strand breaks (DSBs). We also found that pre-treatment with imetelstat led to increased number and size of 53BP1 foci after ionizing radiation. The increase of 53BP1 foci number was especially pronounced during the first 1h of repair whereas the increase of foci size was prominent later on. This study supports the potential of imetelstat as a therapeutic agent for the treatment of esophageal cancer.

Tissue microarray and digital image analysis: a methodological study with special reference to the microenvironment in Hodgkin lymphoma.

AIM: Cancer research has moved from solely investigating the tumour cells to also including analysis of the tumour microenvironment; however, the methods utilized have not been evaluated for this change. The aim of this study was to compare tissue microarrays (TMA) to whole tissue sections (WS) with regard to cells in the tumour microenvironment. Manual evaluation and digital image analyses (DIA) were utilized and also compared. METHODS AND RESULTS: TMA slides from 117 Hodgkin lymphoma patients were immunostained for forkhead box protein 3 (FoxP3) [identifying regulatory T cells (T(reg) )], and 39 corresponding WS were also analysed. Manual evaluation and DIA were utilized for all patients on both the TMA and the WS. A correlation coefficient of 0.83 was obtained for the proportion of T(reg) in TMA versus WS using manual evaluation and a correlation coefficient of 0.77 with DIA. T(reg) counts using manual evaluation correlated in turn with DIA, with a coefficient of 0.79 for the 117 TMA sections and 0.65 for the 39 WS. CONCLUSION: Because a high correlation was observed between TMA and WS, TMA can be utilized when evaluating cells in the tumour microenvironment. DIA appears to provide a reliable measurement method, provided that manual control of the tumour slides is conducted.

Predictors of histology, tissue eosinophilia and mast cell infiltration in Hodgkin's lymphoma--a population-based study.

OBJECTIVE: Classical Hodgkin's lymphoma (HL) lesions comprise few tumour cells, surrounded by numerous inflammatory cells. Like in other malignancies, the microenvironment is presumed to be clinically important in HL; however, microenvironment predictors remain poorly characterised. The aim of this study was to investigate how selected patient characteristics and genetic factors affect HL phenotype, in particular tissue eosinophilia, mast cell counts and HL histological subtype. METHODS: In a population-based study, patients with HL were interviewed about potential HL risk factors. Available tumours, n=448, were classified histologically; the number of eosinophils and mast cells were estimated, and eosinophil cationic protein (ECP) and eosinophil protein-x (EPX) gene polymorphisms were determined. Associations were assessed in regression models. RESULTS: Self-reported history of asthma was predictive of having tumour eosinophilia [≥200 eosinophils/10 high power fields, univariate odds ratio (OR)=2.22, 95% CI 1.06-4.64, P=0.03]. High numbers of eosinophils were predominantly seen in patients carrying the genotype ECP434GG [multivariate relative levels (RLs)=1.84, 95% CI 1.02-3.30, P=0.04]. Lower number of eosinophils was seen in Epstein-Barr virus (EBV)-positive tumours (univariate RL=0.52, 95% CI 0.3-0.9, P=0.02) and in older patients (univariate RL=0.85, 95% CI 0.73-0.99, P=0.03). Well-known factors such as young age, female sex and EBV-negative status predicted nodular sclerosis histology. CONCLUSION: The number of eosinophils in HL tumours is influenced by patient traits such as asthma, ECP genotype and EBV status. EBV status was predictive of histology.

Epidermal Keratinocyte Depletion during Five Weeks of Radiotherapy is Associated with DNA Double-Strand Break Foci, Cell Growth Arrest and Apoptosis: Evidence of Increasing Radioresponsiveness and Lack of Repopulation; the Number of Melanocytes Remains Unchanged.

During fractionated radiotherapy, epithelial cell populations are thought to decrease initially, followed by accelerated repopulation to compensate cell loss. However, previous findings in skin with daily 1.1 Gy dose fractions indicate continued and increasing cell depletion. Here we investigated epidermal keratinocyte response with daily 2 Gy fractions as well as accelerated and hypofractionation. Epidermal interfollicular melanocytes were also assessed. Skin-punch biopsies were collected from breast cancer patients before, during and after mastectomy radiotherapy to the thoracic wall with daily 2 Gy fractions for 5 weeks. In addition, 2.4 Gy radiotherapy four times per week and 4 Gy fractions twice per week for 5 weeks, and two times 2 Gy daily for 2.5 weeks, were used. Basal keratinocyte density of the interfollicular epidermis was determined and immunostainings of keratinocytes for DNA double-strand break (DSB) foci, growth arrest, apoptosis and mitosis were quantified. In addition, interfollicular melanocytes were counted. Initially minimal keratinocyte loss was observed followed by pronounced depletion during the second half of treatment and full recovery at 2 weeks post treatment. DSB foci per cell peaked towards the end of treatment. p21-stained cell counts increased during radiotherapy, especially the second half. Apoptotic frequency was low throughout radiotherapy but increased at treatment end. Mitotic cell count was significantly suppressed throughout radiotherapy and did not recover during weekend treatment gaps, but increased more than threefold compared to unexposed skin 2 weeks post-radiotherapy. The number of melanocytes remained constant over the study period. Germinal keratinocyte loss rate increased gradually during daily 2 Gy fractions for 5 weeks, and similarly for hypofractionation. DSB foci number after 2 Gy irradiation revealed an initial radioresistance followed by increasing radiosensitivity. Growth arrest mediated by p21 strongly suggests that cells within or recruited into the cell cycle during treatment are at high risk of loss and do not contribute significantly to repopulation. It is possible that quiescent (G0) cells at treatment completion accounted for the accelerated post-treatment repopulation. Recent knowledge of epidermal tissue regeneration and cell cycle progression during genotoxic and mitogen stress allows for a credible explanation of the current finding. Melanocytes were radioresistant regarding cell depletion.

Low-dose hypersensitive gammaH2AX response and infrequent apoptosis in epidermis from radiotherapy patients.

BACKGROUND AND PURPOSE: A low-dose hypersensitivity to radiation can be observed in vitro for many human cell types in terms of increased cell kill per dose unit for doses below 0.5Gy. Quantification of the double-strand break marker gammaH2AX in samples taken in clinical radiotherapy practice has the potential to provide important information about how induction and repair of severe DNA damage and apoptosis are linked to low-dose hypersensitivity. MATERIAL AND METHODS: The effects of exposure to low doses (0.05-1.1Gy) were investigated in skin biopsies taken from prostate cancer patients undergoing the first week of radiotherapy. gammaH2AX foci and apoptotic cells were visualised by immunohistochemistry and quantified by image analysis. RESULTS: The gammaH2AX foci pattern in biopsies taken 30min after a single fraction revealed a low-dose hypersensitivity below 0.3Gy (p=0.0009). The result was consistent for repeated fractions (p=0.00001). No decrease in foci numbers could be detected when comparing biopsies taken 30min and 2h after single fractions of 0.4 and 1.2Gy. The result was consistent for repeated fractions. Only 43 of 168,000 cells in total were identified as apoptotic, yet a dose dependency could be detected after 1week of radiotherapy (p=0.003). CONCLUSIONS: We describe a method based on gammaH2AX to study DNA damage response and apoptosis in a clinical setting. A gammaH2AX hypersensitive response to low doses can be observed in epidermal skin, already 30min following delivered fraction. A very low frequency of apoptosis in normal epithelium suggests that this effect is not an important part of the in vivo response to low doses.

Double strand break induction and kinetics indicate preserved hypersensitivity in keratinocytes to subtherapeutic doses for 7weeks of radiotherapy.

BACKGROUND AND PURPOSE: Previously we reported that hyper-radiosensitivity (HRS) was evidenced by quantifying DNA double strand break (DSB) foci in epidermis biopsies collected after delivering radiotherapeutic one and five dose fractions. The aim of this study was to determine whether HRS was preserved throughout a 7-week radiotherapy treatment, and also to examine the rate of foci decline and foci persistence between dose fractions. MATERIALS AND METHODS: 42 patients with prostate cancer received 7-week fractionated radiotherapy treatment (RT) with daily dose fractions of 0.05-1.10Gy to the skin. Before RT, and at several times throughout treatment, skin biopsies (n=452) were collected at 30min, and 2, 3, 24, and 72h after dose fractions. DSB-foci markers, γH2AX and 53BP1, were labelled in epidermal keratinocytes with immunofluorescence and immunohistochemical staining. Foci were counted both with digital image analysis and manually. RESULTS: HRS in keratinocytes was evidenced by the dose-response relationships of DSB foci, observed throughout the treatment course, independent of sampling time and quantification method. Foci observed at 24h after dose fractions indicated considerable DSB persistence. Accordingly, foci significantly accumulated after 5 consecutive dose fractions. For doses below 0.3Gy, persistent foci could be observed even at 72h after damage induction. A comparison of γH2AX and 53BP1 quantifications in double-stained biopsies showed similar HRS dose-response relationships. CONCLUSIONS: These results represented the first evidence of preserved HRS, assessed by γH2AX- and 53BP1-labelled DSB foci, throughout a 7-week treatment course with daily repeated subtherapeutic dose fractions.

Multicellular tumour spheroid as a model for evaluation of [18F]FDG as biomarker for breast cancer treatment monitoring.

BACKGROUND: In order to explore a pre-clinical method to evaluate if [18F]FDG is valid for monitoring early response, we investigated the uptake of FDG in Multicellular tumour spheroids (MTS) without and with treatment with five routinely used chemotherapy agents in breast cancer. METHODS: The response to each anticancer treatment was evaluated by measurement of the [18F]FDG uptake and viable volume of the MTSs after 2 and 3 days of treatment. RESULTS: The effect of Paclitaxel and Docetaxel on [18F]FDG uptake per viable volume was more evident in BT474 (up to 55% decrease) than in MCF-7 (up to 25% decrease). Doxorubicin reduced the [18F]FDG uptake per viable volume more noticeable in MCF-7 (25%) than in BT474 MTSs. Tamoxifen reduced the [18F]FDG uptake per viable volume only in MCF-7 at the highest dose of 1 microM. No effect of Imatinib was observed. CONCLUSION: MTS was shown to be appropriate to investigate the potential of FDG-PET for early breast cancer treatment monitoring; the treatment effect can be observed before any tumour size changes occur.The combination of PET radiotracers and image analysis in MTS provides a good model to evaluate the relationship between tumour volume and the uptake of metabolic tracer before and after chemotherapy. This feature could be used for screening and selecting PET-tracers for early assessment of treatment response. In addition, this new method gives a possibility to assess quickly, and in vitro, a good preclinical profile of existing and newly developed anti-cancer drugs.

Automated classification of glandular tissue by statistical proximity sampling.

Due to the complexity of biological tissue and variations in staining procedures, features that are based on the explicit extraction of properties from subglandular structures in tissue images may have difficulty generalizing well over an unrestricted set of images and staining variations. We circumvent this problem by an implicit representation that is both robust and highly descriptive, especially when combined with a multiple instance learning approach to image classification. The new feature method is able to describe tissue architecture based on glandular structure. It is based on statistically representing the relative distribution of tissue components around lumen regions, while preserving spatial and quantitative information, as a basis for diagnosing and analyzing different areas within an image. We demonstrate the efficacy of the method in extracting discriminative features for obtaining high classification rates for tubular formation in both healthy and cancerous tissue, which is an important component in Gleason and tubule-based Elston grading. The proposed method may be used for glandular classification, also in other tissue types, in addition to general applicability as a region-based feature descriptor in image analysis where the image represents a bag with a certain label (or grade) and the region-based feature vectors represent instances.

DNA double strand break quantification in skin biopsies.

BACKGROUND AND PURPOSE: Following induction of double strand breaks the histone H2AX is rapidly phosphorylated at regions flanking the breaks resulting in nuclear gamma H2AX foci. The purpose of this study was to use this endogenous signalling system to quantify the in vivo response to radiation in normal tissue. PATIENTS AND METHODS: Skin biopsies were taken from prostate cancer patients undergoing radiotherapy with a curative intent. Biopsies were taken at locations corresponding to 5 different doses in the range below 1.1 Gy per fraction. Biopsies were taken from patients 30 min following the first fraction and then once again following the fraction given after the first weekend break in the treatment course. The DNA double strand breaks were visualised as gamma H2AX foci using immunohistochemistry. Images were acquired using a CCD-camera and a fluorescence microscope and the gamma H2AX foci were quantified using digital image analysis including the basic procedures of top-hat transformation, threshold setting and labelling. RESULTS: Repeated assessments of the biopsies showed a high reproducibility in quantifying the number of foci per DNA area of the nucleated cells in epidermis. The reproducibility was equally good for the two biopsy occasions. A linear dose response was observed for the epidermis in the dose region 0-1 Gy. CONCLUSIONS: We have established a method to measure the relative amount of DNA double strand breaks by detecting gamma H2AX foci in patients exposed to radiotherapy. The method provides a tool to study induction and repair of DNA double strand breaks and has the potential to predict individual radiosensitivity.

Image segmentation and identification of paired antibodies in breast tissue.

Comparing staining patterns of paired antibodies designed towards a specific protein but toward different epitopes of the protein provides quality control over the binding and the antibodies' ability to identify the target protein correctly and exclusively. We present a method for automated quantification of immunostaining patterns for antibodies in breast tissue using the Human Protein Atlas database. In such tissue, dark brown dye 3,3'-diaminobenzidine is used as an antibody-specific stain whereas the blue dye hematoxylin is used as a counterstain. The proposed method is based on clustering and relative scaling of features following principal component analysis. Our method is able (1) to accurately segment and identify staining patterns and quantify the amount of staining and (2) to detect paired antibodies by correlating the segmentation results among different cases. Moreover, the method is simple, operating in a low-dimensional feature space, and computationally efficient which makes it suitable for high-throughput processing of tissue microarrays.

Low expression of microRNA-129-5p predicts poor clinical outcome in diffuse large B cell lymphoma (DLBCL).

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous group of B cell lymphomas. MicroRNA expression provides a new and interesting tool for understanding the biology and clinical course of DLBCL. The present study presents microRNA-129-5p expression data from DLBCL patients treated with CHOP or R-CHOP. Patients with low microRNA-129-5p expression had a median survival of 23 months and a significantly shorter overall survival (P = 0.0042) compared to patients with high microRNA-129-5p expression, who had a median survival of 58 months. We also found that patients treated with R-CHOP only and displaying low microRNA-129-5p expression had a significantly shorter overall survival compared to patients with high microRNA-129-5p expression; all such patients were still alive at the time of last follow-up (P = 0.043). No significant difference was found among microRNA-129-5p expression in tumor tissue, the tissue surrounding the tumor, and normal controls. To our knowledge, this is the first report to show that the expression of microRNA-129-5p can affect the clinical outcome of DLBCL patients and that microRNA-129-5p may be involved in the biology of DLBCL development, although larger studies are necessary to confirm this. Further investigations may also help to elucidate the biological role of microRNA-129-5p in DLBCL.

One kilometer (1 km) electric solar wind sail tether produced automatically.

We produced a 1 km continuous piece of multifilament electric solar wind sail tether of µm-diameter aluminum wires using a custom made automatic tether factory. The tether comprising 90,704 bonds between 25 and 50 µm diameter wires is reeled onto a metal reel. The total mass of 1 km tether is 10 g. We reached a production rate of 70 m/24 h and a quality level of 1‰ loose bonds and 2‰ rebonded ones. We thus demonstrated that production of long electric solar wind sail tethers is possible and practical.

Automated classification of immunostaining patterns in breast tissue from the human protein atlas.

BACKGROUND: The Human Protein Atlas (HPA) is an effort to map the location of all human proteins (http://www.proteinatlas.org/). It contains a large number of histological images of sections from human tissue. Tissue micro arrays (TMA) are imaged by a slide scanning microscope, and each image represents a thin slice of a tissue core with a dark brown antibody specific stain and a blue counter stain. When generating antibodies for protein profiling of the human proteome, an important step in the quality control is to compare staining patterns of different antibodies directed towards the same protein. This comparison is an ultimate control that the antibody recognizes the right protein. In this paper, we propose and evaluate different approaches for classifying sub-cellular antibody staining patterns in breast tissue samples. MATERIALS AND METHODS: The proposed methods include the computation of various features including gray level co-occurrence matrix (GLCM) features, complex wavelet co-occurrence matrix (CWCM) features, and weighted neighbor distance using compound hierarchy of algorithms representing morphology (WND-CHARM)-inspired features. The extracted features are used into two different multivariate classifiers (support vector machine (SVM) and linear discriminant analysis (LDA) classifier). Before extracting features, we use color deconvolution to separate different tissue components, such as the brownly stained positive regions and the blue cellular regions, in the immuno-stained TMA images of breast tissue. RESULTS: We present classification results based on combinations of feature measurements. The proposed complex wavelet features and the WND-CHARM features have accuracy similar to that of a human expert. CONCLUSIONS: Both human experts and the proposed automated methods have difficulties discriminating between nuclear and cytoplasmic staining patterns. This is to a large extent due to mixed staining of nucleus and cytoplasm. Methods for quantification of staining patterns in histopathology have many applications, ranging from antibody quality control to tumor grading.

A low-dose hypersensitive keratinocyte loss in response to fractionated radiotherapy is associated with growth arrest and apoptosis.

BACKGROUND AND PURPOSE: The existence of a hypersensitive radiation response to doses below 0.5Gy is well established for many normal and tumour cell lines. There is also evidence for hypersensitive tissue responses in acute skin damage and kidney function in mice. Recently, we have identified that a hypersensitive gammaH2AX response exists in human epidermis. The aim of this study was to investigate the dose-response of basal clonogenic keratinocytes in normal skin to fractionated radiotherapy with low dose fractions. MATERIALS: Skin punch biopsies were taken before and during radiotherapy from prostate cancer patients undergoing radiotherapy with a curative intent. Areas of epidermis receiving daily fractions of approximately 0.1, 0.2, 0.45 and 1.1Gy were biopsied on the same occasion to determine dose-response for each individual patient. In total, 89 cases were assessed either at 1, 2.5, 3, 4, 5 or 6.5 weeks in the treatment course. Biopsy sampling of another 25 patients was performed from areas receiving 0.45 and 1.1Gy per fraction at regular intervals throughout the 7-week treatment period. The number of basal keratinocytes per mm of the interfollicular epidermis was determined. The DNA damage response of the basal keratinocytes was investigated by immunohistochemical staining for molecular markers of growth arrest, mitosis and cell death, using p21, phospho-H3 and gammaH2AX, respectively. The number of stained keratinocytes in the basal layer was counted manually. The p21 staining was also quantified by digital image analysis. RESULTS: The individual dose-response relationships revealed a low-dose hypersensitivity for reduction of basal keratinocytes throughout 7 weeks of radiotherapy (p<0.01). Growth arrest and cell proliferation assessed at 1 week and 6.5 weeks showed, in both cases, hypersensitive increase of p21 (p<0.01) and hypersensitive depression of mitosis (p<0.01). Manual counting and digital image analysis of p21 showed good agreement. Cell death was infrequent but increased significantly between 1 and 6.5 weeks and displayed a hypersensitive dose-response at the end of the treatment period. CONCLUSIONS: A low-dose hypersensitivity in basal skin keratinocyte reduction is present throughout 7 weeks of radiotherapy. A persistent hypersensitive growth arrest response and cell killing mediate this effect.