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This paper addresses digital staining and classification of the unstained white blood cell images obtained with a differential contrast microscope. We have data coming from multiple domains that are partially labeled and partially matching across the domains. Using unstained images removes time-consuming staining procedures and could facilitate and automatize comprehensive diagnostics. To this aim, we propose a method that translates unstained images to realistically looking stained images preserving the inter-cellular structures, crucial for the medical experts to perform classification. We achieve better structure preservation by adding auxiliary tasks of segmentation and direct reconstruction. Segmentation enforces that the network learns to generate correct nucleus and cytoplasm shape, while direct reconstruction enforces reliable translation between the matching images across domains. Besides, we build a robust domain agnostic latent space by injecting the target domain label directly to the generator, i.e., bypassing the encoder. It allows the encoder to extract features independently of the target domain and enables an automated domain invariant classification of the white blood cells. We validated our method on a large dataset composed of leukocytes of 24 patients, achieving state-of-the-art performance on both digital staining and classification tasks.

Many phytochemicals possess antioxidant and cancer-preventive properties, some putatively through antioxidant response element-mediated phase II metabolism, entailing mutagen/oxidant quenching. In our recent studies, however, most candidate phytochemical agents were not potent in inducing phase II genes in normal human lung cells. In this study, we applied a messenger RNA (mRNA)-specific gene expression-based high throughput in vitro screening approach to discover new, potent plant-derived phase II inducing chemopreventive agents. Primary normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cells (HBECs) were exposed to 800 individual compounds in the MicroSource Natural Products Library. At a level achievable in humans by diet (1.0 μM), 2,3-dihydroxy-4-methoxy-4'-ethoxybenzophenone (DMEBP), triacetylresveratrol (TRES), ivermectin, sanguinarine sulfate, and daunorubicin induced reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1 (NQO1) mRNA and protein expression in NHBE cells. DMEBP and TRES were the most attractive agents as coupling potency and low toxicity for induction of NQO1 (mRNA level, ≥3- to 10.8-fold that of control; protein level, ≥ two- to fourfold that of control). Induction of glutathione S-transferase pi mRNA expression was modest, and none was apparent for glutathione S-transferase pi protein expression. Measurements of reactive oxygen species and glutathione/oxidized glutathione ratio showed an antioxidant effect for DMEBP, but no definite effect was found for TRES in NHBE cells. Exposure of NHBE cells to H(2)O(2) induced nuclear translocation of nuclear factor erythroid 2-related factor 2, but this translocation was not significantly inhibited by TRES and DMEBP. These studies show that potency and low toxicity may align for two potential NQO1-inducing agents, DMEBP and TRES.

SummaryKaposi’s sarcoma-associated herpesvirus (KSHV) / human herpesvirus-8 is the causative agent of the endothelial cell-derived tumour Kaposi’s sarcoma. Herpesviruses possess large complex genomes which provide many options to regulate cellular physiology during the viral life cycle and in the course of tumourigenicity. Novel techniques of systems biology and reverse genetics are increasingly applied to dissect the complex interaction of KSHV with endothelial cells. This review will outline novel results and pitfalls of these technologies in the elucidation of KSHV pathogenicity.

Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interrogations of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)–non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP-microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcinomas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p

Epstein-Barr virus (EBV) requires at a minimum membrane-associated glycoproteins gB, gH, and gL for entry into host cells. B-cell entry additionally requires gp42, which binds to gH/gL and triggers viral entry into B cells. The presence of soluble gp42 inhibits membrane fusion with epithelial cells by forming a stable heterotrimer of gH/gL/gp42. The interaction of gp42 with gH/gL has been previously mapped to residues 36 to 81 at the N-terminal region of gp42. In this study, we further mapped this region to identify essential features for binding to gH/gL by use of synthetic peptides. Data from fluorescence polarization, cell-cell fusion, and viral infection assays demonstrated that 33 residues corresponding to 44 to 61 and 67 to 81 of gp42 were indispensable for maintaining low-nanomolar-concentration gH/gL binding affinity and inhibiting B-cell fusion and epithelial cell fusion as well as viral infection. Overall, specific, large hydrophobic side chain residues of gp42 appeared to provide critical interactions, determining the binding strength. Mutations of these residues also diminished the inhibition of B-cell and epithelial cell fusions as well as EBV infection. A linker region (residues 62 to 66) between two gH/gL binding regions served as an important spacer, but individual amino acids were not critical for gH/gL binding. Probing the binding site of gH/gL and gp42 with gp42 peptides is critical for a better understanding of the interaction of gH/gL with gp42 as well as for the design of novel entry inhibitors of EBV and related human herpesviruses.

Human herpesvirus 8 (HHV-8), also called Kaposi's sarcoma-associated herpesvirus, is the causative agent of Kaposi's sarcoma, an endothelial-cell-derived tumor which is characterized by neoangiogenesis and infiltration of inflammatory cells, and of the lymphoproliferative diseases primary effusion lymphoma (PEL) and multicentric Castleman's disease (6-8, 12, 14, 23, 34, 47, 54). The HHV-8 genome is approximately 170 kbp in length and contains more than 80 genes (40). Open reading frames with homology to genes of herpesvirus saimiri (HVS) were numbered according to their position on the HVS genome. HHV-8 genes without homologous counterparts in HVS were numbered separately and given the prefix “K” (“K genes”) (16, 36, 40, 43). Infection with HHV-8 constitutively activates the transcription factor nuclear factor-kappa B (NF-κB) in endothelial cells and lymphocytes (25, 44). The activation of NF-κB is crucial for the development and progression of HHV-8-associated diseases. It protects HHV-8-infected cells against spontaneous apoptosis (25) and maintains the latent viral life cycle (5, 67). The latter is mandatory for the establishment of viral persistence. In agreement with this, the inhibition of NF-κB signaling delays the growth of HHV-8-associated lymphomas in a mouse model (24) and regulates the production of infectious HHV-8 virions (5, 50). Only a few HHV-8 genes have been studied for their impact on NF-κB signaling. The genes K13 (9), K15 (4), and ORF74 (48) were described as activators and K10.5 (49) as an inhibitor of NF-κB, whereas conflicting results were obtained on the activity of K1 (28, 39). Presently, it is not known whether these genes are the only HHV-8 genes which act on NF-κB or whether different HHV-8 genes cooperate positively or negatively in the regulation of this important signaling pathway. Systematic analyses of all HHV-8 genes for the effects of single genes and gene combinations on NF-κB activity are not available due to the large genome of HHV-8, containing at least 86 genes (36, 43). Systematic analyses of all single-gene and pairwise-combination effects of HHV-8 genes on NF-κB require almost 4,000 transfection experiments. Therefore, this approach demands high-throughput transfection technology, which has only recently become available. In 2001, Ziauddin and Sabatini succeeded in scaling down high-throughput gene function analysis to the microarray level (70). Different cDNA expression plasmids are spotted onto slides by using a microarray robot. The dried slides are exposed to a transfection reagent, placed in a culture dish, and covered with adherent mammalian cells in medium. Alternatively, DNA and transfection reagent can be mixed at once and applied to the slide (70). Both methods of application create microarrays of cell clusters simultaneously transfected with different plasmids in distinct and defined areas in a lawn of cells. The procedure creating a microarray of clusters of transfected cells was called transfected cell microarray. The transfection method was named reverse transfection, because, in contrast to conventional transfection protocols, DNA was “seeded” first and the cells were added subsequently. Reverse-transfected cell microarrays (RTCM), also called “cell chip analyses,” allow the carrying out of several hundred to several thousand transfection experiments in parallel using eukaryotic cells on a single glass slide. Cotransfections of appropriate reporter plasmids can be used to establish quantitative measures of gene effects on signaling pathways (for a review, see reference 53). For the work reported herein, we used the RTCM technology as an unbiased systems biology approach to determine the effects of all HHV-8 genes and of selected combinations of genes (all K and latent genes) on NF-κB activation in HEK 293T cells. Through the performance of more than 14,000 transfections and the subsequent biostatistical analyses, K13 and ORF75 were identified as the major activators of NF-κB. Both genes cooperated in the activation of NF-κB in HHV-8-infected cells. In addition, K1 was demonstrated to be a novel inhibitor of K13- and ORF75-induced NF-κB activation.

Kaposi's sarcoma (KS) is a multifocal tumor that appears in four epidemiologically distinct forms: classical, iatrogenic, AIDS-associated, and African endemic KS (63). KS lesions evolve over time from patch- or plaque-like lesions at early stages to nodular lesions characteristic of later stages (for a review, see references 21 and 63). Histologic hallmarks that characterize all stages of KS include a prominent microvasculature and a large inflammatory compartment of CD8+ T cells and monocytes (for a review, see references 21 and 63). Especially in later stages of development, bundles of spindle-shaped cells become the dominant cellular feature. These so-called KS spindle cells (KSC) express markers of lymph vessel endothelial cells and are regarded as the tumor cells of KS (17, 76). As yet, it is unclear whether KSC are derived from lymph or blood vessel endothelial cells, or from both (32, 60, 73). Human herpesvirus 8 (HHV-8), also known as KS-associated herpesvirus, is regarded as the etiological agent of KS (8). In addition, HHV-8 is associated with primary effusion lymphoma and multicentric Castleman's disease (1). In KS lesions, only a few lytically HHV-8-infected cells are detected (6), whereas more than 70% of KSC are latently infected (58, 59). During latency, only a few of the more than 80 viral genes are expressed (22, 53). These genes are crucial to establish latent infection and to protect infected cells from apoptosis (62). The latter is specifically important considering the inflammatory conditions present in KS, which may induce apoptosis in infected cells by cell-mediated cytotoxicity and/or the formation of high concentrations of reactive oxygen species (ROS) (56, 77). A growing body of evidence suggests that the inhibition of cell death rather than increased proliferation may be key for the growth of the lesions. For example, KSC exhibit longer doubling times than normal endothelial cells in vitro (14, 57), which is in agreement with the fact that generally low proliferation rates of KSC are observed in all stages of KS in vivo (14, 57). Most importantly, the numbers of apoptotic cells in KS decrease from early to late stages, and this is inversely related to increasing numbers of HHV-8-infected cells (62). The K13 gene of HHV-8 is a latent gene that is expressed in almost all KSC in vivo (62). This gene encodes a viral Fas-associated death domain-like interleukin-1β (IL-1β)-converting enzyme-inhibitory protein (vFLIP/K13) with homology to cellular FLIP (cFLIP) molecules (cFLIP-long, -short, and -R) and with certain caspases (67, 71, 78). It is well established that vFLIP/K13 has antiapoptotic activity (20, 64) and plays an important role in the pathogenesis of HHV-8-associated tumors, including KS, primary effusion lymphoma, and multicentric Castleman's disease (15, 28, 62). In contrast, the mechanisms of vFLIP/K13 antiapoptotic activities are controversial and continue to be debated. vFLIP/K13 harbors a death effector domain, suggesting that it may bind to caspase 8 and inhibit its activation, which may protect cells from death receptor-induced apoptosis (9, 67). This hypothesis was initially supported by a report showing that vFLIP/K13 and caspase 8 can bind to each other (5). However, these results were questioned in subsequent studies (10, 24, 44). Presently, evidence is accumulating that vFLIP/K13 can interact with and activate the regulatory components of the IκB kinase complex (3, 23, 39). It has been shown that this activates NF-κB signaling (2, 9, 27, 44) and induces the expression of cellular proteins, including antiapoptotic factors, such as cellular inhibitor of apoptosis (cIAP) and cFLIP (28, 64). As yet, the effect of vFLIP/K13 on cellular-gene expression has been investigated only for selected genes. It is not clear whether the respective genes may also encode the proteins most abundantly induced by vFLIP/K13 and whether additional factors may mediate the antiapoptotic activity of vFLIP/K13 in endothelial cells. Using two-dimensional difference gel electrophoresis (2D-DIGE) analysis, we identified manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant and an important antiapoptotic enzyme, as the protein most strongly upregulated by vFLIP/K13 in primary human endothelial cells. Evidence is provided that MnSOD may be an important mediator of the antiapoptotic activity of vFLIP/K13.

One strategy for lung cancer chemoprevention focuses on the use of agents to modulate the metabolism and disposition of tobacco, environmental and endogenous carcinogens through up-regulation of detoxifying phase II enzymes. However, in studies to date, most candidate agents are not active in human lung cells. Therefore we previously applied a gene expression-based high-throughput screening approach to investigate n=800 plant-derived polyphenolic- and flavenoid- predominant compounds in the MicroSource Natural Products Library as to their effect on inducing the phase II metabolic enzymes glutathione S-transferases (GSTP1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) in primary normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cells (HBEC). We had identified 2, 3-dihydroxy-4-methoxy-4’-ethoxybenzophenone (DMEBP, putative source arctic sponge) and triacetylresveratrol (TRES, putative source plant myrtle) as new inducers for NQO1 mRNA expression. In the current study, in order to confirm our results, NHBE and HBEC cells in culture were exposed to diet-achievable levels of TRES and DMEBP (0.5, 1.0, 2.0 µM), as well as a panel of conventionally-recognized phytopreventive agents [epigallocatechin gallate (EGCG), resveratrol (RES) and sulforaphane (SFN)] for 24 h, 48 h, and 6 d, respectively. Quantitative RNA-specific RT-PCR showed an up-regulation of NQO1 mRNA expression of up to 10.8 fold in DMEBP-treated NHBE cells and an up-regulation of up to 4.6 fold in TRES-treated NHBE cells. NQO1 protein expression increased up to 3.4 fold in DMEBP-treated and up to 1.8 fold in TRES-treated NHBE cells. HBEC cells showed similar results for mRNA expression — an up-regulation of NQO1 mRNA expression of up to 9.7 fold in DMEBP-treated HBEC cells and an up-regulation of up to 3.1 fold in TRES-treated HBEC cells — whereas none of the tested compounds had an effect on protein expression in HBEC cells. DMEBP had a narrow therapeutic window, and was notably toxic to lung cells at 1-2 µM. We then measured accumulation of reactive oxygen species (ROS) in NHBE cell using DFA, and total GSH assays. Our ROS data so far show a slight anti-oxidant effect for DMEBP, but no effect yet found for TRES. Our data show two new agents have some effect on the up-regulation of the detoxifying enzyme NQO1 at mRNA and protein levels, but the effect was modest, and none was observed for GSTP1. Among the newly discovered agents, TRES is the most plausible candidate resulting from this search, and compares favorably, to conventional plant-derived polyphenolics/flavenoids tested. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A49.