Z-stack Analysis Research Articles

IL-1β Inhibition Partially Negates the Beneficial Effects of Diet-Induced Atherosclerosis Regression in Mice.

Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. Smooth muscle cell lineage-tracing Apoe-/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1β or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis. Lipid lowering by switching Apoe-/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1β antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1β antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap. Taken together, IL-1β appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells.

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Carboxymethyl cellulose-alginate interpenetrating hydroxy ethyl methacrylate crosslinked polyvinyl alcohol reinforced hybrid hydrogel templates with improved biological performance for cardiac tissue engineering.

Cardiac tissue engineering is an emerging approach for cardiac regeneration utilizing the inherent healing responses elicited by the surviving heart using biomaterial templates. In this study, we aimed to develop hydrogel scaffolds for cardiac tissue regeneration following myocardial infarction (MI). Two superabsorbent hydrogels, CAHA2A and CAHA2AP, were developed employing interpenetration chemistry. CAHA2A was constituted with alginate, carboxymethyl cellulose, (hydroxyethyl) methacrylate, and acrylic acid, where CAHA2AP was prepared by interpenetrated CAHA2A with polyvinyl alcohol. Both hydrogels displayed superior physiochemical characteristics, as determined by attenuated total reflection infrared spectroscopy spectral analysis, differential scanning calorimetrymeasurements, tensile testing, contact angle, water profiling, dye release, and conductivity. In vitro degradation of the hydrogels displayed acceptable weight composure and pH changes. Both hydrogels were hemocompatible, and biocompatible as evidenced by direct contact and MTT assays. The hydrogels promoted anterograde and retrograde migration as determined by the z-stack analysis using H9c2 cells grown with both gels. Additionally, the coculture of the hydrogels with swine epicardial adipose tissue cells and cardiac fibroblasts resulted in synchronous growth without any toxicity. Also, both hydrogels facilitated the production of extracellular matrix by the H9c2 cells. Overall, the findings support an appreciable in vitro performance of both hydrogels for cardiac tissue engineering applications.

An Evaluation of Computational Learning-based Methods for the Segmentation of Nuclei in Cervical Cancer Cells from Microscopic Images.

The manual segmentation of cellular structures on Z-stack microscopic images is time-consuming and often inaccurate, highlighting the need to develop auto-segmentation tools to facilitate this process. This study aimed to compare the performance of three different machine learning architectures, including random forest (RF), AdaBoost, and multi-layer perceptron (MLP), for the autosegmentation of nuclei in proliferating cervical cancer cells on Z-Stack cellular microscopy proliferation images provided by the HCS Pharma. The impact of using post-processing techniques, such as the StarDist plugin and majority voting, was also evaluated. The RF, AdaBoost, and MLP algorithms were used to auto-segment the nuclei of cervical cancer cells on microscopic images at different Z-stack positions. Post-processing techniques were then applied to each algorithm. The performance of all algorithms was compared by an expert to globally generated ground truth by calculating the accuracy detection rate, the Dice coefficient, and the Jaccard index. RF achieved the best accuracy, followed by the AdaBoost and then the MLP. All algorithms achieved good pixel classifications except in regions whereby the nuclei overlapped. The majority voting and StarDist plugin improved the accuracy of the segmentation but did not resolve the nuclei overlap issue. The Z-Stack analysis revealed similar segmentation results to the Z-stack layer used to train the image. However, a worse performance was noted for segmentations performed on different Z-stack positions, which were not used to train the algorithms. All machine learning architectures provided a good segmentation of nuclei in cervical cancer cells but did not resolve the problem of overlapping nuclei and Z-stack segmentation. Further research should therefore evaluate the combined segmentation techniques and deep learning architectures to resolve these issues.

Abstract 4522: An in vitro 3D immune exclusion tumor model engineered in a layer-by-layer fashion

Abstract Recent immunotherapy failures in clinical trials have elucidated the affects of the tumor stroma on attenuating therapeutic response. In some cancers, the stroma consists of a collagen-rich fibrotic zone on the tumor periphery that traps infiltrating CD8+ T cells, allowing the tumor to evade immune cell-mediated death. Typically, animal models are employed to study complex physiology. However, known challenges to these methods include cost, duration and throughput. Thus, we sought to develop a 3D in vitro model that recapitulates the immune exclusion phenotype in a high throughput manner. We utilized a novel 3D cell culture platform, VersaGel and Symphony, to pattern tumor-stroma-immune compartments in a layer-by-layer fashion in 96-well plates. VersaGel is a chemically modified extracellular matrix that is amenable to light-induced crosslinking using the Symphony apparatus. Moreover, Symphony allows for discrete layers to be formed with any VersaGel/cell mixture, with defined 3D gel thicknesses (eg. 100µm, 250µm, 500µm) and areas in the plate (eg. 2mm, 4mm, or 8mm diameter discs). GFP-labeled MC38-ova (ovalbumin antigen) colon carcinoma cells were grown in 100µm thick 3D VersaGel with or without CFP-labeled mouse embryonic fibroblasts (MEF) layered on top for 4 days. After, OT-1-RFP CD8+ cells were added to the culture medium to assess their penetration depth and tumor cell death over 3 days. 3D cell analysis through the optically clear VersaGel was performed in situ using high content confocal microscopy, effectively quantifying RFP/CFP/GFP-labeled cells and DRAQ7-labeled dead cells using Z-stack analysis. In the control (tumor-only condition), OT-1 cells fully penetrated the tumor layer and induced cell death. In the experimental condition (with fibroblasts), it was found that the fibrotic stromal layer limited OT-1 penetration to the periphery of the 3D tumor, and, moreover, the total OT-1 cells increased relative to the control. Future analysis may include the utility of wild-type T cells and novel therapeutics targeting the stroma to activate the immune response. The results suggest a novel 3D immune exclusion model that integrates with high content confocal analysis and is amenable to immunotherapeutic screening and translational assessment. Citation Format: Kolin C. Hribar, Tracy Kleinheinz, Joanna Klementowicz, Susan Kaufman, Robert A. Blake, Celine Eidenschenk. An in vitro 3D immune exclusion tumor model engineered in a layer-by-layer fashion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4522.

Evaluation of Pigment Distribution and Depth Analysis Methods for Decorative Soft Contact Lenses.

This study evaluates pigment component distribution and depth in decorative soft contact lenses (DSCLs) using a variety of analytical methods. We sampled 18 DSCLs using optical microscopy, optical coherence tomography analysis, Z-stack analysis, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDX), and time-of-flight secondary ion mass spectrometry (TOF-SIMS) to evaluate the distribution and depth of pigment components. Pigment distribution in DSCLs was easily observed with optical methods including Z-stack analysis. X-ray photoelectron spectroscopy, SEM/EDX, and TOF-SIMS were used to evaluate the level of pigment exposure on the lens surface and the results showed significant differences between the methods. Pigment components were detected in 16 samples by SEM/EDX, but not by XPS. Pigment components were only detected in eight samples using TOF-SIMS. It may be necessary to show that a nanometer-thick monomolecular film does not exist on the surface of DSCLs, to demonstrate the exposure of a pigment particle. Taking into account the principle behind each of the measurement methods and the resolution and sensitivity of each of the analytical methods compared, TOF-SIMS may be the most appropriate method to accurately judge pigment exposure on DSCLs. The Z-stack method may be useful for estimating the depth of pigment components in DSCLs.

Quantitative Algorithm-Based Paired Imaging Measurement for Antibody-Triggered Endocytosis in Cultured Cells

Antibody-triggered endocytosis (ATE) is a biological mechanism on which many therapeutic strategies are grounded, such as delivery of antibody–drug conjugates (ADCs). Current methods monitoring ATE include confocal Z-stack analysis, acid wash, antibody quenching, and pH-sensitive dye labeling. However, those generate less quantifiable results with low throughput. Here we report a new method referred to as “paired imaging measurement” to analyze ATE using a quantitative algorithm in conjunction with high-content imaging. With two sequential measurements of cell surface antibody employing live cell staining and total antibody by immunostaining before and after cell permeabilization, intracellular antibody undergoing endocytosis can be quantified indirectly. Antibodies against CD98 and transferrin receptor were tested on hCMEC/D3 and hiPSC-derived endothelial cells. The maximal response and potency of endocytosed antibodies were generated with good assay robustness (Z′ > 0.6) and >5-fold signal/background ratio. Antibody endocytosis response ranking is consistent between batches (R2 > 0.9). The obtained results were confirmed by other traditional methods. In conclusion, we have developed a novel method using a quantitative imaging algorithm in conjunction with live cell staining for high-throughput investigation of ATE.

Development of a Nanostructured Platform for Identifying HER2-Heterogeneity of Breast Cancer Cells by Surface-Enhanced Raman Scattering.

Biosensor technology has great potential for the detection of cancer through tumor-associated molecular biomarkers. In this work, we describe the immobilization of the recombinant humanized anti-HER2 monoclonal antibody (trastuzumab) on a silver nanostructured plate made by pulsed laser deposition (PLD), over a thin film of Au(111). Immobilization was performed via 4-mercapto benzoic acid self-assembled monolayers (4-MBA SAMs) that were activated with coupling reagents. A combination of immunofluorescence images and z-stack analysis by confocal laser scanning microscopy (CLSM) allowed us to detect HER2 presence and distribution in the cell membranes. Four different HER2-expressing breast cancer cell lines (SKBR3 +++, MCF-7 +/−, T47D +/−, MDA-MB-231 −) were incubated during 24 h on functionalized silver nanostructured plates (FSNP) and also on Au(111) thin films. The cells were fixed by means of an ethanol dehydration train, then characterized by atomic force microscopy (AFM) and surface-enhanced Raman scattering (SERS). SERS results showed the same tendency as CLSM findings (SKBR3 > MCF-7 > T47D > MDA-MB-231), especially when the Raman peak associated with phenylalanine amino acid (1002 cm−1) was monitored. Given the high selectivity and high sensitivity of SERS with a functionalized silver nanostructured plate (FSNP), we propose this method for identifying the presence of HER2 and consequently, of breast cancer cells.

Confocal microscopic analysis of erythrocytes and erythroplastids from Major Mitchell’s cockatoos (Lophochroa leadbeateri)

The majority of avian erythrocytes retain their nucleus throughout the circulatory lifespan of the cell; however, a small proportion of avian erythrocytes are enucleated to form erythroplastids. Previously, we have reported a Major Mitchell’s cockatoo that had a much greater proportion of erythroplastids than typically observed in avian blood. To further classify the erythrocytes of this bird, we employed fluorescent laser scanning confocal microscopy. Fluorescence of the cytoplasm of erythroid cells, including erythroplastids, facilitated assessment including z-stack analysis. Erythroplastids had a mean volume of 118 fL and were predominantly elliptical with minor depressions and infolding of cell surfaces.

Burdock root extracts limit quorum-sensing-controlled phenotypes and biofilm architecture in major urinary tract pathogens.

Bacterial biofilms are serious concern in patients infected with urinary tract infections, complicated urinary tract infections and other device-associated infections. Microbes within the biofilms are effectively shielded from antibiotics and host immune cells, hence can be treated only with agents which has the potential to disassemble the biofilms. The study is focused on the root extracts of Arctium lappa Linn. as a source for complementary medicine against three major biofilm forming clinical isolates of Escherichia coli, Proteus mirabilis, and Serratia marcescens. Methanol extracts of burdock roots (BR) showed no bactericidal activity (p > 0.05) against the uropathogens, whereas restrained the biofilms (p < 0.05) on polystyrene and glass surfaces at a biofilm inhibitory concentration of 100 µg/mL. The 3D confocal laser scanning microscopy was used to analyze the biofilm architecture which showed significant reduction in the surface area. Z-stack analysis has also revealed substantial reduction in the biofilm thickness (E. coli-50.79%, P. mirabilis-69.49%, and S. marcescens-75.84%). Further, BR extracts also inhibited quorum-sensing (QS)-controlled cellular phenotypes such as violacein, prodigiosin, swarming motility, and cell surface hydrophobicity. LC-MS/MS analysis of BR extracts identified the presence of two major quercetin derivatives (miquelianin and peltatoside) along with few other constituent components. Exploring such phytocompounds will provide potential agents to treat infections caused by biofilm forming uropathogens. The antibiofilm and anti-QS agents will ultimately serve as armor, facilitating the host immune system to fight infections.

Synergy of flavonoids with HDAC inhibitor: new approach to target Candida tropicalis biofilms

Candida biofilms on various implanted medical devices and living tissues are serious concern in several hospital-acquired infections. The study was conducted to employ a new therapeutic strategy to inhibit the formation of Candida tropicalis biofilms. No significant antifungal or antibiofilm activity was observed when the sodium butyrate (SB), quercetin, and kaempferol were used as a lone drug. Significant decline (P<0·05) in biofilm formation was noted when sub-lethal concentration of SB (30 mM) was used in combination with the flavonoids (450 μg/ml). Z-stack analysis using 3D-confocal laser scanning microscopy (CLSM) also showed substantial reduction in the biofilm thickness in treated glass slides. In conclusion, this is the first report to our knowledge on implementing a combination therapy using flavonoids and histone deacetylase (HDAC) inhibitor.

Histone Deacetylase 6 (HDAC6) Deacetylates Survivin for Its Nuclear Export in Breast Cancer

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.

Characterization of the Niemann-Pick C pathway in alveolar type II cells and lamellar bodies of the lung

The Niemann-Pick C (NPC) pathway plays an essential role in the intracellular trafficking of cholesterol by facilitating the release of lipoprotein-derived sterol from the lumen of lysosomes. Regulation of cellular cholesterol homeostasis is of particular importance to lung alveolar type II cells because of the need for production of surfactant with an appropriate lipid composition. We performed microscopic and biochemical analysis of NPC proteins in isolated rat type II pneumocytes. NPC1 and NPC2 proteins were present in the lung, isolated type II cells in culture, and alveolar macrophages. The glycosylated and nonglycosylated forms of NPC1 were prominent in the lung and the lamellar body organelles. Immunocytochemical analysis of isolated type II pneumocytes showed localization of NPC1 to the limiting membrane of lamellar bodies. NPC2 and lysosomal acid lipase were found within these organelles, as confirmed by z-stack analysis of confocal images. All three proteins also were identified in small, lysosome-like vesicles. In the presence of serum, pharmacological inhibition of the NPC pathway with compound U18666A resulted in doubling of the cholesterol content of the type II cells. Filipin staining revealed a striking accumulation of cholesterol within lamellar bodies. Thus the NPC pathway functions to control cholesterol accumulation in lamellar bodies of type II pneumocytes and, thereby, may play a role in the regulation of surfactant cholesterol content.

Hematopoietic Cells Undergo Homotypic Cell Fusion: A Novel Platform to Study Genomic Instability and Leukemogenesis.

AbstractAbstract 1196Gene copy number variation (CNV) and translocation-derived gene fusion products are commonly observed in hematological malignancies. Mechanisms precipitating CNV include failed cytokinesis or spontaneous cell fusion. Heterotypic fusion of hematopoietic cells with non-hematopoeitic cells has been reported following injury in diverse tissues. Cell fusion involves blending membranes, intracellular material, and nuclear material from two parental cells to form a genetically and immunophenotypically distinct (often hyperdiploid) single daughter cell. We therefore hypothesized that if we could generate genetically chimeric, hematopoietic cells via cell-cell fusion, they might provide an instructive model for determining the role CNV plays in leukemogenesis. To generate genetically abnormal hematopoietic cells in vivo, a transplantation model with radiation induction was employed in which donor and host mice possess numerous unique cell surface and genomic markers. Fusion was determined by co-expression of donor and host markers among hematopoietic cells isolated from recipient BM, spleen, and thymus by serial fluorescence activated cell sorting. The presence of ‘bona fide’ fused cells was confirmed by performing rigorous immunophenotypic and genotypic analyses of individual cells, including immunofluoresence staining and z-stack analysis, single nucleotide polymorphism (SNP) PCR, and fluorescent in situ hybridization (FISH). Fused cell populations were detected in both mature myeloid and lymphoid lineages. Consistent with the proposed model of studying early, initiating leukemogenic events, cell fusion was also detected in the progenitor cell populations including colony forming unit (CFU-C) myeloid progenitors, and c-kit+, sca-1+, lin- (KSL) cells. Moreover, transplanted fused hematopoietic cells could reconstitute lethally irradiated secondary hosts >16 weeks after transplant. Thus, hematopoietic progenitor cells are capable of homotypic cell fusion, and subsequent to cell fusion, these cells are further able to divide, differentiate, and are competent to contribute to functional hematopoiesis. Additional assays are underway to more definitively address the functional significance of hematopoietic cell fusion as a repair mechanism, or during homeostasis. Importantly, we detected evidence of genomic instability in fused hematopoietic cells using a variety of assays. Fused cells with loss of specific donor and/or host marker genes were identified by immunofluorescence and genomic DNA analysis. DNA strand breakage, evaluated by single cell gel electrophoresis (comet assay) showed the presence of a significantly larger tail moment in fused cells suggesting a higher degree of DNA damage in these cells. Studies for evaluation of p53 status and histone H2AX activation are underway. In summary, our data not only provide novel evidence that homotypic cell fusion occurs between hematopoietic cells following injury, but also that fused cells retain their replicative potential in vitro and in vivo and exhibit greater genomic instability compared to non-fused counterparts. Because most existing murine models of leukemogenesis focus on events in the promotion of disease, we believe fusion and reductive division of hematopoietic cells shown here will serve as a novel platform to study the role of CNV as a leukemia-initiating event. Disclosures:No relevant conflicts of interest to declare.

Cell Surface Biliverdin Reductase Mediates Biliverdin-induced Anti-inflammatory Effects via Phosphatidylinositol 3-Kinase and Akt

Biliverdin reductase A (BVR) catalyzes the reduction of biliverdin (BV) to bilirubin (BR) in all cells. Others and we have shown that biliverdin is a potent anti-inflammatory molecule, however, the mechanism by which BV exerts its protective effects is unclear. We describe and elucidate a novel finding demonstrating that BVR is expressed on the external plasma membrane of macrophages (and other cells) where it quickly converts BV to BR. The enzymatic conversion of BV to BR on the surface by BVR initiates a signaling cascade through tyrosine phosphorylation of BVR on the cytoplasmic tail. Phosphorylated BVR in turn binds to the p85alpha subunit of phosphatidylinositol 3-kinase and activates downstream signaling to Akt. Using bacterial endotoxin (lipopolysaccharide) to initiate an inflammatory response in macrophages, we find a rapid increase in BVR surface expression. One of the mechanisms by which BV mediates its protective effects in response to lipopolysaccharide is through enhanced production of interleukin-10 (IL-10) the prototypical anti-inflammatory cytokine. IL-10 regulation is dependent in part on the activation of Akt. The effects of BV on IL-10 expression are lost with blockade of Akt. Inhibition of surface BVR with RNA interference attenuates BV-induced Akt signaling and IL-10 expression and in vivo negates the cytoprotective effects of BV in models of shock and acute hepatitis. Collectively, our findings elucidate a potentially important new molecular mechanism by which BV, through the enzymatic activity and phosphorylation of surface BVR (BVR)(surf) modulates the inflammatory response.

Donor-Derived Squamous Cell Carcinoma after Allogeneic Bone Marrow Transplantation.

Long-term survivors after allogeneic hematopoietic stem cell transplantation (HSCT) develop solid tumors. Chronic graft-versus-host disease (GvHD) increases the risk of squamous cell carcinoma (SCC). Five of 24 patients with HSCT and SCC, had sex-mismatched HSCT. In 3 of them, tumor sex karyotype studied by combined XY FISH and double immunostaining on the same SCC section, with 3D Z-stack analysis, was compatible with a cancer originating from donor hematopoietic cells. After control by short tandem repeats analysis (PCR-STR) of laser micro-dissected tumor and normal epidermal cells from the recipient and donor or PCR-STR and FISH analysis of a tumoral cell line from one SCC, a recipient origin was found in one case but in the two other cases the donor genotype of the tumor was confirmed. In a third step, PCR-STR of laser micro-dissected tumor in 2 cases after sex-matched transplant proved SCC to be of the donor origin. We thus obtain conclusive evidence that 4 epithelial tumors among 8 studied arose from the engrafted donor cells. Tissue stem cells were proposed to exhibit an unexpected level of plasticity, although issues on cell fusions have lead to some controversies. Only transplantation experiments using genetically distinct recipients and donors can unequivocally demonstrate these changes in cell fate. We have analyzed a unique set of squamous cell carcinomas arising in long terms survivors of allogeneic stem cell transplantation. In these patients, chronic graft-versus host disease greatly favors development of solid tumors, possibly as a consequence of mucosal inflammation and immunosuppression. Thus, donor-derived hematopoietic stem cells recruited to sites of chronic inflammation yielded epithelial tumors.

Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus

In infected cells, hepatitis C virus (HCV) core protein is targeted to lipid droplets, which serve as intracellular storage organelles. Using a tissue culture system to generate infectious HCV, we have shown that the coating of lipid droplets by the core protein occurs in a time-dependent manner and coincides with higher rates of virus production. At earlier times, the protein was located at punctate sites in close proximity to the edge of lipid droplets. Investigations by using Z-stack analysis have shown that many lipid droplets contained a single punctate site that could represent positions where core transfers from the endoplasmic reticulum membrane to droplets. The effects of lipid droplet association on virus production were studied by introducing mutations into the domain D2, the C-terminal region of the core protein necessary for droplet attachment. Alteration of a phenylalanine residue that was crucial for lipid droplet association generated an unstable form of the protein that could only be detected in the presence of a proteasome inhibitor. Moreover, converting two proline residues in D2 to alanines blocked coating of lipid droplets by core, although the protein was directed to punctate sites that were indistinguishable from those observed at early times for wild-type core protein. Neither of these virus mutants gave rise to virus progeny. By contrast, mutation at a cysteine residue positioned 2 aa upstream of the phenylalanine residue did not affect lipid droplet localization and produced wild-type levels of infectious progeny. Taken together, our findings indicate that lipid droplet association by core is connected to virus production.

Stroke Injury in Rats Causes an Increase in Activin A Gene Expression Which is Unaffected by Oestradiol Treatment

Activins are members of the transforming growth factor-beta superfamily that exert neurotrophic and neuroprotective effects on various neuronal populations. To determine the possible function of activin in stroke injury, we assessed which components of the activin signalling pathway were modulated in response to middle cerebral artery occlusion (MCAO). Furthermore, because oestradiol replacement protects against MCAO-induced cell death, we explored whether oestradiol replacement influences activin gene expression. Female Sprague-Dawley rats underwent permanent MCAO and the expression of activins and their corresponding receptors was determined by semiquantitative reverse transcriptase-polymerase chain reaction at 24 h after onset of ischaemia. We observed up-regulation of activin betaA and activin type I receptor A mRNA in response to injury. Dual-label immunocytochemistry followed by confocal z-stack analysis showed that the activin A expressing cells comprised neurones. Next, we monitored the time course of activin betaA mRNA expression in oestradiol- or vehicle-treated rats at 4, 8, 16 and 24 h after MCAO via in situ hybridisation. Starting at 4 h after injury, activin betaA mRNA was up-regulated in cortical and striatal areas in the ipsilateral hemisphere. Activin betaA mRNA levels in the cortex increased dramatically with time and were highest at 24 h after the insult, and oestradiol replacement did not influence this increase.

A Quantitative Method for Determining Polarization of Neutrophil Adhesion Molecules Associated with Ischemia Reperfusion

Ischemia-reperfusion-induced neutrophil adhesion to endothelium is CD18-dependent, but information regarding polarity of CD18 adhesion molecules remains speculative. This study evaluated neutrophil adhesion using an in vitro cell adhesion assay and introduces a quantitative method of measuring CD18 membrane distribution using confocal microscopy. Neutrophils from normal animals were isolated from whole blood and incubated with plasma from rat gracilis muscle flaps with no ischemia and reperfusion (nonischemic control, n = 10) or 4 hours of ischemia and 90 minutes of reperfusion (ischemia/reperfusion, n = 10), on coverslips pretreated with and without (phosphate-buffered saline) soluble intercellular adhesion molecules. Coverslips without intercellular adhesion molecules represented a negative control (intercellular adhesion molecules were required for adhesion). Percent adherence to intercellular adhesion molecules was expressed as a ratio of adherent cells/total cells. CD18 polarization was assessed by staining neutrophils with fluorescein isothiocyanate-labeled anti-CD11b, followed by confocal microscopy and Z-stack analysis. Membrane-associated CD18 was expressed as fluorescence intensity units in three equal areas of the cell membrane. Capping was defined as twice as much fluorescence in 33 percent of the cell membrane as in the remaining 67 percent. Neutrophils exposed to ischemia and reperfusion plasma showed a significant increase in adhesion (0.8 +/- 0.1 percent versus 16.7 +/- 2.2 percent, p < 0.001) and CD18 polarization (6.2 +/- 1.7 percent versus 43.9 +/- 12.2 percent, p = 0.0206) compared with controls. This article describes an in vitro assay that reliably reproduces the neutrophil adhesion phenomenon associated with ischemia-reperfusion injury. Results from confocal microscopy allowed for quantitative estimation of membrane-associated receptor polarization.

Activin gene expression increases after stroke injury: Modulation by estradiol

Activins are members of the Transforming Growth Factor-β (TGF-β) superfamily that are involved in regulation of hormone secretion, wound repair, cell differentiation, and organogenesis. Within the central nervous system, activins exert neurotrophic and neuroprotective effects on various neuronal populations. To determine a possible function of activin in stroke injury, we assessed which components of the activin signaling pathway were modulated in response to middle cerebral artery occlusion (MCAO). Female Sprague-Dawley rats underwent permanent MCAO and the expression of activins and their corresponding receptors was determined by semiquantitative RT-PCR at 24h after onset of ischemia. Activin A mRNA increased tenfold in the ipsilatal cortex and 4.5 fold in the ipsilateral striatum compared to the uninjured contralateral side. ActRIA mRNA expression was induced 1.6 fold in the cortex and 2.4 fold in the striatum. To monitor the effect of estradiol treatment on the injury-induced activin A gene expression, we determined the time course of activin A mRNA induction in vehicle- and estradiol-treated animals. Brains were collected 4h, 8h, 16h, and 24h following stroke injury and activin A mRNA levels were determined by in situ hybridization. Starting at 4h after injury, activin A mRNA expression was induced in cortical and striatal areas ipsilateral to the injury. Activin A mRNA levels were highest at 24h after the insult and not modulated by estradiol treatment. Immunocytochemistry revealed that changes in mRNA expression were paralleled by changes in protein expression. Dual-label immunocytochemistry followed by confocal z-stack analysis identified neurons as the activin A expressing cells. Upregulation of activin A has been demonstrated in various in vivo paradigms of brain injury and exogeneous application of activin A has been shown to rescue neurons from a variety of toxic insults. Thus, our results provide evidence for a neuroprotective action of activin A in stroke injury.