Cryo Sections Research Articles

FC091: Changes in the Gut Microbiome and Systemic Metabolome in an In Vivo Model of Peritoneal Dialysis Supplemented with Alanyl-Glutamine

Abstract BACKGROUND AND AIMS Long-term usage of peritoneal dialysis (PD) leads to morphological and functional changes—such as progressive thickening, vascularization and vasculopathy—of the peritoneum, thereby limiting PD efficiency. Vasculopathy induces changes in permeability of the endothelial barrier, which has been suggested to influence the gut microbiome (gMB). Microbial dysbiosis in turn can potentiate inflammation, a risk factor for cardiovascular disease (CVD). The complex interplay of molecules associated with CKD and PD with the gMB is hypothesized to be in causal relationship with occurrences of cardiovascular events and progression of CVD. Novel PD fluids that slow or prevent these processes are in need. The immunomodulatory and cytoprotective compound alanyl-glutamine (AG) has been shown in other model systems to improve the endothelial barrier function, supposedly protecting the gMB from the extraintestinal environment. Our aim is to identify changes of the gMB and the systemic metabolome caused by the addition of AG to PD under conditions of CKD. METHOD Mice (C57/Bl6 NCrl) underwent a 5/6 nephrectomy to induce uraemia. Via a subcutaneously implanted catheter the animals received five times per week a daily injection of 2 mL PD fluid (3.86% glucose with/without 8 mM AG) for six weeks to a total of 30 injections. During the experiment, mice received standard chow and tap water ad libitum. At the end of the experiment, fecal samples from three different intestinal sites and stool were collected and a serum sample was drawn from each mouse. Tissue samples from parietal and visceral peritoneum and relevant organs were preserved for paraffin and cryo sections. Peritoneal effluent cells were spun down and snap-frozen in liquid nitrogen and the supernatant was stored on −80°C. Bacterial ribosomal RNA (rRNA) was isolated from each fecal sample and the V4 region of the 16S rRNA was amplified. Assessment of the plasma metabolome was performed by targeted mass spectrometry (MxP Quant 500, Biocrates, Austria). The experiment was approved by the institutional animal ethics committee. RESULTS Elevated creatinine levels after subtotal nephrectomy confirmed the uremic status of the mice. Descriptive analysis of the gMB showed decreased Shannon diversity, indicating reduced bacterial diversity, in uremic and PD fluid receiving mice over time, whereas AG receiving and control mice retained a stable Shannon diversity. Furthermore, the gMB of mice exposed to PD fluid with AG showed five bacterial classes and 13 bacterial orders to be differently abundant versus mice exposed to PD fluid without AG. In the colon of uremic mice Clostridia was decreased versus control. Serum concentrations of 630 metabolites were assessed by our metabolomics approach. A significant increase in alanine and glutamine serum concentrations was seen in mice exposed to AG supplemented PD fluid. Further, 17 amino acids and four biogenic amines showed to be differently abundant in mice exposed to AG supplemented PD fluid versus without AG. Lastly, plasma of uremic mice showed a significant increase of the toxic amino acid symmetric dimethyl arginine (SDMA) and citrulline. CONCLUSION Exposure to PD fluid and having chronic kidney disease both affect the gut microbiome and systemic metabolome. Supplementation of AG to PD fluid showed changes at overall microbiome diversity and serum metabolites. Further analysis of these changes in the context of tissue alterations will clarify functional differences of the cytoprotective additive AG. Preservation of the mesothelial and endothelial barrier function may represent a mechanistic explanation for the observed improvement in protein loss and systemic inflammation in a recent phase II clinical trial in PD patients. Better understanding of the gut-peritoneal barrier may open up new therapeutic targets for improving clinical outcome of PD treatment and its long-term efficacy.

A One-Stop Protocol to Assess Myocardial Fibrosis in Frozen and Paraffin Sections.

Masson’s Trichrome Staining (MTS) is a useful tool for analyzing fibrosis in a plethora of disease pathologies by differential staining of tissue components. It is used to identify collagen fibers in different tissues like heart, lung, skin, and muscles. Especially in cardiac fibrosis, MTS stains the collagen fibers (blue color), which helps in the distinction of scar area versus the healthy area (red color). However, there are several challenges to stain both paraffin-embedded sections and frozen (cryosections) using a single protocol. Therefore, the goal of this study was to develop a simple short protocol to assess cardiac fibrosis in both paraffin-embedded and cryo heart sections. MTS uses three different stains, i.e., Weigert’s Iron Hematoxylin, Biebrich scarlet-acid fuchsin, and aniline blue to detect nuclei, cytoplasm, and collagen, respectively. In this study, we developed a simple short protocol that can be adapted by any lab to easily assess cardiac fibrosis in paraffin and frozen heart sections. Furthermore, we have addressed the challenges that are commonly faced during the immunostaining process and troubleshooting techniques. Overall, we have successfully developed a simple one-step protocol to assess myocardial fibrosis in paraffin-embedded and frozen cryosections.

Cartilage matrix remodelling differs by disease state and joint type.

Dramatic alterations in mechanical properties have been documented for osteoarthritic (OA) cartilage. However, the matrix composition underlying these changes has not been mapped and their aetiology is not entirely understood. We hypothesised that an understanding of the cartilage matrix heterogeneity could provide insights into the origin of these OA-related alterations. We generated serial transverse cryo sections for 7 different cartilage conditions: 2 joint sites (knee and hip), 2 disease states (healthy and OA) and 3 tissue depths (superficial, middle and deep). By laser capture microscopy, we acquired ~200 cartilage matrix specimens from territorial (T) and interterritorial (IT) regions for all 7 conditions. A standardised matrix area was collected for each condition for a total of 0.02 ± 0.001 mm3 (corresponding to 20 µg of tissue) from a total of 4800 specimens. Extracted proteins were analysed for abundance by targeted proteomics. For most proteins, a lower IT/T ratio was observed for the OA disease state and knee joint type. A major cause of the altered IT/T ratios was the decreased protein abundance in IT regions. The collagenase-derived type III collagen neo-epitope, indicative of collagen proteolysis, was significantly more abundant in OA cartilage. In addition, it was enriched on average of 1.45-fold in IT relative to T matrix. These results were consistent with an elevated proteolysis in IT regions of OA cartilage, due to degenerative influences originating from synovial tissue and/or produced locally by chondrocytes. In addition, they offered direct evidence for dynamic remodelling of cartilage and provided a cogent biochemical template for understanding the alterations of matrix mechanical properties.

Successful Cryo Electron Tomography of Vitreous Cryo Sections

Successful Cryo Electron Tomography of Vitreous Cryo Sections

P 114 - High cholesterol diet-mediated unfolded protein response activation enhances autophagic cell death in heart tissue

The role of ER in the proper synthesis and correct folding of protein, to establish the homeostasis of organism, is well documented by various studies. Following excessive oxidative stress, the accumulation of unfolded/misfolded proteins increase in the lumen of ER which blocks the inhibitory effect of GRP78 on UPR regulatory proteins; IRE1, PERK and ATF6. It is clear that ER stress mediated UPR is a potent inducer of proteasomal and autophagic activities which serves as a degradation process for maintaining stress tolerance, limited damage and viability under stress conditions. Interestingly, aberrantly activation of autophagy can result in destroying major portions of the cytosol and organelles, which will lead to autophagic death of cardiac myocytes, and development of cardiac failure. In the present study Grp78, Grp94, pIRE1, pPERK, derlin-1, proteasome β5 subunit, VCP, beclin-1 and LC3-II, well known markers of ER stress, proteasomal and autophagic activities, are investigated in the heart tissue of rabbits. Furthermore, autophagic activity in heart tissue is observed in hypercholesterolemic vs control rabbits by electron microscopy. Additionally, colocalization studies of mitochondria and p62 in cryo sections of heart tissue were applied to understand the levels of mitochondria degradation by autophagy.

A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Immunodetection of NETs in Paraffin-Embedded Tissue.

The pathogenic potential of neutrophil extracellular traps (NETs) was recently described, and their detection in tissue could serve as a prognostic marker. NETs are delicate and filigree structures; hence good tissue preservation is essential for their detection. Indeed, analysis of paraffin-embedded tissue has proven superior to the study of cryo sections. Though, under favorable conditions, the presence of NETs can be detected in tissue sections stained with histological dyes, definitive identification of NETs needs the colocalization of immunofluorescent signals for both nuclear and granular (or cytoplasmic) NET components. We tested diverse antigen retrieval methods and various combinations of commercially available antibodies and present here staining protocols to detect NETs in human and murine tissue sections.

Astrocyte migration in injectable gelatin-hydroxyphenyl propionic acid matrices for neuronal guidance in spinal cord injury

Event Abstract Back to Event Astrocyte migration in injectable gelatin-hydroxyphenyl propionic acid matrices for neuronal guidance in spinal cord injury Josna Joseph1*, Kazuhide Hayakawa2*, Motoichi Kurisawa3*, Li Shan Wang3*, Wanting Niu1 and Myron Spector1 1 VA Boston Healthcare System, Brigham&Women's Hospital, Harvard Medical School, Tissue Engineering, United States 2 Massachusetts General Hospital, Neuroprotection Research laboratory, Department of Neurology& Radiology, United States 3 Institute for Bioengineering and Nanotechnology, Singapore Introduction: The glial scar that is formed around residual cavities after certain spinal cord injuries is a physical and physiological impediment to neuronal regeneration. Gelatin–hydroxyphenyl propionic acid (Gtn-HPA), a novel injectable matrix with tuneable cross-linking properties[1], could provide the missing stroma in such defects, enabling migration of astrocytes from the glial scar into the matrix-filled defect and their guidance of neurons into the lesion[2]. The principal objective of this study was to evaluate astrocyte migration and activation in an in vitro ‘tissue simulant’ model using Gtn-HPA incorporating select growth factors. Materials and Methods: Cortical astrocytes were isolated from postnatal rats, and grown in DMEM high glucose medium. Astrocyte proliferation in Gtn-HPA gels at days 2, 4 and 7 was analysed by counting live cells stained by Calcein-AM under fluorescence microscopy. A three-dimensional migration assay model was set up in 3% agarose-coated 24-well culture plates by surrounding Gtn-HPA cores (1cm diameter) containing EGF (50 or 200 ng/ul) or TGF-β1 (50 or 200 ng/ul) with an annular tissue simulant of 2 wt. % Gtn-HPA gel (cross-linked with 0.1 U/ml horseradish peroxidase and 1.2 mM H202) containing 3*105 cells/ml. Cellular migration into the growth factor-laden inner core was analysed quantitatively by counting cells and measuring the migration distance (n=5 fields) from the gel interface to the farthest cell body (n=4 gels) under phase contrast microscopy. Immunostaining of 30-mm thick cryo sections of gel construct was carried out to detect the presence of GFAP-positive astrocytes. GFAP expression by migrated cells was quantified by Western blot by lysing the gel with collagenase 1 (500U/ml). The results were tested for statistical significance by ANOVA and post-hoc tests. Results and Discussion: Astrocytes migrated in response to chemotactic gradients in Gtn-HPA gels in contrast to the blank gels which displayed virtually no migration. The migration distance was significantly higher (p<0.001) in TGF-50 ng (TGFL) group when compared to EGF and blank Gtn-HPA control group (Fig.1). The number of cells infiltrating the inner core and GFAP expression was significantly higher in the EGF-50 ng (EGFL) group (Fig.2). This indicates that astrocytes migrate well in response to TGF-β1 and are activated by EGF at low concentration, whereas higher conc. of EGF has no specific effect on GFAP expression. Conclusion: This study shows that growth factor-laden Gtn-HPA hydrogels provide a supportive environment for astrocyte migration, proliferation and activation. Gtn-HPA hydrogels laden with optimum combination of EGF and TFG-β1 could effectively guide astrocyte infiltration and subsequent neurite outgrowth in a spinal cord injury defect with diminished glial scarring. J. Joseph acknowledges Fulbright Fellowship from United States India Educational Foundation. Gtn-HPA was provided by the Institute of Bioengineering and Nanotechnology, Singapore.

Improved Hilbert phase contrast for transmission electron microscopy

Hilbert phase contrast has been recognized as a means of recording high resolution images with high contrast using a transmission electron microscope. This imaging mode could be used to image typical phase objects such as unstained biological molecules or cryo sections of biological tissue. According to the original proposal by (Danev et al., 2002) the Hilbert phase plate applies a phase shift of π to approximately half the focal plane (for example the right half excluding the central beam) and an image is recorded at Gaussian focus. After correction for the inbuilt asymmetry of differential phase contrast this image will have an almost perfect contrast transfer function (close to 1) from the lowest spatial frequency up to a maximum resolution determined by the wave length and spherical aberration of the microscope. In this paper I present theory and simulations showing that this maximum spatial frequency can be increased considerably almost without loss of contrast by using a Hilbert phase plate of half the thickness, leading to a phase shift of π/2, and recording images at Scherzer defocus. The maximum resolution can be improved even more by imaging at extended Scherzer defocus, though at the cost of contrast loss at lower spatial frequencies.

Targeted nano analysis of water and ions in the nucleus using cryo-correlative microscopy.

The cell nucleus is a crowded volume in which the concentration of macromolecules is high. These macromolecules sequester most of the water molecules and ions which, together, are very important for stabilization and folding of proteins and nucleic acids. To better understand how the localization and quantity of water and ions vary with nuclear activity, it is necessary to study them simultaneously by using newly developed cell imaging approaches. Some years ago, we showed that dark-field cryo-Scanning Transmission Electron Microscopy (cryo-STEM) allows quantification of the mass percentages of water, dry matter, and elements (among which are ions) in freeze-dried ultrathin sections. To overcome the difficulty of clearly identifying nuclear subcompartments imaged by STEM in ultrathin cryo-sections, we developed a new cryo correlative light and STEM imaging procedure. This combines fluorescence imaging of nuclear GFP-tagged proteins to identify, within cryo ultrathin sections, regions of interest which are then analyzed by STEM for quantification of water and identification and quantification of ions. In this chapter we describe the new setup we have developed to perform this cryo-correlative light and STEM imaging approach, which allows a targeted nano analysis of water and ions in nuclear compartments.

Histological control of tumor surgical margins with three-dimensional histology.

Three-dimensional (3D) histology provides a complete 3D representation of the margins of a tumor specimen on histological slides. This eliminates diagnostic gaps and provides maximum sensitivity in identifying tumor outgrowths. The method is used to confirm tumor-free margins or, in case of an incomplete tumor resection, to make even very small infiltrated areas visible with topographic orientation. The basic principle of 3D histology is the conversion from the 3D structure of the tumor-specimen margins into a two-dimensional view of histological sections. The 3D margins are flattened with their outside down and then cut into pieces, suitable for histopathological procedures of cryo or paraffin sections. The management of a full 3D histology needs a tight communication between the surgeon and the pathologist. Clear and safe rules are necessary to enable both the surgeon and the pathologist to point out areas of incomplete tumor excisions very precisely. Using a circle as representation of the separation between tumor base and margin, the pathologist can point out the exact position of an outgrowth by referring to different positions on the circle in clock times. This system will be sufficient to point out tumor outgrowths. The advantage is that it is very simple to communicate and easy implemented in any electronic communication.

Oil Red O-assessed macrosteatosis in liver transplant donor biopsies predicts ischemia–reperfusion injury and clinical outcome

Steatosis in donor livers is an accepted adverse prognostic factor after liver transplantation. While its semiquantitative assessment shows varying reproducibility, it is questioned as a standard method. Additionally, the influence of hepatic steatosis on ischemia/reperfusion injury (I/R injury) has not been evaluated in biopsies after reperfusion. We compared different staining and analyzing methods for the assessment of donor liver steatosis in order to predict I/R injury and clinical outcome after transplantation. To do this, 56 paired pre- and post-reperfusion liver biopsies were analyzed for macro- (MaS)/micro- (MiS) and total steatosis in cryo and permanent sections by special fat (Oil Red O or ORO) and standard stains. Computerized morphometrical analyses were compared to the semiquantitative assessment by a pathologist. I/R injury was determined histopathologically and by M30 immunohistochemistry. We found ORO to be more sensitive in detecting hepatic steatosis with higher reproducibility for MaS. Semiquantitative analyses were highly reproducible and not inferior to computerized morphometry. Categorized MaS as determined by ORO correlated with the extent of I/R injury, initial poor function, liver enzymes, and survival. Therefore fat stains like ORO are a reliable and easy method comprising significant advantages in the evaluation of hepatic steatosis and are thereby of prognostic value. Computerized analysis is a precise tool though not superior to semiquantitative analyses.

Continuous digital hypothermia initiated after the onset of lameness prevents lamellar failure in the oligofructose laminitis model

Prophylactic digital hypothermia reduces the severity of acute laminitis experimentally but there is no evidence for its efficacy as a treatment once lameness has already developed. To investigate the therapeutic effects of digital hypothermia, applied after the onset of lameness, in an experimental acute laminitis model. Randomised, controlled (within subject), blinded, experimental trial. Eight Standardbred horses underwent laminitis induction using the oligofructose model. Once lameness was detected at the walk, one forelimb was continuously cooled (CRYO), with the other forelimb maintained at ambient temperature (NON-RX). Dorsal lamellar sections (proximal, middle and distal) harvested 36 h after the onset of lameness/initiation of cryotherapy were analysed by 2 blinded observers: laminitis pathology was scored (0 [normal] to 4 [severe]) and morphometric analyses performed. Median (interquartile range) histological scores were greater (P<0.05) in NON-RX (proximal 2.8 [2.5-4]; middle 3.5 [2-4]; distal 2.5 [2-3.8]) compared with CRYO limbs (proximal 0.5 [0.5-1.4]; middle 1 [0.6-1]; distal 0.75 [0.5-1]). There was complete physical separation of lamellar dermis from epidermis (score of 4) in 4 of the NON-RX feet at one or more section level(s), which was not observed in any CRYO sections. Histomorphometry was thus limited to sections that remained intact; there was a trend of increased total (TELL) and secondary (SELL) epidermal lamellar length and decreased secondary epidermal lamellar width (SELW) in NON-RX limbs compared with CRYO at all 3 levels; differences were significant (P<0.05) for SELL and SELW in the distal sections. Digital hypothermia reduced the severity of lamellar injury and prevented lamellar structural failure (complete dermoepidermal separation) when initiated at the detection of lameness in an acute laminitis model. This study provides the first evidence to support the use of therapeutic digital hypothermia as a treatment for acute laminitis.

A Unique Immunofluorescence Protocol to Detect Protein Expression in Vascular Tissues: Tacking a Long Standing Pathological Hitch.

Autofluorescence induced interference is one of the major drawbacks in immunofluorescence analysis of formalin-fixed paraffin-embedded tissues, as it decreases the signal-to-noise ratio of specific labeling. Apart from aldehyde-fixation induced artifacts; collagen and elastin, red blood cells and endogenous fluorescent pigment lipofuscin are prime sources of autofluorescence in vascular and aging tissues. We describe herein, an optimized indirect-immunofluorescence method for archival formalin-fixed paraffin-embedded tissues tissues and cryo sections, using a combination of 3-reagents in a specific order, to achieve optimal fluorescence signals and imaging. Human telomerase reverse transcriptase, a protein implicated as a proliferation marker, was chosen relevant to its expression in solid tumors along with 3 other intracellular proteins exhibiting nuclear and/or cytoplasmic expression. Staining was performed on 10 glioma tissue sections along with 5 of their cryo sections, 5 sections each of hepatocellular, lung, papillary-thyroid and renal cell carcinoma, with 10 non-malignant brain tissue samples serving as control. Specimens were imaged using epifluorescence microscopy, followed by software-based quantification of fluorescence signals for statistical analysis and validation. We observed that the combined application of sodium-borohydride followed by crystal violet before antigen retrieval and a Sudan black B treatment after secondary antibody application proved to be most efficacious for masking autofluorescence/non-specific background in vascular tissues. This unique trio-methodology provides quantifiable observations with maximized fluorescence signal intensity of the target protein for longer retention time of the signal even after prolonged storage. The results can be extrapolated to other human tissues for different protein targets.

463 CELL VIABILITY IN HUMAN SAMPLES OF HERNIATED INTERVERTEBRAL DISC: IMPLICATIONS FOR BIOLOGICAL THERAPIES

Purpose: The intervertebral disc is dependent on the extracellular matrix for its function, which is produced and maintained by the resident cells. However, disc degeneration is common and may present early on in life, being associated with major clinical problems such as herniation and back pain. Previous work suggests that cells isolated from the degenerate intervertebral disc have a very limited potential for regeneration, with a number of the cells necrotic and/or senescent. Never-the-less cells are currently being used in the clinic for cell therapy which have been isolated from routine discectomy patients. Since regulatory authorities are now placing more emphasis on cell therapies being classed as pharmaceutical products, a deeper knowledge of the source and quality of cells used is required. Thus, this study was designed to assess the viability of cells present in surgical samples of degenerate intervertebral disc tissue.Methods: The technique for live/dead differentiation of cells used in this study utilises the fluorescent probes chloromethyl fluoresce in diacetate (CMFDA) and ethidium homodimer (EH). Samples of herniated discs were obtained from patients undergoing routine discectomy and processed within 2 hours of excision. A small piece of each sample, approximately5mm2, was incubated in 1 ml of 25 mM CMFDA/2mM EH solution in the dark for 2 hours at 37°C. The tissue was then blotted dry and snap frozen. Cryo sections were taken at various points throughout the thickness of the tissue sample, mounted with Vectashield containing DAPI and visualised with a fluorescent microscope. Cell viability was analysed by counting at least 200 cells from a minimum of five fields of view.Results: To date 21 surgical disc samples from patients, aged 17–57 years,have been examined. Preliminary results show that greater than 80%of cells are alive after excision. Cells in these discs commonly occur in clusters (approximately 54% of disc cells are clustered). Clustered cells exhibited a higher proportion of live cells compared to single cells with in the same tissue section, e.g. 93% of clustered cells were viable compared to 82% of single cells. Cell viability appeared consistent across all locations of the disc samples. Dead cells, when they did occur, were predominantly seen around the periphery of the disc sections which may be derived from trauma during dissection.Conclusions: In contrast to reports in the literature, these results indicate that the viability of cells from the herniated disc may be adequate to consider for utilising in biological therapies. Interestingly, a greater proportion of clustered cells appear to be viable compared to single cells.Additionally we plan to investigate whether there is any relationship between cell viability and the pathology, duration of symptoms or age of patient, as this may serve as an indicator of the suitability of degenerate discs for cell therapy.

Imaging Specific Protein Labels on Eukaryotic Cells in Liquid with Scanning Transmission Electron Microscopy

Understanding the structure and dynamics of the protein complexes that underlie cellular function is a central scientific challenge. Biochemical techniques used to identify such complexes would be enhanced by the imaging of specific molecular positions in the context of intact cells, with protein-scale resolution (on the order of a few nanometers). Currently, though, nanometer resolution can only be achieved at the cost of less-direct imaging of the unperturbed cell. Cellular ultrastructure is traditionally studied by transmission electron microscopy (TEM), which yields nanometer resolution on embedded and stained sections, or cryo sections. These cellular samples are neither intact nor in their native liquid state. Light microscopy is used to image protein distributions in fluorescently labeled cells in liquid to investigate cellular function, but even recent improvements in resolution by nanoscopy techniques are still insufficient to resolve the individual constituents of protein complexes. Thus, development of techniques capable of high-resolution imaging in native cellular states would contribute significantly to our understanding of cellular function at the molecular level. The development of liquid compartments that include electron-transparent silicon nitride membrane windows has led to the introduction of a novel concept to achieve nanometer resolution on tagged proteins in cells.

Application of cryo-compatible antibodies to human placenta paraffin sections

The hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) -fixation and paraffin embedding technique has been described to expand possibilities for immuno-labellings due to low denaturation of proteins. In this study, the issue was addressed as to whether the HOPE technique could be a useful tool in placenta tissue-based studies when only cryo-compatible antibodies are available. Such antibodies can be used on cryostat sections only, giving results of considerably inferior morphological detail as compared to routinely fixed paraffin embedded tissue sections. Commercially available, only cryo-compatible, monoclonal antibodies against a conformational epitope of HLA-G (clone MEM-G/9) and leukocyte differentiation antigens CD56, CD163 and CD34 III were selected and applied to frozen sections, routinely formalin-fixed and HOPE-fixed paraffin sections. All tested antibodies immunolocalized their antigen on cryo sections and on HOPE-fixed but not formalin-fixed paraffin sections. The HOPE technique provides an excellent preservation of protein antigenicity together with well presented morphological details in paraffin embedded placenta tissues. The detection of native or conformation-dependent epitopes in paraffin sections expands the immunolocalization possibilities in placenta research and reproductive immunology.

Semenogelins in the human retina: Differences in distribution and content between AMD and normal donor tissues

The two cellular targets of interest in age-related macular degeneration (AMD) are the photoreceptors and the RPE. However, the mechanisms involved in AMD pathology are not yet fully understood. In the present report, we extend our previous studies on semenogelin proteins (Sgs) in normal human retina and compare these with the distribution in retinas from AMD donor eyes. Semenogelins I (SgI) and II (SgII) are the major structural protein components of semen coagulum, but have been recently found in non-genital tissues as well. Cryo and paraffin sections of human retina were processed for both immunofluorescence and DAB reaction with a specific antibody. The presence of SgI was analyzed in retina and RPE total lysates and SgI was detected by western blot in human retina and RPE. The intensity of immunoreactivity was significantly reduced in the AMD eyes. SgI is expressed in the normal human retina and in the retina of AMD donor eyes, where localization was detected in the photoreceptors and in a few ganglion cells. We find the distribution of SgI in the AMD retinas substantially lower than observed in normal retina. SgI localization to photoreceptors and the RPE suggests a possible function related to the ability of these cells to sequester zinc.

Abstract 623: Role Of Cd73-derived Adenosine In Atherosclerosis

We recently reported that mice with targeted deletion of ecto-5′-nucleotidase (CD73) are characterized by enhanced platelet activation and increased adherence of monocytes to the endothelium. Wire-induced injury of the carotid artery resulted in enhanced neointima formation associated with increased macrophage content and VCAM-1 expression in CD73−/−mice. In order to study a possible modulation of atherogenesis and vascular inflammation by CD73-derived adenosine, we generated ApoE−/−/CD73−/− double mutant mice. Quantification and characterisation of atherosclerotic lesions was performed in WT, ApoE−/−, and ApoE−/− / CD73−/− mice on chow diet after 6 and 12 month, respectively, by macroscopic analysis of the descending thoracic aorta after oil red staining and by histological stainings for cholesterol, hyaloronic acid, and collagen in cryo sections of the aortic sinus. Immunohistological analysis of lesion morphology included primary antibodies for ICAM-1, VCAM-1, CD4, CD73, and CD11b. Determination of the plaque score by oil red staining revealed enhanced development of atherosclerotic lesions over the entire thoracic aorta after 6 month in the double mutant compared with ApoE−/− (2.5-fold increased, n=8, P&lt;0.05). Atherosclerotic plaques in the aortic sinus were substantially enlarged as compared to ApoE−/− mice. Changes in extracellular matrix composition were not detected. However, we found enhanced VCAM-1 expression in ApoE−/−/CD73−/− mice after 6 month, which was accompanied by an increased infiltration of monocytes and macrophages but unchanged T-cells. We also noted that in atherosclerotic plaques of ApoE−/− mice the expression and activity of CD73 was significantly increased compared to WT. Measurements of cytokines in plasma support the notion of an increased inflammatory state in the double knockout. Interestingly, after 12 months all double mutant mice showed multiple scattered myocardial infarcts associated with myocardial hypertrophy and fibrosis. Our findings demonstrate that CD73-derived adenosine acts as an endogenous modulator protecting against chronic vascular inflammation and monocyte recruitment. Thus in the murine model, extracellular adenosine appears to limit the progression of atherosclerosis.

Elevated Expression and Genetic Association Links the SOCS3 Gene to Atopic Dermatitis

In a systematic analysis of global gene-expression patterns, we found that SOCS3 messenger RNA was significantly more highly expressed in skin from patients with atopic dermatitis than in skin from healthy controls, and immunohistochemical analysis confirmed a similar elevation of SOCS3 protein. Furthermore, we found a genetic association between atopic dermatitis and a haplotype in the SOCS3 gene in two independent groups of patients (P<.02 and P<.03). These results strongly suggest that SOCS3, located in a chromosomal region previously linked to the disease (17q25), is a susceptibility gene for atopic dermatitis.