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The aim of this review is to give an outline of the blood clearance function of the liver sinusoidal endothelial cells (LSECs) in health and disease. Lining the hundreds of millions of hepatic sinusoids in the human liver the LSECs are perfectly located to survey the constituents of the blood. These cells are equipped with high-affinity receptors and an intracellular vesicle transport apparatus, enabling a remarkably efficient machinery for removal of large molecules and nanoparticles from the blood, thus contributing importantly to maintain blood and tissue homeostasis. We describe here central aspects of LSEC signature receptors that enable the cells to recognize and internalize blood-borne waste macromolecules at great speed and high capacity. Notably, this blood clearance system is a silent process, in the sense that it usually neither requires or elicits cell activation or immune responses. Most of our knowledge about LSECs arises from studies in animals, of which mouse and rat make up the great majority, and some species differences relevant for extrapolating from animal models to human are discussed. In the last part of the review, we discuss comparative aspects of the LSEC scavenger functions and specialized scavenger endothelial cells (SECs) in other vascular beds and in different vertebrate classes. In conclusion, the activity of LSECs and other SECs prevent exposure of a great number of waste products to the immune system, and molecules with noxious biological activities are effectively “silenced” by the rapid clearance in LSECs. An undesired consequence of this avid scavenging system is unwanted uptake of nanomedicines and biologics in the cells. As the development of this new generation of therapeutics evolves, there will be a sharp increase in the need to understand the clearance function of LSECs in health and disease. There is still a significant knowledge gap in how the LSEC clearance function is affected in liver disease.

BackgroundLiver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs; liver resident macrophages) form the body’s most effective scavenger cell system for the removal of harmful blood-borne substances, ranging from modified self-proteins to pathogens and xenobiotics. Controversies in the literature regarding the LSEC phenotype pose a challenge when determining distinct functionalities of KCs and LSECs. This may be due to overlapping functions of the two cells, insufficient purification and/or identification of the cells, rapid dedifferentiation of LSECs in vitro, or species differences. We therefore characterized and quantitatively compared expressed gene products of freshly isolated, highly pure LSECs (fenestrated SE-1/FcγRIIb2+) and KCs (CD11b/c+) from Sprague Dawley, Crl:CD (SD), male rats using high throughput mRNA-sequencing and label-free proteomics.ResultsWe observed a robust correlation between the proteomes and transcriptomes of the two cell types. Integrative analysis of the global molecular profile demonstrated the immunological aspects of LSECs. The constitutive expression of several immune genes and corresponding proteins of LSECs bore some resemblance with the expression in macrophages. LSECs and KCs both expressed high levels of scavenger receptors (SR) and C-type lectins. Equivalent expression of SR-A1 (Msr1), mannose receptor (Mrc1), SR-B1 (Scarb1), and SR-B3 (Scarb2) suggested functional similarity between the two cell types, while functional distinction between the cells was evidenced by LSEC-specific expression of the SRs stabilin-1 (Stab1) and stabilin-2 (Stab2), and the C-type lectins LSECtin (Clec4g) and DC-SIGNR (Clec4m). Many immune regulatory factors were differentially expressed in LSECs and KCs, with one cell predominantly expressing a specific cytokine/chemokine and the other cell the cognate receptor, illustrating the complex cytokine milieu of the sinusoids. Both cells expressed genes and proteins involved in antigen processing and presentation, and lymphocyte co-stimulation.ConclusionsOur findings support complementary and partly overlapping scavenging and immune functions of LSECs and KCs. This highlights the importance of including LSECs in studies of liver immunity, and liver clearance and toxicity of large molecule drugs and nano-formulations.

Infectious keratoconjunctivitis (IKC) is a common transmissible ocular disease in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus). In large outbreaks, IKC may affect tens of animals in a herd, with the most severe cases often requiring euthanasia due to the destruction of the affected eyes and permanent blindness. An experimental inoculation with cervid herpesvirus 2 (CvHV2), alone or in combination with Moraxella bovoculi, demonstrated that CvHV2 has the ability to cause clinical signs of IKC in previously unexposed reindeer. Tissues collected from upper and lower eyelids, lacrimal gland and cornea, were processed for light and transmission electron microscopy. Histopathological analysis of the eyes inoculated with CvHV2 showed widespread and severe pathological findings. Mucosal tissues from these eyes showed fibrinous and purulent exudates, hyperemia, hemorrhages, necrosis, vascular thrombosis, vascular necrosis, infiltration of mononuclear cells and neutrophils, and lymphoid follicle reaction, which matches the described histopathology of IKC in reindeer. Characteristic alpha-herpesvirus particles matching the size and morphology of CvHV2 were identified by transmission electron microscopy in the conjunctival tissue. The quantification of viral particles by qPCR revealed high copy numbers of viral DNA in all CvHV2 inoculated eyes, but also in the non-inoculated eyes of the same animals. The histopathology of eye tissues obtained from the CvHV2 inoculated reindeer and the lack of inflammation from bacterial infection, together with the detection of CvHV2 DNA in swabs from the inoculated and non-inoculated eyes of the same animals, verified that CvHV2 was the primary cause of the observed histopathological changes.

The liver is constantly exposed to dietary antigens, viruses, and bacterial products with inflammatory potential. For decades cellular uptake of virus has been studied in connection with infection, while the few studies designed to look into clearance mechanisms focused mainly on the role of macrophages. In recent years, attention has been directed towards the liver sinusoidal endothelial cells (LSECs), which play a central role in liver innate immunity by their ability to scavenge pathogen- and damage-associated molecular patterns. Every day our bodies are exposed to billions of gut-derived pathogens which must be efficiently removed from the circulation to prevent inflammatory and/or immune reactions in other vascular beds. Here, we have used GFP-labelled Enterobacteria phage T4 (GFP-T4-phage) as a model virus to study the viral scavenging function and metabolism in LSECs. The uptake of GFP-T4-phages was followed in real-time using deconvolution microscopy, and LSEC identity confirmed by visualization of fenestrae using structured illumination microscopy. By combining these imaging modalities with quantitative uptake and inhibition studies of radiolabelled GFP-T4-phages, we demonstrate that the bacteriophages are effectively degraded in the lysosomal compartment. Due to their high ability to take up and degrade circulating bacteriophages the LSECs may act as a primary anti-viral defence mechanism.

The following article, Larsen, A.K., Nymo, I.H., Sørensen, K.K., Seppola, M., Rødven, R., Jiménez de Bagüés, M.P., ... Godfroid, J. (2018). Concomitant Temperature Stress and Immune Activation may Increase Mortality Despite Efficient Clearance of an Intracellular Bacterial Infection in Atlantic Cod. Frontiers in Microbiology, 9(2963), can be accessed at https://doi.org/10.3389/fmicb.2018.02963. Licensed CC BY-NC-ND 4.0. The environmental temperature has profound effects on biological systems of marine aquatic organisms and plays a critical role in species distribution and abundance. Particularly during the warmer seasons, variations in habitat temperature may introduce episodes of stressful temperatures which the organisms must adapt to and compensate for to maintain physiological homeostasis. The marine environment is changing and predicted raises in water temperatures will affect numerous marine species. Translocation of pathogens follow migration of species and alternations in physical environmental parameters may have influence upon the virulence of pathogens, as well as the hosts immune responses. While pathogenicity of many true pathogens is expected to increase following climate induced temperature stress, the impact from environmental stressors on the occurrence and severity of opportunistic infections is unknown. Here we describe how thermal stress in the cold-water species Atlantic cod influenced the fish immune responses against an opportunistic intracellular bacterium. Following experimental infection with Brucella pinnipedialis at normal water temperature (6°C) and sub-optimal temperature (15°C), cod cleared the intracellular bacteria more rapidly at the highest temperature. The overall immune response was faster and of higher amplitude at 15°C, however, a significant number of cod died at this temperature despite efficient clearance of infection. An increased growth rate not affected by infection was observed at 15°C, confirming multiple energy demanding processes taking place. Serum chemistry suggested that general homeostasis was influenced by both infection and increased water temperature, highlighting the cumulative stress responses (allostatic load) generated by simultaneous stressors. Our results suggest a trade-off between resistance and tolerance to survive infection at sub-optimal temperatures and raise questions concerning the impact of increased water temperatures on the energetic costs of immune system activation in aquatic ectotherms.

Triethylene glycol dimethacrylate (TEGDMA) is commonly used in polymer resin-based dental materials. This study investigated the molecular mechanisms of TEGDMA toxicity by identifying its time- and dose-dependent effects on the proteome of human THP-1 monocytes. The effects of different concentrations (0.07–5 mM) and exposure times (0–72 h) of TEGDMA on cell viability, proliferation, and morphology were determined using a real-time viability assay, automated cell counting, and electron microscopy, and laid the fundament for choice of exposure scenarios in the proteomic experiments. Solvents were not used, as TEGDMA is soluble in cell culture medium (determined by photon correlation spectroscopy). Cells were metabolically labeled [using the stable isotope labeled amino acids in cell culture (SILAC) strategy], and exposed to 0, 0.3 or 2.5 mM TEGDMA for 6 or 16 h before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Regulated proteins were analyzed in the STRING database. Cells exposed to 0.3 mM TEGDMA showed increased viability and time-dependent upregulation of proteins associated with stress/oxidative stress, autophagy, and cytoprotective functions. Cells exposed to 2.5 mM TEGDMA showed diminished viability and a protein expression profile associated with oxidative stress, DNA damage, mitochondrial dysfunction, and cell cycle inhibition. Altered expression of immune genes was observed in both groups. The study provides novel knowledge about TEGDMA toxicity at the proteomic level. Of note, even low doses of TEGDMA induced a substantial cellular response. publishedVersion

The liver sinusoidal endothelial cell (LSEC) forms the fenestrated wall of the hepatic sinusoid and functions as a control post regulating and surveying the trafficking of molecules and cells between the liver parenchyma and the blood. The cell acts as a scavenger cell responsible for removal of potential dangerous macromolecules from blood, and is increasingly acknowledged as an important player in liver immunity. This review provides an update of the major functions of the LSEC, including its role in plasma ultrafiltration and regulation of the hepatic microcirculation, scavenger functions, immune functions, and role in liver aging, as well as issues that are either undercommunicated or confusingly dealt with in the literature. These include metabolic functions, including energy metabolic interplay between the LSEC and the hepatocyte, and adequate ways of identifying and distinguishing the cells.

Aging is characterized by progressive loss of metabolic and biochemical functions and accumulation of metabolic by-products, including advanced glycation end products (AGEs), which are observed in several pathological conditions. A number of waste macromolecules, including AGEs are taken up from the circulation by endocytosis mainly into liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs). However, AGEs still accumulate in different tissues with aging, despite the presence of this clearance mechanism. The aim of the present study was to determine whether the efficiency of LSECs and KCs for disposal of AGEs changes through aging. Results After intravenous administration of 14 C-AGE-albumin in pre-pubertal, young adult, middle aged and old mice, more than 90% of total recovered 14 C-AGE was liver associated, irrespective of age. LSECs and KCs represented the main site of uptake. A fraction of the 14 C-AGE degradation products ( 14 C-AGE-DPs) was stored for months in the lysosomes of these cells after uptake. The overall rate of elimination of 14 C-AGE-DPs from the liver was markedly faster in pre-pubertal than in all post-pubertal age groups. The ability to eliminate 14 C-AGE-DPs decreased to similar extents after puberty in LSECs and KCs. A rapid early removal phase was characteristic for all age groups except the old group, where this phase was absent. Conclusions Removal of AGE-DPs from the liver scavenger cells is a very slow process that changes with age. The ability of these cells to dispose of AGEs declines after puberty. Decreased AGE removal efficiency early in life may lead to AGE accumulation.

Source at http://dx.doi.org/10.1186/s13028-017-0291-2 Background: Infectious keratoconjunctivitis (IKC) is a transmissible disease in semi-domesticated Eurasian reindeer (Rangifer tarandus tarandus). It is regarded as multifactorial and a single causative pathogen has not yet been identified. From clinical outbreaks we have previously identified Cervid herpesvirus 2 (CvHV2) and Moraxella bovoculi as candidates for experimental investigations. Eighteen reindeer were inoculated in the right eye with CvHV2 (n = 5), M. bovoculi (n = 5), CvHV2 and M. bovoculi (n = 5) or sterile saline water (n = 3; controls). Results: All animals inoculated with CvHv2, alone or in combination with M. bovoculi, showed raised body temperature, increased lacrimation, conjunctivitis, excretion of pus and periorbital oedema; clinical signs that increased in severity from day 2 post inoculation (p.i.) and throughout the experiment, until euthanasia 5–7 days p.i. Examination after euthanasia revealed corneal oedema, and three animals displayed a corneal ulcer. CvHV2 could be identified in swab samples from both the inoculated eye and the control eye from most animals and time points, indicating a viral spread from the inoculation site. Conclusions: This study showed that CvHV2 alone and in combination with M. bovoculi was able to cause the characteristic clinical signs of IKC in reindeer, whereas inoculation of M. bovoculi alone, originally isolated from a reindeer with IKC, did not produce clinical signs. Previous studies have suggested that herding procedures, animal stress and subsequent reactivation of latent CvHV2 infection in older animals is a plausible mechanism for IKC outbreaks among reindeer calves and young animals in reindeer herds. However, further studies are needed to fully understand the infection biology and epidemiology associated with IKC in reindeer. Keywords: Alphaherpesvirus, Eye disease, IKC, Moraxella, Ophthalmology, Reindeer, Wildlife

General comprehension of terms and confounding factors associated with in vitro experiments can maximize the potential of in vitro testing of substances. In this systematic review, we present an overview of the terms and methods used to determine low-dose effects of matrix constituents in polymer resin-based dental materials in cell-culture studies and discuss the findings in light of how they may influence the comprehension and interpretation of results. Articles published between 1996 and 2015 were identified by searches in the Scopus, Web of Science, MEDLINE, PubMed, and Embase databases using keywords associated with low-dose effects, polymer resin-based materials, in vitro parameters, and dental materials. Twenty-nine articles were included. Subtoxic (n = 11), sublethal (n = 10), and nontoxic (n = 6) were the terms most commonly used to describe the low-dose effects of methacrylates. However, definition of terms varied. Most (82%) studies employed only one method to define the exposure scenario, and no agreement was seen between studies on the use of solvents. Prophylactic use of antibiotics was widespread, and mycoplasma screening was not reported. In conclusion, cell-culture conditions and tests used to define exposure scenarios have changed little in the last decades, despite development in recommendations. Nomenclature alignment is needed for a better understanding of possible biohazards of methacrylates.

To maintain homeostasis, the animal body is equipped with a powerful system to remove circulating waste.1This review presents evidence that the scavenger endothelial cell (SEC) is responsible for the clearance of blood-borne waste macromolecules in vertebrates. SECs express pattern-recognition endocytosis receptors (mannose and scavenger receptors), and in mammals, the endocytic Fc gamma-receptor IIb2. This cell type has an endocytic machinery capable of super-efficient uptake and degradation of physiological and foreign waste material, including all major classes of biological macromolecules. In terrestrial vertebrates, most SECs line the wall of the liver sinusoid. In phylogenetically older vertebrates, SECs reside instead in heart, kidney, or gills. SECs, thus, by virtue of their efficient nonphagocytic elimination of physiological and microbial substances, play a critical role in the innate immunity of vertebrates. In major invertebrate phyla, including insects, the same function is carried out by nephrocytes. The concept of a dual-cell principle of waste clearance is introduced to emphasize that professional phagocytes (macrophages in vertebrates; hemocytes in invertebrates) eliminate larger particles (>0.5 μm) by phagocytosis, whereas soluble macromolecules and smaller particles are eliminated efficiently and preferentially by clathrin-mediated endocytosis in nonphagocytic SECs in vertebrates or nephrocytes in invertebrates. Including these cells as important players in immunology and physiology provides an additional basis for understanding host defense and tissue homeostasis.

Atherogenesis is associated with elevated levels of low-density lipoprotein (LDL) and its oxidized form (oxLDL) in the blood. The liver is an important scavenger organ for circulating oxLDLs. The present study aimed to examine endocytosis of mildly oxLDL (the major circulating form of oxLDLs) in liver sinusoidal endothelial cells (LSECs) and the involvement of the scavenger receptors stabilin-1 and stabilin-2 in this process. Freshly isolated LSECs, Kupffer cells (KCs), and stabilin-1- and stabilin-2-transfected human embryonic kidney cells were incubated with fluorescently labeled or radiolabeled oxLDLs [oxidized for 3 h (oxLDL3), 6 h, or 24 h (oxLDL24)] to measure endocytosis. The intracellular localization of oxLDLs and stabilins in LSECs was examined by immunofluorescence and immunogold electron microscopy. Whereas oxLDL24was endocytosed both by LSECs and KCs, oxLDL3(mildly oxLDL) was taken up by LSECs only. The LSEC uptake of oxLDLs was significantly inhibited by the scavenger receptor ligand formaldehyde-treated serum albumin. Uptake of all modified LDLs was high in stabilin-1-transfected cells, whereas stabilin-2-transfected cells preferentially took up oxLDL24, suggesting that stabilin-1 is a more important receptor for mildly oxLDLs than stabilin-2. Double immunogold labeling experiments in LSECs indicated interactions of stabilin-1 and stabilin-2 with oxLDL3on the cell surface, in coated pits, and endocytic vesicles. LSECs but not KCs endocytosed mildly oxLDL. Both stabilin-1 and stabilin-2 were involved in the LSEC endocytosis of oxLDLs, but experiments with stabilin-transfected cells pointed to stabilin-1 as the most important receptor for mildly oxLDL.

Liver sinusoidal endothelial cells (LSECs) are largely responsible for the removal of circulating lysosomal enzymes (LE) via mannose receptor (MR)-mediated endocytosis. We hypothesized that LSECs rely on this uptake to maintain their extraordinarily high degradation capacity for other endocytosed material. Circulatory half-life studies of 125I-cathepsin-D in MR knockout (MR−/−) and wild-type mice, and endocytosis studies in LSEC cultures, showed a total dependence on the MR for effective clearance of cathepsin-D. Radioiodinated formaldehyde-treated serum albumin, a ligand for the LSEC scavenger receptors, was used to study catabolism of endocytosed material in MR−/− and wild-type mice. The plasma clearance, liver uptake, and the starting point for release of degradation products to blood, were similar in both experimental groups, indicating normal endocytosis and intracellular transport of scavenger receptor ligands in MR−/− mice. However, the rate of formaldehyde-treated serum albumin catabolism in the liver of the MR deficient animals was reduced to approximately 50% of wild-type values. A similar reduction in intracellular degradation was recorded in LSEC cultures from MR−/− mice compared to wild-type controls. In accordance with this, MR−/− LSECs had markedly and significantly reduced enzyme activities for four out of five LE tested, i.e., cathepsin-D, α-mannosidase, β-hexosaminidase and arylsulfatase, but not acid phosphatase, compared to wild-type controls. Immunoblot analysis showed that the content of pro-cathepsin-D relative to total cathepsin-D in wild-type LSECs was less than one-fifth of that in hepatocytes, indicating lower endogenous LE production in the LSECs. Conclusion: We show for the first time that LSEC depend on MR-mediated recruitment of LE from their surroundings for effective catabolism of endocytosed macromolecules. (HEPATOLOGY 2008;48:2007–2015.)

The liver sinusoidal endothelial cell (LSEC) influences the transfer of substrates between the sinusoidal blood and hepatocytes and has a major role in endocytosis; therefore, changes in the LSEC have significant implications for hepatic function. There are major morphological changes in the LSEC in old age called pseudocapillarization. These changes include increased LSEC thickness and reduced numbers of pores in the LSEC, which are called fenestrations. Pseudocapillarization has been found in old humans, rats, mice, and nonhuman primates. In addition, old age is associated with impaired LSEC endocytosis and increased leukocyte adhesion, which contributes to reduced hepatic perfusion. Given that fenestrations in the endothelium allow passage of some lipoproteins, including chylomicron remnants, age-related reduction in fenestrations impairs hepatic lipoprotein metabolism. In old rats, caloric restriction was associated with complete preservation of LSEC morphology and fenestrations. In conclusion, pseudocapillarization of the LSEC is a newly discovered aging change that, through its effects on lipoproteins, contributes to the association between old age, dyslipidemia, and vascular disease.

The advent of optical nanoscopy has provided an opportunity to study fundamental properties of nanoscale biological functions, such as liver sinusoidal endothelial cells (LSEC) and their fenestrations. The fenestrations are nano-pores (50-200 nm) on the LSEC plasma membrane that allow free passage of molecules through cells. The fenestrated LSEC also hase a voracious appetite for waste molecules, viruses and nanoparticles. LSEC daily remove huge amounts of waste, nanoparticles and virus from the blood. Pharmaceuticals also need to pass through these fenestrations to be activated (e.g. cholesterol reducing statins) or detoxified by hepatocytes. And, when we age, our LSEC fenestrations become smaller and fewer. Today, we study these cells and structures using either conventional light microscopy on living cells, or high-resolution (but static) methods such as transmission and scanning electron microscopy on fixed (i.e. dead) tissue. Such methods, while very powerful, yield no real time information about the uptake of virus or nanoparticles, nor any information about fenestration dynamics. Therefore, to study LS-SEC, we are now using optical nanoscopy methods, and developing our own, to map their functions in 4 dimensions. Attaining this goal will shed new light on the cell biology of the liver and how it keeps us alive. This paper describes the challenges of studying LS-SEC with light microscopy, as well as current and potential solutions to this challenge using optical nanoscopy. © (2015) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Studies over the last two decades have shown that mammalian nonmacrophagic liver endothelial cells clear the blood from numerous physiological and foreign waste macromolecules, such as polysaccharides and proteins released during extracellular matrix turnover, intracellular macromolecules, modified serum proteins, and bacterial and fungal proteins [Smedsrød, B., Pertoft, H., Gustafson, S. & Laurent, T. C. (1990) Biochem. J. 266, 313–327]. These macromolecules are released daily in gram-amounts in a normal human body and are effectively taken up and degraded by the liver endothelial cells. Recent studies show that bony fishes harbor a similar system of specialized nonmacrophagic scavenger endothelial cells in either kidney [Smedsrød, B., Gjøen, T., Sveinbjørnsson, B. & Berg, T. (1993) J. Fish Biol. 42, 279–291] or heart [Sørensen, K. K., Melkko, J. & Smedsrød, B. (1998) J. Exp. Biol. 201, 1707–1718], but not in liver. Using specific and extremely effective endocytosis, these fish scavenger endothelial cells function as their mammalian counterpart to eliminate soluble waste macromolecules from the circulation. We show here that species from all seven vertebrate classes carry a population of nonmacrophagic scavenger endothelial cells that efficiently eliminate an array of circulating waste macromolecules. Thus representing an important part of the vertebrate innate immune system, these scavenger endothelial cells display the following distribution in the different vertebrate classes: Gills in Agnatha and Chondrichtyes; heart or kidney in Osteichtyes; and liver in Amphibia, Reptilia, Aves, and Mammalia.

Liver sinusoidal endothelial cells (LSEC) are an important class of endothelial cells facilitating the translocation of lipoproteins and small molecules between the liver and blood. A number of clinical conditions, especially metabolic and aging-related disorders, are implicated by improper function of LSECs. Despite their importance, research into these cells is limited because the primary ultrastructures involved in their function are transcellular pores, called fenestrations, with diameters in a size range between 50-200 nm, i.e. well below the optical diffraction limit. Here, we show that we are able to resolve fenestrations with a spatial resolution of similar to 20 nm by direct stochastic optical reconstruction microscopy (dSTORM). The cellular plasma membrane was labeled at high fluorophore density with CellMask Deep Red and imaged using a reducing buffer system. We compare the higher degree of structural detail that dSTORM provides to results obtained by 3D structured illumination microscopy (3D-SIM). Our results open up a path to image these physiologically important cells in vitro using highly resolving localization microscopy techniques that could be implemented on non-specialized fluorescence microscopes, enabling their investigation in most biomedical laboratories without the need for electron microscopy.

The fate of the major connective tissue polysaccharide hyaluronan, as it appears after release from the matrix, was studied in the Atlantic cod by use of subcutaneous administration of hyaluronan conjugated with fluorescein isothiocyanate (FITC) and labelled with 125I. After administration, the ligand was transported to the heart, which contained 58% of the recovered label after 22 h, whereas 36% remained at the injection site. Uptake in other organs was low. Results from intravenous co-injection studies showed that 125I-FITC-hyaluronan and native hyaluronan were in competition for uptake by the same receptor in cod heart. Fluorescence microscopy revealed that FITC-hyaluronan accumulated in spherical structures and discrete vesicles in endocardial endothelial cells lining the muscular trabeculae of both heart chambers. Immunoelectron microscopy showed that, in these cells, the ligand lined the limiting membrane of endosomes and filled the lumen of late endosomes or lysosomes. We conclude that, in the cod, heart endothelial cells are essential for the turnover of hyaluronan. Atrial endothelial cells were also able to ingest 2-microm latex beads, although these were far more effectively phagocytosed by head kidney macrophages. The present results strengthen the notion that the cod endocardium consists of specialized scavenger endothelial cells, resembling sinusoidal endothelial cells of salmonid kidney and mammalian liver. These cells should therefore be regarded as an important part of the cod reticulo-endothelial system.

Oxidized low-density lipoproteins (oxLDLs) are involved in proinflammatory and cytotoxic events in different microcirculatory systems. The liver is an important scavenger organ for circulating oxLDLs. However, the interaction of oxLDL with the hepatic microcirculation has been poorly investigated. The present study was conducted to examine the effects of differently modified oxLDLs on the hepatic microvasculature. C57Bl/6J mice were injected intravenously with low-density lipoprotein (LDL), or LDL oxidized for 3 h (oxLDL3) or 24 h (oxLDL24), at doses resembling oxLDL plasma levels in cardiovascular disease patients. Radioiodinated ligands were used to measure blood decay and organ distribution, and nonlabeled ligands to evaluate microcirculatory responses, examined by in vivo microscopy 30–60 min after ligand injection, immunohistochemistry, and scanning and transmission electron microscopy. Mildly oxLDL (oxLDL3) was cleared from blood at a markedly slower rate than heavily oxLDL (oxLDL24), but significantly faster than LDL ( P < 0.01). Injected oxLDLs distributed to liver. OxLDL effects were most pronounced in central areas of the liver lobules where oxLDL3elicited a significant ( P < 0.05) reduction in perfused sinusoids, and both oxLDL3and oxLDL24significantly increased the numbers of swollen endothelial cells and adherent leukocytes compared with LDL ( P < 0.05). OxLDL-treated livers also exhibited increased intercellular adhesion molecule (ICAM)-1 centrilobular staining. Electron microscopy showed a 30% increased thickness of the liver sinusoidal endothelium in the oxLDL3group ( P < 0.05) and a reduced sinusoidal fenestration in centrilobular areas with increased oxidation of LDL ( P for linear trend

Liver sinusoidal endothelial cells (LSECs) play an essential role in systemic waste clearance by effective endocytosis of blood-borne waste macromolecules. We aimed to study LSECs' scavenger function during aging, and whether age-related morphological changes (eg, defenestration) affect this function, in F344/BN F1 rats. Endocytosis of the scavenger receptor ligand formaldehyde-treated serum albumin was significantly reduced in LSECs from old rats. Ligand degradation, LSEC protein expression of the major scavenger receptors for formaldehyde-treated serum albumin endocytosis, stabilin-1 and stabilin-2, and their staining patterns along liver sinusoids, was similar at young and old age, suggesting that other parts of the endocytic machinery are affected by aging. Formaldehyde-treated serum albumin uptake per cell, and cell porosity evaluated by electron microscopy, was not correlated, indicating that LSEC defenestration is not linked to impaired endocytosis. We report a significantly reduced LSEC endocytic capacity at old age, which may be especially important in situations with increased circulatory waste loads.

Cervid herpesvirus 2 (CvHV2) has been isolated from reindeer ( Rangifer tarandus tarandus ), and serological data indicate that in reindeer this virus is endemic in Fennoscandia, Alaska, Canada, and Greenland. CvHV2 has been described as a cause of subclinical genital infections in reindeer, but little information on primary infections exists. In this study, six seronegative and presumably pregnant reindeer were allocated to one of two groups. Two animals were inoculated with CvHV2 intratracheally, and two animals intravaginally, with one control animal in each group receiving sterile water. Mild hyperthermia and serous discharges from the vagina and nose were observed. No abortions were recorded, but one calf died shortly after birth. Inoculated animals seroconverted and had neutralizing antibodies after days 7 to 10 postinfection. CvHV2 was detected by PCR in nasal and vaginal swabs from animals in both groups but could be isolated only from nasal swabs in the respiratory group and from vaginal swabs in the genital group. CvHV2 was detected by PCR in various organs and tissues postmortem. In control animals, the virus could not be isolated in spite of PCR-positive nasal and vaginal swab samples and some degree of positive immunostaining. One of the animals that were inoculated intratracheally developed a hemorrhagic, necrotizing bronchopneumonia, which was CvHV2 positive by PCR and immunohistochemistry. We conclude that CvHV2 can cause systemic infection, that both genital and respiratory inoculations can lead to virus shedding, and that the virus can infect the fetus in utero.

Age-related changes in Bruch's membrane (BM), situated between the retina and the choroid, include the accumulation of advanced glycation end-products (AGEs) and other modified macromolecules. An important clearance mechanism for such molecules is endocytosis via macrophages and specialized endothelial cells. This study examined the endocytic clearance of AGEs in choriocapillaris endothelial (CCE) cells, which are strategically located beneath the BM.Bovine CCE cultures were incubated with radiolabeled or fluorescently labeled AGE-modified bovine serum albumin (AGE-BSA). Cells and tissues were examined for the expression of two AGE-binding scavenger receptors, stabilin-1 and -2, by immunohistochemistry and Western blotting, and with antibody inhibition studies.CCE cells effectively endocytosed AGE-BSA via a scavenger receptor-mediated pathway. A polyclonal antibody against stabilin-2 significantly inhibited endocytosis (40%). Tissue sections stained positive for stabilin-2 and -1 in the choriocapillaris layer, which was confirmed by immunoblots of cell extracts. Colocalization of stabilin-1 and -2 and internalized AGE-BSA was seen in early endosomes.CCEs actively endocytose AGE-BSA and express the scavenger receptors, stabilin-1 and -2, of which at least stabilin-2 is involved in AGE-BSA uptake. Impairment of this stabilin-mediated clearance function may contribute to depositions of waste macromolecules in BM and subsequent retinopathy.

Morphological changes in the hepatic sinusoid with old age are increasingly recognized. These include thickening and defenestration of the liver sinusoidal endothelial cell, sporadic deposition of collagen and basal lamina in the extracellular space of Disse, and increased numbers of fat engorged, nonactivated stellate cells. In addition, there is endothelial up-regulation of von Willebrand factor and ICAM-1 with reduced expression of caveolin-1. These changes have been termed age-related pseudocapillarization. The effects of old age on Kupffer cells are inconsistent, but impaired responsiveness is likely. There are functional implications of these aging changes in the hepatic sinusoid. There is reduced sinusoidal perfusion, which will impair the hepatic clearance of highly extracted substrates. Blood clearance of a variety of waste macromolecules takes place in liver sinusoidal endothelial cells (LSECs). Previous studies indicated either that aging had no effect, or reduced the endocytic capacity of LSECs. However, a recent study in mice showed reduced endocytosis in pericentral regions of the liver lobules. Reduced endocytosis may increase systemic exposure to potential harmful waste macromolecules such as advanced glycation end products Loss of fenestrations leads to impaired transfer of lipoproteins from blood to hepatocytes. This provides a mechanism for impaired chylomicron remnant clearance and postprandial hyperlipidemia associated with old age. Given the extensive range of substrates metabolized by the liver, age-related changes in the hepatic sinusoid and microcirculation have important systemic implications for aging and age-related diseases.

Background In a project to determine the causes of winter mortality in reindeer in Finnmark County, northern Norway, the most frequent diagnosis turned out to be complete emaciation, despite several of the reindeer having been given silage for up to 4 weeks before they died. The present paper describes autopsy results and other findings in these animals. Methods Autopsies were made of 32 reindeer carcasses, and 28 of these were diagnosed as completely emaciated based on lack of visible fat and serous atrophy of subepicardial and bone marrow fat. Other investigations of the carcasses included histology, bacteriology, parasitology (counting of macro parasites and faecal egg counting), analysis of vitamin E and selenium in liver, chemical and botanical analysis of rumen content, analysis of lipid content in femur bone marrow and estimation of muscle atrophy by use of a muscle index. Results Main findings were: Low carcass weight, severe muscle atrophy, hemosiderosis in liver and spleen, subcutaneous oedema (18%) and effusions to body cavities (18%). Two types of lipofuscin granula were identified in the liver: One type occurred in liver endothelial cells of all carcasses, while the other type occurred in hepatocytes, and prevailed in adult animals. Abomasal haemorrhages, consistent with previously described stress lesions, was present in 68% of the carcasses. Diarrhoea occurred in 2 cases, and loose faecal consistency was associated with silage feeding. Rumen content was low in crude protein. Grass dominated rumen content in silage-fed carcasses, while reindeer on natural pastures had mainly woody plants, mosses and litter in rumen. Stem dominated the grass fraction in rumens with high grass content, indicating ruminal indigestion as a cause of emaciation in silage fed animals. Some cases had heavy infestation of parasites such as warble fly larvae (Hypoderma tarandi), throat bot larvae (Cephenemyiae trompe) and lung nematodes. Conclusion Lack of appropriate amounts and/or appropriate quality of feed has been the main cause of emaciation, though heavy infestation of parasites may have contributed to the emaciation in some cases.

The purpose of this study was to identify the receptor responsible for endocytosis of denatured collagen from blood. The major site of clearance of this material (at least 0.5 g/day in humans) is a receptor on liver sinusoidal endothelial cells (LSECs). We have now identified an 180-kDa endocytic receptor on LSECs, peptide mass fingerprinting of which revealed it to be the mannose receptor. Challenge of mannose-receptor knockout mice and their cultured LSECs revealed significantly reduced blood clearance and a complete absence of LSEC endocytosis of denatured collagen. Organ analysis of wild-type versus knockout mice after injection of denatured collagen revealed significantly reduced liver uptake in the knockout mice. Clearance/endocytosis of ligands for other receptors in these animals was as that for wild-type mice, and denatured collagen uptake in wild-type mice was not affected by other ligands of the mannose receptor, namely mannose and mannan. Furthermore, unlike that of mannose and mannan, endocytosis of denatured collagen by the mannose receptor is calcium independent. This suggests that the binding site for denatured collagen is distinct from that for mannose/mannan. Mannose receptors on LSECs appear to have less affinity for circulating triple helical type I collagen. Conclusion: The mannose receptor is the main candidate for being the endocytic denatured collagen receptor on LSECs. (HEPATOLOGY 2007.)

Aging of the liver is associated with impaired metabolism of drugs, adverse drug interactions, and susceptibility to toxins. Since reduced hepatic blood flow is suspected to contribute this impairment, we examined age-related alterations in hepatic microcirculation. Livers of C57Bl/6 mice were examined at 0.8 (pre-pubertal), 3 (young adult), 14 (middle-aged), and 27 (senescent) months of age using in vivo and electron microscopic methods. The results demonstrated a 14% reduction in the numbers of perfused sinusoids between 0.8 and 27 month mice associated with 35% reduction in sinusoidal blood flow. This was accompanied by an inflammatory response evidenced by a fivefold increase in leukocyte adhesion in 27 month mice, up-regulated expression of ICAM-1, and increases in intrahepatic macrophages. Sinusoidal diameter decreased 6-10%. Liver sinusoidal endothelial cell (LSEC) dysfunction was seen as early as 14 months when there was a threefold increase in the numbers of swollen LSEC. The endocytotic capacity of LSEC also was found to be reduced in older animals. The sinusoidal endothelium in 27 month old mice exhibited pseudocapillarization. In conclusion, the results suggest that leukocyte accumulation in the sinusoids and narrowing of sinusoidal lumens due to pseudocapillarization and dysfunction of LSEC reduce sinusoidal blood flow in aged livers.

During March to May 2000, 48 carcasses of semi-domesticated reindeer (Rangifer tarandus tarandus) were collected on winter pastures and calving grounds from two herds in western Finnmark and two herds in eastern Finnmark, northern Norway. The animals were autopsied and blood and tissue samples were collected for serology (alphaherpes- and pestivirus; virus neutralization test) and polymerase chain reaction (PCR; parapoxvirus; B2L gene) investigations. Autopsy revealed that 39 of 48 animals (81%) had died of emaciation. Parapoxvirus-specific DNA was detected in samples from 6 of 48 animals (12.5%; liver, parotid salivary gland and/or pulmonary lymph nodes). A DNA sequence of 376 base pairs from a PCR amplicon obtained from a liver sample from one animal showed 98-99% identity with orf virus strain Orf-11 and reindeer parapoxvirus isolates from Norway and Finland (1992 and 1994), 92-93% similarity with pseudocowpoxvirus and 87% similarity with bovine papular stomatitis virus. Alphaherpes- and pestivirus antibodies were detected in 10% and 33% of the animals, respectively. These results indicates that parapoxvirus, presumably orf-virus, is present among reindeer also in Finnmark, although contagious ecthyma has never been reported in reindeer in this important reindeer herding area. Furthermore, they show that herpes- and pestiviruses are still endemic in reindeer herds in Finnmark. The nature of these viruses and their impact on reindeer health and reproduction and reindeer herding economy should be further addressed, as well as the possibility that these viruses may be transferred between reindeer and domestic animals in this region.Abstract in Norwegian / Sammendrag: I løpet av perioden mars-mai 2000 ble 48 reinsdyrkadavre (Rangifer tarandus tarandus) samlet inn fra vinterbeiter og kalvingsområder fra to flokker i Vest-Finnmark og to i Øst-Finnmark, Norge. Dyrene ble obdusert, og blod og vevsprøver ble samlet for påvisning av antistoffer mot alfaherpes- og pestivirus i blod(serologi) og tilstedeværelse av parapoxvirus-DNA i vev (Polymerase kjedereaksjon, PCR; parapoxvirus B2L genet). Obduksjonen viste at 39 av de 48 dyrene (81%) hadde dødd av avmagring. Parapoxvirus-spesifikt DNA ble funnet i prøver av lever, spyttkjertel (Gl. parotis) og/eller lungelymfeknuter fra 6 av de 48 dyrene (12,5%). En DNA sekvens på 376 basepar fra PCR-oppformeringsproduktet fra en leverprøve hadde 98-99% likhet med orf-virus (Orf-11) og parapoxvirus isolert fra reinsdyr i Norge og Finland (1992 og 1994), 92-93% likhet med pseudocowpoxvirus og 87% likhet med bovint papulær stomatittvirus, hvorav de to siste parapoxvirusartene er assosiert med storfé. Disse resultatene viser at også reinsdyr i Finnmark er infisert av parapoxvirus, til tross for at sykdommen munnskurv ikke ennå er rapportert hos rein i dette fylket. Alfaherpes- og pestivirus antistoffer ble funnet hos henholdsvis 10% og 33% av dyrene. Dette er i samsvar med tidligere funn på slaktedyr, og viser at disse virusinfeksjonene er endemiske hos rein i Finnmark. Det er viktig å avklare hvilken rolle disse virusinfeksjonene spiller for reinsdyrenes helse og næringens økonomi. Videre bør det avklares i hvilken grad disse virustypene er i stand til å smitte mellom rein og husdyr.

Investigations for Brucella-infections were conducted in 29 hooded seals (Cystophora cristata) caught between Svalbard and Greenland (North Atlantic Ocean; Greenland Sea) autumn 2002, and from 20 ringed seals (Phoca hispida) caught in Billefjord, Svalbard, spring 2003. All animals were apparently healthy and were caught in their natural habitat. Bacteriology on tissue samples from ringed seals was negative, whereas Brucella sp. were recovered in tissues from 11 of the 29 hooded seals (38%), with the highest tissue prevalence in spleen (9/29) and lung lymph nodes (9/24). Anti-Brucella antibodies were detected in sera from 9 hooded seals (31%) (EDTA-modified Slow Agglutination test of Wright, Rose Bengal test, Complement Fixation Test, and Protein-A ELISA). The bacterial isolates all belonged to the genus Brucella according to classical biotyping and PCR analysis based on Insertion Sequence IS711, and were shown to be typical marine mammal strains, based on the occurrence of an IS711 element downstream of the bp26 gene. Their dependency on CO2 for growth, and the presence of one copy each of the omp2a and omp2b gene finally classified them as Brucella pinnipediae. Furthermore, all the hooded seal isolates showed an A+ M+ agglutination profile, which is different from the profile of reference seal strain 2/94 (harbour seal, Phoca vitulina). Thus, these results indicate that B. pinnipediae may contain different biovars. The present results suggest that infection with B. pinnipediae is enzootic in this population. Since the hooded seal is commercially hunted and consumed in Norway, the pathological impact of such infections and their zoonotic potential should be further addressed.

The catabolic fate of circulating hyaluronan and the proteoglycan chondroitin sulphate (CSPG) was studied in the Atlantic cod (Gadus morhua L.). Distribution studies using radio-iodinated ligand demonstrated that CSPG was rapidly eliminated from the blood by the endocardial endothelial cells (EECs) of the heart atrium and ventricle. The presence of excess amounts of hyaluronan or CSPG inhibited uptake of [125I]hyaluronan into cultured atrial EECs (aEECs) by 46 % and 84 %, respectively. Neither formaldehyde-treated serum albumin (FSA) nor mannose inhibited this uptake. The presence of excess amounts of CSPG and hyaluronan inhibited uptake of [125I]CSPG by 90 % and 42 %, respectively, suggesting that aEECs express a specific hyaluronan binding site that also recognizes CSPG. FSA inhibited endocytosis of [125I]CSPG by 65 %, indicating that CSPG is also recognized by the scavenger receptor. Approximately 17 % and 57 % of added [125I]hyaluronan and 15 % and 65 % of the added [125I]CSPG were endocytosed after 1 and 24 h, respectively. High-performance liquid chromatographic analyses of the spent medium after endocytosis of hyaluronan and CSPG serglycin labelled biosynthetically with 3H in the acetyl groups identified labelled the low-molecular-mass degradation products as [3H]acetate, indicating that aEECs operate anaerobically. These findings suggest that acetate released from cod EECs following catabolism of endocytosed hyaluronan and CSPG represents a high-energy metabolite that may fuel cardiomyocytes.

Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72 – 96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary.