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Background & Aims: The SALVE Histopathology Group (SHG) developed and validated a grading and staging system for the clinical and full histological spectrum of alcohol-related liver disease (ALD) and evaluated its prognostic utility in a multinational cohort of 445 patients., Methods: SALVE grade was described by semiquantitative scores for steatosis, activity (hepatocellular injury and lobular neutrophils) and cholestasis. The histological diagnosis of steatohepatitis due to ALD (histological ASH, hASH) was based on the presence of hepatocellular ballooning and lobular neutrophils. Fibrosis staging was adapted from the Clinical Research Network staging system for non-alcoholic fatty liver disease and the Laennec staging system and reflects the pattern and extent of ALD fibrosis. There are 7 SALVE fibrosis stages (SFS) ranging from no fibrosis to severe cirrhosis., Results: Interobserver κ-value for each grading and staging parameter was >0.6. In the whole study cohort, long-term outcome was associated with activity grade and cholestasis, as well as cirrhosis with very broad septa (severe cirrhosis) (p <0.001 for all parameters). In decompensated ALD, adverse short-term outcome was associated with activity grade, hASH and cholestasis (p = 0.038, 0.012 and 0.001, respectively), whereas in compensated ALD, hASH and severe fibrosis/cirrhosis were associated with decompensation-free survival (p = 0.011 and 0.001, respectively). On multivariable analysis, severe cirrhosis emerged as an independent histological predictor of long-term survival in the whole study cohort. Severe cirrhosis and hASH were identified as independent predictors of short-term survival in decompensated ALD, and also as independent predictors of decompensation-free survival in compensated ALD., Conclusion: The SALVE grading and staging system is a reproducible and prognostically relevant method for the histological assessment of disease activity and fibrosis in ALD., Lay Summary: Patients with alcohol-related liver disease (ALD) may undergo liver biopsy to assess disease severity. We developed a system to classify ALD under the microscope by grading ALD activity and staging the extent of liver scarring. We validated the prognostic performance of this system in 445 patients from 4 European centers., Competing Interests: Conflicts of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: Observational instruments are preferred for assessment of cultural competence. The aim of the current study is to identify observational instruments to assess cultural competence in healthcare providers and dieticians specifically and assess their psychometric properties., Methods: A systematic review was conducted in Cinahl, Cochrane, EMBASE, PsycInfo, Pubmed, and Web of Science using search terms related to cultural competency and measurement properties. Methodological quality of the selected studies of observational cultural competence instruments in dieticians, other healthcare professionals and psychological counsellors and the measurement properties of instruments were assessed using the COSMIN checklist., Results: From 11,913 articles, six articles on five instruments were selected. Instruments were targeted at health professionals and counsellors only, and designed for face-to-face communication (n = 4) or verbal responses to videotaped simulated interactions (n = 1). The instruments' content varied largely, with main focus on attitude, and little on knowledge and skills. The measurement properties were suboptimal., Conclusion: No observational instrument are available to evaluate cultural competence of dieticians. Studies on psychometric properties of instruments targeted at other health professionals lack methodological rigour., Practice Implications: Future work should focus on developing an instrument that encompasses both 'general' cultural competences necessary for all healthcare professionals and dietetic specific competences., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background & Aims: Cholangiopathies are chronic liver diseases in which damaged cholangiocytes trigger a proinflammatory and profibrotic reaction. The nuclear vitamin D receptor (VDR) is highly expressed in cholangiocytes and exerts immune-regulatory functions in these cells. In the present study, we examined the protective function of VDR and other vitamin D signaling pathways in chronic cholangiopathy and cholangiocytes., Methods: Vdr was invalidated in Abcb4 knockout mice, a widely used animal model of chronic cholangiopathy. The impact of vitamin D signaling on cholangiopathy features was examined in vivo and in cholangiocytes (primary and cell lines)., Results: Cholangiopathy features (i.e, cholestasis, ductular reaction and fibrosis) were aggravated in Vdr;Abcb4 double knockout mice compared to the Abcb4 simple knockout, and associated with an overexpression of proinflammatory factors. The proinflammatory phenotype of cholangiocytes was also exacerbated following VDR silencing in vitro. The expression of proinflammatory factors and the severity of cholangiopathy were reduced in the double knockout mice treated with the vitamin D analog calcipotriol or with vitamin D. In vitro, the inflammatory response to TNFα was significantly reduced by calcipotriol in biliary cells silenced for VDR, and this effect was abolished by co-silencing the plasma membrane receptor of vitamin D, protein disulfide-isomerase A3 (PDIA3)., Conclusions: Our results demonstrate an anti-inflammatory role of VDR signaling in cholangiocytes and cholangiopathy. They also provide evidence for PDIA3-mediated anti-inflammatory effects of vitamin D and vitamin D analog in these settings., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Two goats (6 months old and 5 years old) were evaluated for neurological signs including laboured breathing, stiffness and obtundation. Solitary masses were noted in the brainstem and spinal cord, respectively. Histopathology of both cases revealed the lesions were composed of a mixture of glial and neuronal cells, consistent with glioneuronal hamartomas. The cause of death was attributed to the mass in the 6-month-old, while the cause of death in the 5-year-old was attributed to listeriosis. Hamartomas of neural origin are rarely described in veterinary species, and this report represents the first report of glioneuronal hamartomas in goats., Competing Interests: Conflict of Interest Statement The authors declare no potential conflicts of interest with respect to the research, authorship or publication of this article., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The tentacular system of Clytia hemisphaerica medusa (Cnidaria, Hydrozoa) has recently emerged as a promising experimental model to tackle the developmental mechanisms that regulate cell lineage progression in an early-diverging animal phylum. From a population of proximal stem cells, the successive steps of tentacle stinging cell (nematocyte) elaboration, are spatially ordered along a "cellular conveyor belt". Furthermore, the C. hemisphaerica tentacular system exhibits bilateral organisation, with two perpendicular polarity axes (proximo-distal and oral-aboral). We aimed to improve our knowledge of this cellular system by combining RNAseq-based differential gene expression analyses and expression studies of Wnt signalling genes. RNAseq comparisons of gene expression levels were performed (i) between the tentacular system and a control medusa deprived of all tentacles, nematogenic sites and gonads, and (ii) between three samples staggered along the cellular conveyor belt. The behaviour in these differential expression analyses of two reference gene sets (stem cell genes; nematocyte genes), as well as the relative representations of selected gene ontology categories, support the validity of the cellular conveyor belt model. Expression patterns obtained by in situ hybridisation for selected highly differentially expressed genes and for Wnt signalling genes are largely consistent with the results from RNAseq. Wnt signalling genes exhibit complex spatial deployment along both polarity axes of the tentacular system, with the Wnt/β-catenin pathway probably acting along the oral-aboral axis rather than the proximo-distal axis. These findings reinforce the idea that, despite overall radial symmetry, cnidarians have a full potential for elaboration of bilateral structures based on finely orchestrated deployment of an ancient developmental gene toolkit., (Copyright © 2019. Published by Elsevier Inc.)

Background: The aim of the study was to explore the correlation between clinical signs and confirmatory tests for cow's milk allergy (CMA) in the neonatal period and their relation to family history and the occurrence of food allergies in the postneonatal period., Methods: The medical documentation of 361 newborns with suspected CMA and exclusion of other comorbidities was analyzed. The correlations between clinical signs and methods to confirm CMA [elevated levels of total immunoglobulin E (IgE) and/or specific IgE for cow's milk, improvement after the introduction of a cow's milk-free diet and positive challenge procedure] were studied. In 90 children, the data were additionally analyzed in relation to outcome (at the age of 1-11 years), evaluated by questionnaires, which inquired about signs and symptoms of food allergy, methods of CMA confirmation, and the presence of other food allergies., Results: There was a positive correlation between exanthema and confirmed CMA in the neonatal period (R = 0.184; p = <0.001; n = 361), and hematochezia and confirmed CMA in the neonatal (R = 0.203; p < 0.001; n = 361) and postneonatal period (R = 0.215; p = 0.042; n = 90). Additional food allergies in the postneonatal period were positively correlated with neonatal CMA (R = 0.275; p = 0.009; n = 90). No correlation was found between a positive family history of food allergies and CMA in the neonatal (R = -0.66; p = 0.398; n = 165) and postneonatal periods (R = 0.00; p = 1.000; n = 116)., Conclusion: Neonatal exanthema and hematochezia were the predominant clinical signs in neonates with CMA. Allergies to other food allergens appeared more frequently in children with CMA in the neonatal period. Neonatal CMA did not occur more frequently in families with food allergies., (Copyright © 2017. Published by Elsevier B.V.)

Objective: In psychotherapy clients' I don't know-responses (IDK-responses) to therapists' questions are typically considered to be non-cooperating behaviors. How therapists actually handle these behaviors remains unclear. This study therefore aims to assess client-therapist interactions following IDK-responses., Methods: Data were collected in a Dutch child and adolescent mental healthcare service by observing Dialectical Behavior Therapy aimed at adolescents with severe emotional distress. Eighteen individual psychotherapy sessions involving two therapists with six clients were video-recorded and transcribed. Stand-alone IDK-responses were selected (n=77) and analyzed using conversation analysis., Results: Adolescents' IDK-responses led to varying actions of therapists. We identified five categories of continuations after IDK-responses: no IDK-related continuation; redoing of the question; proposing a candidate answer; employing therapy-specific techniques; and meta-talk on the problematic nature of the IDK-response., Conclusions: Therapists treat IDK-responses not just as non-cooperative behavior on the part of the client; IDK-responses are also used as a starting point to collaboratively enhance clients' insights in their own thoughts, emotions, and behaviors., Practice Implications: After IDK-responses therapists can use a variety of continuation strategies with varying therapeutic functions. These strategies can be embedded in training of therapists to deal with potentially non-cooperative behavior., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Ctenophores are non-bilaterian metazoans of uncertain phylogenetic position, some recent studies placing them as sister-group to all other animals whereas others suggest this placement is artefactual and ctenophores are more closely allied with cnidarians and bilaterians, with which they share nerve cells, muscles and gut. Available information about developmental genes and their expression and function in ctenophores is reviewed. These data not only unveil some conserved aspects of molecular developmental mechanisms with other basal metazoan lineages, but also can be expected to enlighten the genomic and molecular bases of the evolution of ctenophore-specific traits, including their unique embryonic development, complex anatomy and high cell type diversity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Using high-speed imaging we assessed Streptococcus mutans biofilm-fluid interactions during exposure to a 60-ms microspray burst with a maximum exit velocity of 51m/s. S. mutans UA159 biofilms were grown for 72h on 10mm-length glass slides pre-conditioned with porcine gastric mucin. Biofilm stiffness was measured by performing uniaxial-compression tests. We developed an in-vitro interproximal model which allowed the parallel insertion of two biofilm-colonized slides separated by a distance of 1mm and enabled high-speed imaging of the removal process at the surface. S. mutans biofilms were exposed to either a water microspray or an air-only microburst. High-speed videos provided further insight into the mechanical behaviour of biofilms as complex liquids and into high-shear fluid-biofilm interaction. We documented biofilms extremely transient fluid behaviour when exposed to the high-velocity microsprays. The presence of time-dependent recoil and residual deformation confirmed the pivotal role of viscoelasticity in biofilm removal. The air-only microburst was effective enough to remove some of the biofilm but created a smaller clearance zone underlying the importance of water and the air-water interface of drops moving over the solid surface in the removal process. Confocal and COMSTAT analysis showed the high-velocity water microspray caused up to a 99.9% reduction in biofilm thickness, biomass and area coverage, within the impact area., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Environmental exposure to manganese (Mn) results in several toxic effects, mainly neurotoxicity. This study investigated associations among Mn exposure, neuropsychological performance, biomarkers of oxidative damage and early kidney dysfunction in children aged 6-12 years old. Sixty-three children were enrolled in this study, being 43 from a rural area and 20 from an urban area. Manganese was quantified in blood (B-Mn), hair (H-Mn) and drinking water using inductively coupled plasma mass spectrometry (ICP-MS). The neuropsychological functions assessed were attention, perception, working memory, phonological awareness and executive functions - inhibition. The Intelligence quotient (IQ) was also evaluated. The biomarkers malondialdehyde (MDA), protein carbonyls (PCO), δ-aminolevulinate dehydratase (ALA-D), reactivation indexes with dithiothreitol (ALA-RE/DTT) and ZnCl2 (ALA-RE/ZnCl2), non-protein thiol groups, as well as microalbuminuria (mALB) level and N-acetyl-β-D-glucosaminidase (NAG) activity were assessed. The results demonstrated that Mn levels in blood, hair and drinking water were higher in rural children than in urban children (p<0.01). Adjusted for potential confounding factors, IQ, age, gender and parents' education, significant associations were observed mainly between B-Mn and visual attention (β=0.649; p<0.001). Moreover, B-Mn was negatively associated with visual perception and phonological awareness. H-Mn was inversely associated with working memory, and Mn levels from drinking water with written language and executive functions - inhibition. Rural children showed a significant increase in oxidative damage to proteins and lipids, as well as alteration in kidney function biomarkers (p<0.05). Moreover, significant associations were found between B-Mn, H-Mn and Mn levels in drinking water and biomarkers of oxidative damage and kidney function, besides between some oxidative stress biomarkers and neuropsychological tasks (p<0.05). The findings of this study suggest an important association between environmental exposure to Mn and toxic effects on neuropsychological function, oxidative damage and kidney function in children., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The flow field and local hydrodynamics of high-velocity water microdrops impacting the interproximal (IP) space of typodont teeth were studied experimentally and computationally. Fourteen-day old Streptococcus mutans biofilms in the IP space were treated by a prototype AirFloss delivering 115 µL of water at a maximum exit-velocity of 60 ms(-1) in a 33-ms burst. Using high-speed imaging, footage was generated showing the details of the burst, and demonstrating the removal mechanism of the biofilms. Footage was also generated to characterize the viscoelastic behavior of the biofilms when impacted by an air-only burst, which was compared to the water burst. Image analysis demonstrated the importance of fluid forces on the removal pattern of interdental biofilms. X-ray micro-Computed Tomography (µ-CT) was used to obtain 3D images of the typodont and the IP spaces. Computational Fluid Dynamics (CFD) simulations were performed to study the effect of changing the nozzle position and design on the hydrodynamics within the IP space. Results confirmed our previous data regarding the wall shear stress generated by high-velocity water drops which dictated the efficacy of biofilm detachment. Finally, we showed how CFD models could be used to optimize water drop or burst design towards a more effective biofilm removal performance., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: To examine adolescents' attributed relevance and experiences regarding communication, and whether discrepancies in these are associated with clients' participation and learning processes in psychosocial care., Methods: Adolescents receiving psychosocial care (n=211) completed measures of communication in three domains: affective communication, information provision, and shared decision-making. Participation involved clients' attendance and adherence (professional-reported). Learning processes involved clients' improved understanding and improved confidence (client and professional-reported)., Results: Important but less often experienced affective communication was associated with low adherence (odds ratio, 95% confidence interval: 2.8, 1.1-6.8), less improvement in understanding (3.7, 1.5-9.0), and less improvement in confidence (4.5, 1.8-11.6). If information provision or shared decision-making was important but less often experienced, adolescents were more likely to demonstrate less improvement in understanding (3.1, 1.1-8.5; 4.2, 1.7-10.8). The combination "less important but experienced" only had an effect regarding affective communication; these adolescents were more likely to demonstrate less improvement in confidence (6.0, 2.3-15.4)., Conclusion: Discrepancies between attributed relevance and experiences frequently occur. These discrepancies negatively affect adolescents' participation and their learning processes, although the pattern differs across communication domains., Practice Implications: Care professionals should pay considerable attention to their clients' communication preferences and adapt their communication style when necessary., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Objective: To describe the safety of ulipristal acetate in emergency contraception., Study Design: Postmarketing pharmacovigilance data collection., Results: A total of 553 women experienced 1049 adverse drug reactions. The most frequent (n,%) were pregnancies (282, 6.8%); nausea, abdominal pain and vomiting (139, 13.3%); headache, dizziness (67, 6.4%); and metrorrhagia, menses delay and breast symptoms (84, 8.0%). Including data from clinical trials, 376 pregnancies have been reported in total, 232 (62%) with a known outcome: 28 live births (29 newborns), 34 miscarriages, 151 induced abortions, 4 ectopics and 15 which are ongoing., Conclusions: No safety concern emerges from a sizable database of reported adverse reactions following ulipristal acetate exposure among varying ethnicities and regions. Postapproval data confirm the safety profile described during the clinical trials., Implications: Use of ulipristal acetate for emergency contraception in a variety of settings and among diverse populations indicate that it is safe and without unexpected or serious adverse events., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cerebral amyloid angiopathy (CAA) is a key histopathological hallmark of Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). CAA is characterized by amyloid-beta (Aβ) depositions and remodeling of the extracellular matrix (ECM) in brain vessels and plays an important role in the development and progression of both AD and HCHWA-D. Tissue transglutaminase (tTG) modulates the ECM by molecular cross-linking of ECM proteins. Here, we investigated the distribution pattern, cellular source, and activity of tTG in CAA in control, AD, and HCHWA-D cases. We observed increased tTG immunoreactivity and colocalization with Aβ in the vessel wall in early stage CAA, whereas in later CAA stages, tTG and its cross-links were present in halos enclosing the Aβ deposition. In CAA, tTG and its cross-links at the abluminal side of the vessel were demonstrated to be either of astrocytic origin in parenchymal vessels, of fibroblastic origin in leptomeningeal vessels, and of endothelial origin at the luminal side of the deposited Aβ. Furthermore, the ECM proteins fibronectin and laminin colocalized with the tTG-positive halos surrounding the deposited Aβ in CAA. However, we observed that in situ tTG activity was present throughout the vessel wall in late stage CAA. Together, our data suggest that tTG and its activity might play a differential role in the development and progression of CAA, possibly evolving from direct modulation of Aβ aggregation to cross-linking of ECM proteins resulting in ECM restructuring., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The separation of the germ line from the soma is a classic concept in animal biology, and depending on species is thought to involve fate determination either by maternally localized germ plasm ("preformation" or "maternal inheritance") or by inductive signaling (classically termed "epigenesis" or "zygotic induction"). The latter mechanism is generally considered to operate in non-bilaterian organisms such as cnidarians and sponges, in which germ cell fate is determined at adult stages from multipotent stem cells. We have found in the hydrozoan cnidarian Clytia hemisphaerica that the multipotent "interstitial" cells (i-cells) in larvae and adult medusae, from which germ cells derive, express a set of conserved germ cell markers: Vasa, Nanos1, Piwi and PL10. In situ hybridization analyses unexpectedly revealed maternal mRNAs for all these genes highly concentrated in a germ plasm-like region at the egg animal pole and inherited by the i-cell lineage, strongly suggesting i-cell fate determination by inheritance of animal-localized factors. On the other hand, experimental tests showed that i-cells can form by epigenetic mechanisms in Clytia, since larvae derived from both animal and vegetal blastomeres separated during cleavage stages developed equivalent i-cell populations. Thus Clytia embryos appear to have maternal germ plasm inherited by i-cells but also the potential to form these cells by zygotic induction. Reassessment of available data indicates that maternally localized germ plasm molecular components were plausibly present in the common cnidarian/bilaterian ancestor, but that their role may not have been strictly deterministic., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: To describe the clinical presentations and treatment modalities of a series of BB gun-related perforating ocular injuries., Methods: Clinical records of all consecutive cases of perforating BB gun injuries to the globe seen between September 2004 and September 2008 were reviewed retrospectively. At the time of the trauma and after final treatment, all patients underwent a complete ocular examination, including visual acuity,applanation tonometry for intraocular pressure, slit lamp biomicroscopy, indirect ophthalmoscopy and fundus photography, if possible. In all cases, primary globe repair was performed in the first session, and then appropriate surgery took place based on the individual situation., Results: In this study, 13 patients (11 males and 2 females) with a mean age of 20.8 years (range 9–50 years) were enrolled. The mean follow-up period was 7.2 4.3 months (range 1–25 months). Initial visual acuity (VA) ranged from no-light perception (NLP) to finger counting (CF). Vitreous haemorrhage and retinal detachment were present in all involved eyes. Hyphema (30.76%), uveal and retinal prolapse (30.8%), retinalin carceration (30.8%) and retinal haemorrhage (53.8%) were other ocular findings. VA remained stable in 46.2% of the patients (6 cases). The best achieved final VA was CF at 2 min one case after 6 months follow-up.After several surgical procedures, enucleation was necessary in only 2/13 (15.4%) cases., Conclusion: Despite several surgical procedures which decreased the number of enucleations, BB gunperforating ocular injuries still lead to a grim visual outcome. This implies the importance of political strategies targeting on education of parents and restriction for children to access to these guns., (2010 Elsevier Ltd. All rights reserved.)

Stem cells are essential for animal development and adult tissue homeostasis, and the quest for an ancestral gene fingerprint of stemness is a major challenge for evolutionary developmental biology. Recent studies have indicated that a series of genes, including the transposon silencer Piwi and the translational activator Vasa, specifically involved in germline determination and maintenance in classical bilaterian models (e.g., vertebrates, fly, nematode), are more generally expressed in adult multipotent stem cells in other animals like flatworms and hydras. Since the progeny of these multipotent stem cells includes both somatic and germinal derivatives, it remains unclear whether Vasa, Piwi, and associated genes like Bruno and PL10 were ancestrally linked to stemness, or to germinal potential. We have investigated the expression of Vasa, two Piwi paralogues, Bruno and PL10 in Pleurobrachia pileus, a member of the early-diverging phylum Ctenophora, the probable sister group of cnidarians. These genes were all expressed in the male and female germlines, and with the exception of one of the Piwi paralogues, they showed similar expression patterns within somatic territories (tentacle root, comb rows, aboral sensory complex). Cytological observations and EdU DNA-labelling and long-term retention experiments revealed concentrations of stem cells closely matching these gene expression areas. These stem cell pools are spatially restricted, and each specialised in the production of particular types of somatic cells. These data unveil important aspects of cell renewal within the ctenophore body and suggest that Piwi, Vasa, Bruno, and PL10 belong to a gene network ancestrally acting in two distinct contexts: (i) the germline and (ii) stem cells, whatever the nature of their progeny., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Most cells with an intact interferon-gamma receptor and signaling pathway are able to express MHC class II molecules when treated with cytokines such as interferon-gamma and tumor necrosis factor-a. Interestingly, primary uveal melanocytes and most ocular melanoma cells are resistant to interferon-gamma mediated induction of class II MHC genes. This unusual phenotype is hypothesized to be germane to the immune-privileged status to the eye. Via a series of experiments, we have probed the molecular basis of this class II MHC resistant phenotype. We have analyzed the methylation status of the gene encoding the class II transactivator (CIITA), and asked whether treatment of class II MHC resistant ocular melanoma cells with the demethylating agent 5'-azacytidine can restore interferon-gamma inducibility of these class II MHC genes in these cells. The data obtained suggest that the specific blockade in cytokine-induced class II MHC gene expression is due to a suppression of the gene encoding the class II transactivator (CIITA). Treatment with 5' azacytidine restores the ability of these cells to express class II MHC genes upon interferon-gamma treatment. Whilst this is reminiscent of what occurs in another immune-privileged tissue--the placental trophoblast--we show here that silencing of the CIITA gene in uveal melanocytes either involves methylation of distinct nucleotides from those detected in trophoblasts, or involves an upstream activator of CIITA gene expression.

The lipid organization in the upper layer of the skin, the stratum corneum (SC), is important for the skin barrier function. This lipid organization, including the characteristic 13 nm lamellar phase, can be reproduced in vitro with mixtures based on cholesterol, free fatty acids and natural as well as synthetic ceramides (CER). In human SC, nine CER classes have been identified (CER1-CER9). Detailed studies on the effect of molecular structure of individual ceramides on the SC lipid organization are only possible with synthetic lipid mixtures, as their composition can be accurately chosen and systematically modified. In the present study, small-angle X-ray diffraction was used to examine the organization in synthetic lipid mixtures of which the synthetic ceramide fraction was prepared with sphingosine-based CER1 or phytosphingosine-based CER9. The latter acylceramide contains an additional hydroxyl group at the sphingoid backbone. The results show that a gradual increase in CER1 level consistently promotes the formation of the 13 nm lamellar phase and that partial replacement of CER1 by CER9 does not affect the phase behavior. Interestingly, complete substitution of CER1 with CER9 reduces the formation of the long periodicity phase and results in phase separation of CER9.

Structural maintenance of chromosomes (SMC) proteins have diverse cellular functions including chromosome segregation, condensation and DNA repair. They are grouped based on a conserved set of distinct structural motifs. All SMC proteins are predicted to have a bipartite ATPase domain that is separated by a long region predicted to form a coiled coil. Recent structural data on a variety of SMC proteins shows them to be arranged as long intramolecular coiled coils with a globular ATPase at one end. SMC proteins function in pairs as heterodimers or as homodimers often in complexes with other proteins. We expect the arrangement of the SMC protein domains in complex assemblies to have important implications for their diverse functions. We used scanning force microscopy imaging to determine the architecture of human, Saccharomyces cerevisiae, and Pyrococcus furiosus Rad50/Mre11, Escherichia coli SbcCD, and S.cerevisiae SMC1/SMC3 cohesin SMC complexes. Two distinct architectural arrangements are described, based on the way their components were connected. The eukaryotic complexes were similar to each other and differed from their prokaryotic and archaeal homologs. These similarities and differences are discussed with respect to their diverse mechanistic roles in chromosome metabolism.

Background/aims: Inflammation in the liver is a complex interaction between parenchymal and non-parenchymal cells, and therefore can not be studied in vitro in pure cultures of these cells., Methods: We investigated whether Kupffer cells in the liver slice are still responsive to an inflammatory stimulus of lipopolysaccharide (LPS), and evoke an inflammatory response in the hepatocytes., Results: TNFalpha, IL-1beta and IL-10 were significantly elevated in culture medium of LPS-stimulated rat liver slices. Nitric oxide (NO) production of LPS-treated slices gradually increased from 5 to 24 h (24 h: 81+/-5 microM vs. 14+/-2 microM in control P < 0.05), paralleled by inducible nitric oxide synthase (iNOS) in the hepatocytes, iNOS mRNA was induced after 3 h. NO production but not iNOS induction was significantly inhibited by NOS inhibitors S-methylisothiourea and N(G)-nitro-L-arginine methylester. Both pentoxifylline and dexamethasone inhibited TNFalpha and IL-1beta production, albeit to a different extent, iNOS induction and, as a result thereof, NO production., Conclusions: These results imply that non-parenchymal cells in liver slices are viable and can be activated by LPS. In addition, it is concluded that the upregulation of iNOS in hepatocytes by LPS is caused by cytokines produced by Kupffer cells because inhibition of TNFalpha and IL-1beta production attenuated iNOS induction.

In the present study we investigated the applicability of the liver slice model to study mechanisms of drug uptake. Four model compounds were investigated that enter hepatocytes via entirely different membrane transport mechanisms. Rhodamine B (RB), which enters hepatocytes by passive diffusion, was homogeneously distributed throughout the rat liver slice (250 microm thickness) within 5 min, indicating that the penetration rate into the slice and the diffusion rate into the cells are rapid. In contrast, lucigenin (LU), which is taken up by hepatocytes through adsorptive endocytosis, was detected in the inner cell layers after 15 min. Digoxin uptake into the slice showed a temperature-dependent component and was stereoselectively inhibited by quinine, which is compatible with the involvement of a carrier-mediated uptake mechanism. The neo-glycoalbumin Lactose(27)-Human Serum Albumin (Lact(27)-HSA) and the negatively charged Succinylated-Human Serum Albumin (Suc-HSA) entered the slices and were taken up temperature-dependently into hepatocytes and endothelial cells, respectively. The liver slice preparation is a valuable tool to investigate the mechanisms of cellular uptake of drugs. Moreover, the precision-cut liver slices offer the unique possibility to study both hepatocyte and endothelial cell function in human and rat liver.

Corneal transplantations may survive when transplanted orthotopically even in the presence of massive corneal neovascularization, while such grafts would be rejected in heterotopic locations. Experiments with murine tissues have shown that cultured corneal tissue as well as iris/ciliary body (I/CB) tissue produce immunosuppressive factors. This was not the case when traumatized corneas were tested. We therefore wondered whether the same difference could be observed when explants of normal or pathological human corneas or I/CB tissues were tested. Supernatant of cultured cornea and I/CB tissue was added to a mixed lymphocyte reaction (BALB/c responder and C57B1/6 stimulator cells) and thymidine incorporation was measured. Culture supernatants from normal corneas and corneas with bullous keratopathy achieved similar levels of MLR inhibition, which could not be blocked by addition of anti-transforming-growth factor-beta 2 antibody or indomethacin. The supernatant from cultured I/CB cells from normal eyes and from eyes containing a posterior segment melanoma did not differ in their immunosuppressive effect. The MLR inhibitory effect of supernatant from I/CB cells was partly blocked by addition of indomethacin. We conclude that MLR inhibiting factors are produced by normal and pathological corneas and by I/CB cells, but the supernatants from these tissues achieve their effect by different means. It is likely that the ability of cornea and I/CB tissues to produce immunosuppressive factors contributes to the success of orthotopic corneal transplants in man.

Atypical nevi and other potential risk factors for uveal melanoma were studied in 109 uveal melanoma patients and 149 controls. Information concerning employment, medical history, drug use, family history of cancer, excess sun exposure, and blistering sunburn before and after the age of 15 was obtained. A total skin examination was performed and skin type, hair color, eye color, freckles, actinic damage, the total number of common acquired nevi, and the number of clinically atypical nevi were noted. More atypical nevi were found in uveal melanoma patients than in controls (age- and sex-adjusted odds ratio of 2.9 [95% confidence interval 1.2-6.3] for one or two atypical nevi versus none; odds ratio of 5.1 [95% CI 1.3-20.0] for three or more atypical nevi versus none). Light skin types and freckling also prevailed in uveal melanoma cases. In our study, atypical nevi are more common in uveal melanoma patients than in controls. Further studies will have to indicate whether risk factors comparable to those for cutaneous melanoma really exist for uveal melanoma.

The reactivity of 33 choroidal and ciliary body melanomas with monoclonal antibody Ki-67, which recognizes a proliferation associated nuclear antigen, has been assessed and compared with clinicopathologic parameters. In 23 cases, 8 Gy irradiation was given 2 days before enucleation. Nonirradiated melanomas had a significantly higher proliferation rate as defined by staining with monoclonal antibody Ki-67 as compared with irradiated tumors (P = 0.007). Similarly, a strong relationship was found between pre-enucleation irradiation and low mitotic activity (P = 0.001). There was no significant correlation between the presence of Ki-67-positive nuclei and histologic classification, largest tumor diameter, localization of the tumor, age, sex, scleral invasion, pigmentation, and lymphocytic infiltration. The relevance of Ki-67 immunohistochemistry for the assessment of the life prognosis of patients with uveal melanoma has to be studied prospectively.

Monocyte specific antigens are relevant in renal and bone marrow transplantation, but a reproducible monocyte-antigen system has not yet been recognized. In order to establish a sensitive test system with reproducible results in monocyte serology, 3 different monocyte cytotoxicity techniques were compared. In our hands the two-colour fluorescence test on post-Ficoll total leukocyte suspensions fulfilled the criteria. This technique was used to screen sera from multiparous women and renal transplant recipients for the presence of monocyte specific antibodies. By testing sera on cells from individuals who were HLA compatible with the serum donors, anti-HLA reactions were excluded. Several promising sera containing monocyte specific antibodies were identified, thus indicating the success of our approach.