Surveillance of Fish Diseases in the Nordic Countries

Due to the increasing importance of disease problems in the fish farming industry and the impact disease may have on both feral and farmed fish in the Nordic countries, monitoring and surveillance on diseases have for many years been considered to be of socioeconomic importance. All the Nordic countries have a national legislation as basis for their surveillance and disease control in aquatic animals and regulations listing notifiable diseases of concern to the countries. The list of diseases vary between the countries. In addition, Denmark, Finland and Sweden are ruled by [3] as regards placing on the market of aquaculture animals and products. The surveillance for viral diseases in all the Nordic countries has mainly been based on the testing procedures given in the EU Commission [2].     

Hypercalcemia induces a proinflammatory phenotype in rat leukocytes and endothelial cells

Endothelial plasma membrane lipid microdomains, so called lipid rafts/caveolae, are rich in neutral glycosphingolipid, globotriaosylceramide, Gb3Cer, or CD77. Several plasma membrane Ca²⁺ channels and pumps are located in lipid rafts/caveolae. Increased Ca²⁺ influx could cause the development of an endothelial proinflammatory phenotype. Therefore, the aim of this study was to estimate the effects of hypercalcemia in rats by determination of CD77 expression on CD34+ endothelial cells in the heart, kidney, and vena cava. In addition, potential proinflammatory calcium effect was estimated by CD11b and CD15s expression on leukocytes. To achieve hypercalcemia, Sprague–Dawley male rats were given CaCl₂ solution with a concentration of 1.5 % elemental calcium during 14 days. CD77 expression on CD34+ endothelial cells in cell suspensions of the heart, kidney, and vena cava, as well as leukocyte expression of CD11b and CD15s in hypercalcemic and control rats were determined by flow cytometry. Ionized calcium concentration in plasma was 1.37?±?0.01 mM in hypercalcemic vs. 1.19?±?0.03 mM in control rats. Hypercalcemic group showed statistically significantly decreased proportion of endothelial CD34+ CD77? cells in the kidney and vena cava in parallel with increase of CD11b and CD15s leukocyte proinflammatory markers. In conclusion, it is tempting to speculate that plasma membranes of glycosphingolipid CD77? endothelial cells are poorer in caveolae lipid microdomains and therefore weaker in controlling of Ca²⁺ influx. The percentage of CD11b+ CD15s+ leukocytes could be a measure of proinflammatory effects of mild hypercalcemia.     

Anti-inflammatory mechanism of exogenous C2 ceramide in lipopolysaccharide-stimulated microglia

Ceramide is a major molecule among the sphingolipid metabolites which are produced in the brain and other organs and act as intracellular second messengers. Although a variety of physiological roles of ceramide have been reported in the periphery and central nervous systems, the role of ceramide in microglial activation has not been clearly demonstrated. In the present study, we examined the effects of exogenous cell permeable short chain ceramides on microglial activation in vitro and in vivo. We found that C2, C6, and C8 ceramide and C8 ceramide-1-phosphate inhibited iNOS and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and rat primary microglia. In addition, the administration of C2 ceramide suppressed microglial activation in the brains of LPS-exposed mice. By HPLC and LC/MS/MS analyses, we found that C2 ceramide on its own, rather than its modified form (i.e. ceramide-1-phosphate or long chain ceramides), mainly work by penetrating into microglial cells. Further mechanistic studies by using the most effective C2 ceramide among the short chain ceramides tested, revealed that C2 ceramide exerts anti-inflammatory effects via inhibition of the ROS, MAPKs, PI3K/Akt, and Jak/STAT pathways with upregulation of PKA and hemeoxygenase-1 expressions. Interestingly, we found that C2 ceramide inhibits TLR4 signaling by interfering with LPS and TLR4 interactions. Therefore, our data collectively suggests the therapeutic potential of short chain ceramides such as C2 for neuroinflammatory disorders such as Alzheimer's disease and Parkinson's disease.     

Biochemical and crystallographic characterization of a glucansucrase from Lactobacillus reuteri 180

Glucansucrases are large extracellular transglycosidases secreted by lactic acid bacteria. Using sucrose as a substrate they synthesize high molecular mass α-glucans or, in the presence of suitable acceptor molecules, low molecular mass oligosaccharides. Although about 60 glucansucrases have been classified in glycoside hydrolase family GH70, no three-dimensional structure has been reported for any. With the aim of solving the first structure of a GH70 glucansucrase, purification and crystallization experiments were performed with a fully active, 117 kDa N-terminally truncated fragment of glucansucrase GTF180 from Lactobacillus reuteri 180 (residues 742–1772). Crystallization experiments yielded crystals that belong to two different triclinic crystal forms (space group P1) and one orthorhombic crystal form (space group P2₁2₁2₁). Native data sets for both triclinic and the orthorhombic crystals were collected at 1.7 and 2.0 Å resolution, respectively. Enzyme activity assays, pH and temperature optima show comparable values for both the full-length and the N-terminally truncated GTF180.     

Regulation of Cell Wall Synthesis by the Clathrin Light Chain Is Essential for Viability in Schizosaccharomyces pombe

The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, β(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization.     

Associations between APOE Genotypes and Disease Susceptibility,Joint Damage and Lipid Levels in Patients with Rheumatoid Arthritis

Objective

Apolipoprotein E (APOE) genotypes are associated with cardiovascular disease (CVD) and lipid levels. In rheumatoid arthritis (RA), an association has been found with disease activity. We examined the associations between APOE genotypes and disease susceptibility and markers of disease severity in RA, including radiographic joint damage, inflammatory markers, lipid levels and cardiovascular markers.

Method

A Norwegian cohort of 945 RA patients and 988 controls were genotyped for two APOE polymorphisms. We examined longitudinal associations between APOE genotypes and C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) as well as hand radiographs (van der Heijde Sharp Score(SHS)) in 207 patients with 10 year longitudinal data. Lipid levels, cardiovascular markers and history of CVD were compared across genotypes in a cross sectional study of 136 patients. Longitudinal radiological data of cohorts from Lund and Leiden were available for replication. (N = 935, with 4799 radiographs).

Results

In the Norwegian cohort, associations between APOE genotypes and total cholesterol (TC) and low-density lipoproteins (LDL) were observed (ε2<ε3/ε3<ε4, p = 0.03 and p = 0.02, respectively). No association was present for acute phase reactant or CVD markers, but a longitudinal linear association between APOE genotypes and radiographic joint damage was observed (p = 0.007). No association between APOE genotypes and the severity of joint destruction was observed in the Lund and Leiden cohorts, and a meta- analysis combining all data was negative.

Conclusion

APOE genotypes are associated with lipid levels in patients with RA, and may contribute to dyslipidemia in some patients. APOE genotypes are not consistently associated with markers of inflammation or joint destruction in RA.     

Linkage disequilibrium in two European F<Subscript>2</Subscript> flint maize populations under modified recurrent full-sib selection

According to quantitative genetic theory, linkage disequilibrium (LD) can hamper the short- and long-term selection response in recurrent selection (RS) programs. We analyzed LD in two European flint maize populations, KW1265 × D146 (A × B) and D145 × KW1292 (C × D), under modified recurrent full-sib selection. Our objectives were to investigate (1) the decay of initial parental LD present in F₂ populations by three generations of intermating, (2) the generation of new LD in four (A × B) and seven (C × D) selection cycles, and (3) the relationship between LD changes and estimates of the additive genetic variance. We analyzed the F₂ and the intermated populations as well as all selection cycles with 104 (A × B) and 101 (C × D) simple sequence repeat (SSR) markers with a uniform coverage of the entire maize genome. The LD coefficient D and the composite LD measure Δ were estimated and significance tests for LD were performed. LD was reduced by intermating as expected from theory. A directional generation of negative LD between favorable alleles could not be observed during the selection cycles. However, considerable undirectional changes in D were observed, which we attributed to genetic sampling due to the finite population size used for recombination. Consequently, a long-term reduction of the additive genetic variance due to negative LD was not observed. Our experimental results support the hypothesis that in practical RS programs with maize, LD generated by selection is not a limiting factor for obtaining a high selection response.     

Are Patients with Erythema Migrans Who Have Leukopenia and/or Thrombocytopenia Coinfected with Anaplasma phagocytophilum or Tick-Borne Encephalitis Virus?

Lyme borreliosis (LB), tick-borne encephalitis (TBE) and human granulocytic anaplasmosis (HGA) are endemic in central part of Slovenia. We tested the hypothesis that patients with erythema migrans (EM) from this region, who have leukopenia and/or thrombocytopenia (typical findings in HGA and in the initial phase of TBE but not in patients with LB) are coinfected with Anaplasma phagocytophilum and/or with TBE virus, i.e. that cytopenia is a result of concomitant HGA or the initial phase of TBE. Comparison of clinical and laboratory findings for 67 patients with EM who disclosed leukopenia/thrombocytopenia with the corresponding results in sex- and age-matched patients with EM and normal blood cell counts revealed no differences. In addition, patients with typical EM and leukopenia and/or thrombocytopenia tested negative for the presence of IgM and IgG antibodies to TBE virus by ELISA as well as for the presence of specific IgG antibodies to A. phagocytophilum antigens by IFA in acute and convalescent serum samples. Thus, none of 67 patients (95% CI: 0 to 5.3%) with typical EM (the presence of this skin lesion attests for early Lyme borreliosis and is the evidence for a recent tick bite) was found to be coinfected with A. phagocytophilum or had a recent primary infection with TBE virus. The findings in the present study indicate that in Slovenia, and probably in other European countries endemic for LB, TBE and HGA, patients with early LB are rarely coinfected with the other tick-transmitted agents.     

Tick-Borne Encephalitis Virus Infects Rat Astrocytes but Does Not Affect Their Viability

Tick-borne encephalitis virus (TBEV) causes one of the most dangerous human neuroinfections in Europe and Asia. To infect neurons it must cross the blood-brain-barrier (BBB), and presumably also cells adjacent to the BBB, such as astrocytes, the most abundant glial cell type. However, the knowledge about the viral infection of glial cells is fragmental. Here we studied whether TBEV infects rat astrocytes. Rats belong to an animal group serving as a TBEV amplifying host. We employed high resolution quantitative fluorescence microscopy to investigate cell entry and cytoplasmic mobility of TBEV particles along with the effect on the cell cytoskeleton and cell survival. We report that infection of astrocytes with TBEV increases with time of exposure to TBEV and that with post-infection time TBEV particles gained higher mobility. After several days of infection actin cytoskeleton was affected, but cell survival was unchanged, indicating that rat astrocytes resist TBEV-mediated cell death, as reported for other mammalian cells. Therefore, astrocytes may present an important pool of dormant TBEV infections and a new target for therapeutic intervention.