Differences in growth between offspring of anadromous and freshwater brown trout Salmo trutta.

In this study, individual growth of juvenile offspring of anadromous and freshwater resident brown trout Salmo trutta and crosses between the two from the River Imsa, Norway, was estimated. The juveniles were incubated until hatching at two temperatures (±S.D.), either 4.4 ± 1.5°C or 7.1 ± 0.6°C. Growth rate was estimated for 22 days in August-September when the fish on average were c. 8 g in wet mass, and the estimates were standardized to 1 g fish dry mass. Offspring of anadromous S. trutta grew better at both 15 and 18°C than offspring of freshwater resident S. trutta or offspring of crosses between the two S. trutta types. This difference appears not to result from a maternal effect because anadromous S. trutta grew better than the hybrids with anadromous mothers. Instead, this appears to be an inherited difference between the anadromous and the freshwater resident fish lending support to the hypothesis that anadromous and freshwater resident S. trutta in this river differ in genetic expression. Egg incubation temperature of S. trutta appeared not to influence the later growth as reported earlier from the studies of Atlantic salmon Salmo salar.

Continuous outmigration and sequential encountering of environmental cues are important for successful homing of hatchery-reared, anadromous brown trout Salmo trutta.

When rehabilitating and reintroducing trout Salmo trutta in rivers, it is a goal that as many as possible survive, home and form self-sustaining populations. Hatchery-reared, anadromous S. trutta have significant lower ability to return to the area where they were raised if (a) transported in a closed tank to sea and released 5 km from the River Imsa, relative to those that were (b) transported when swimming in a partly submerged tank with sea water run-through, while being slowly towed by a boat the same distance or (c) released at the outlet of the River Imsa. Thus, if deprived from environmental cues during part of the way, they lose their ability to home.

Egg incubation temperature affects the timing of the Atlantic salmon Salmo salar homing migration.

Here, we show that adult Atlantic salmon Salmo salar returned about 2 weeks later from the feeding areas in the North Atlantic Ocean to the Norwegian coast, through a phenotypically plastic mechanism, when they developed as embryos in c. 3°C warmer water than the regular incubation temperature. This finding has relevance to changes in migration timing caused by climate change and for cultivation and release of S. salar.

Monocyte Chemoattractant Protein-1 in Antineutrophil Cytoplasmic Autoantibody-Associated Vasculitis: Biomarker Potential and Association with Polymorphisms in the MCP-1 and the CC Chemokine Receptor-2 Gene.

Antineutrophil cytoplasmic autoantibody- (ANCA-) associated vasculitis (AAV) are relapsing-remitting disorders with unpredictable prognosis. There is a need of biomarkers for distinguishing which patients will have a more severe outcome and also for predicting relapses in disease activity. This study confirms the previous results of urinary MCP-1 (uMCP-1) as a prognostic marker and explores its potential as a marker of disease activity. Method. 114 patients with AAV were followed regularly between 2002 and 2011 at Skåne University Hospital. Urine samples, blood samples, and clinical status were registered. The urine samples were analyzed in an in-house-developed ELISA. PCR-RLFP was used to analyze the MCP-1 and CCR2 genes. Results. Patients with severe prognosis had significantly higher levels of uMCP-1 compared to patients with nonsevere prognosis and healthy controls. Patients with renal damage had higher levels compared to patients who did not have renal damage. There was also a tendency of higher uMCP-1 levels in active disease as compared to remission. AA in the -2518 position in the MCP-1 gene was associated with a more severe outcome compared to the A/G or the G/G genotype. The A/A genotype were also associated with higher levels of uMCP-1. No significant associations were seen for the CCR2-V64I. Conclusion. This study confirmed the connection between high uMCP-1 levels and poor prognosis and also disease activity. It also suggests an association of the A/A genotype at position -2518 in the MCP-1 gene and poor prognosis in AAV. uMCP-1 is clearly a candidate biomarker of potential clinical value. The A/A genotype association needs further evaluation.

Reciprocal relationship between contact and complement system activation on artificial polymers exposed to whole human blood.

BACKGROUND: Inappropriate and uncontrolled activation of the cascade systems in the blood is a driving force in adverse inflammatory and thrombotic reactions elicited by biomaterials, but limited data are available on the activation of the contact system by polymers and the present study was undertaken to investigate these mechanisms in established models. METHODS: Polymer particles were incubated in (1) EDTA-plasma (10 mM) to monitor the adsorption of 20 selected proteins; (2) lepirudin-anticoagulated plasma to evaluate contact system activation, monitored by the formation of complexes between the generated proteases factor[F]XIIa, FXIa and kallikrein and the serpins C1-inhibitor [C1INH] and antithrombin [AT]; (3) lepirudin-anticoagulated whole blood to determine cytokine release. RESULTS: Strong negative correlations were found between 10 cytokines and the ratio of deposited FXII/C1INH, generated FXIIa-C1INH complexes, and kallikrein-C1INH complexes. Formation of FXIIa-C1INH complexes correlated negatively with the amount of C3a and positively with deposited IgG. CONCLUSIONS: A reciprocal relationship was found between activation of the contact system and the complement system induced by the polymers studied here. The ratios of FXII/C1INH or C4/C4BP, adsorbed from EDTA-plasma are useful surrogate markers for cytokine release and inflammatory response to materials intended for blood contact.

Thromboinflammation in Therapeutic Medicine.

Thromboinflammation is primarily triggered by the humoral innate immune system, which mainly consists of the cascade systems of the blood, i.e., the complement, contact/coagulation and fibrinolytic systems. Activation of these systems subsequently induces activation of endothelial cells, leukocytes and platelets, finally resulting in thrombotic and inflammatory reactions. Such reactions are triggered by a number of medical procedures, e.g., treatment with biomaterials or drug delivery devices as well as in transplantation with cells, cell clusters or whole vascularized organs. Here, we (1) describe basic mechanisms for thromboinflammation; (2) review thromboinflammatory reactions in therapeutic medicine; and (3) discuss emerging strategies to dampen thromboinflammation.

Efficient replication of recombinant Enterovirus B types, carrying different P1 genes in the coxsackievirus B5 replicative backbone.

Recombination is an important feature in the evolution of the Enterovirus genus. Phylogenetic studies of enteroviruses have revealed that the capsid genomic region (P1) is type specific, while the parts of the genome coding for the non-structural proteins (P2-P3) are species specific. Hence, the genome may be regarded as consisting of two modules that evolve independently. In this study, it was investigated whether the non-structural coding part of the genome in one type could support replication of a virus with a P1 region from another type of the same species. A cassette vector (pCas) containing a full-length cDNA copy of coxsackievirus B5 (CVB5) was used as a replicative backbone. The P1 region of pCas was replaced with the corresponding part from coxsackievirus B3 Nancy (CVB3N), coxsackievirus B6 Schmitt (CVB6S), and echovirus 7 Wallace (E7W), all members of the Enterovirus B species. The replication efficiency after transfection with clone-derived in vitro transcribed RNA was studied and compared with that of pCas. All the recombinant viruses replicated with similar efficiencies and showed threshold cycle (Ct) values, tissue culture infectivity dose 50 %, and plaque-forming unit titers comparable to viruses generated from the pCas construct. In addition to this, a clone without the P1 region was also constructed, and Western Blot and immunofluorescence staining analysis showed that the viral genome could be translated and replicated despite the lack of the structural protein-coding region. To conclude, the replicative backbone of the CVB5 cassette vector supports replication of intraspecies constructs with P1 regions derived from other members of the Enterovirus B species. In addition to this, the replicative backbone can be both translated and replicated without the presence of a P1 region.

Low concentrations of citrate reduce complement and granulocyte activation in vitro in human blood.

BACKGROUND: The use of acetate in haemodialysis fluids may induce negative effects in patients including nausea and increased inflammation. Therefore, haemodialysis fluids where acetate is substituted with citrate have recently been developed. In this study, we investigated the biocompatibility of citrate employing concentrations used in haemodialysis. METHODS: The effects of citrate and acetate were investigated in human whole blood in vitro under conditions promoting biomaterial-induced activation. Complement activation was measured as generation of C3a, C5a and the sC5b-9 complex, and granulocyte activation as up-regulation of CD11b expression. For the experimental set-up, a mathematical model was created to calculate the concentrations of acetate and citrate attained during haemodialysis. RESULTS: Citrate reduced granulocyte activation and did not induce higher complement activation compared with acetate at concentrations attained during haemodialysis. Investigating different citrate concentrations clearly showed that citrate is a potent complement inhibitor already at low concentrations, i.e. 0.25 mM, which is comparable with concentrations detected in the blood of patients during dialysis with citrate-containing fluids. Increased citrate concentration up to 6 mM further reduced the activation of C3a, C5a and sC5b-9, as well as the expression of CD11b. CONCLUSIONS: Our results suggest that citrate is a promising substitute for acetate for a more biocompatible dialysis, most likely resulting in less adverse effects for the patients.

Early cytokine release in response to live Borrelia burgdorferi Sensu Lato Spirochetes is largely complement independent.

AIM: Here we investigated the role of complement activation in phagocytosis and the release of cytokines and chemokines in response to two clinical isolates: Borrelia afzelii K78, which is resistant to complement-mediated lysis, and Borrelia garinii LU59, which is complement-sensitive. METHODS: Borrelia spirochetes were incubated in hirudin plasma, or hirudin-anticoagulated whole blood. Complement activation was measured as the generation of C3a and sC5b-9. Binding of the complement components C3, factor H, C4, and C4BP to the bacterial surfaces was analyzed. The importance of complement activation on phagocytosis, and on the release of cytokines and chemokines, was investigated using inhibitors acting at different levels of the complement cascade. RESULTS: 1) Borrelia garinii LU59 induced significantly higher complement activation than did Borrelia afzelii K78. 2) Borrelia afzelii K78 recruited higher amounts of factor H resulting in significantly lower C3 binding. 3) Both Borrelia strains were efficiently phagocytized by granulocytes and monocytes, with substantial inhibition by complement blockade at the levels of C3 and C5. 4) The release of the pro-inflammatory cytokines and chemokines IL-1ß, IL-6, TNF, CCL20, and CXCL8, together with the anti-inflammatory IL-10, were increased the most (by>10-fold after exposure to Borrelia). 5) Both strains induced a similar release of cytokines and chemokines, which in contrast to the phagocytosis, was almost totally unaffected by complement blockade. CONCLUSIONS: Our results show that complement activation plays an important role in the process of phagocytosis but not in the subsequent cytokine release in response to live Borrelia spirochetes.

Basin-scale phenology and effects of climate variability on global timing of initial seaward migration of Atlantic salmon (Salmo salar).

Migrations between different habitats are key events in the lives of many organisms. Such movements involve annually recurring travel over long distances usually triggered by seasonal changes in the environment. Often, the migration is associated with travel to or from reproduction areas to regions of growth. Young anadromous Atlantic salmon (Salmo salar) emigrate from freshwater nursery areas during spring and early summer to feed and grow in the North Atlantic Ocean. The transition from the freshwater ('parr') stage to the migratory stage where they descend streams and enter salt water ('smolt') is characterized by morphological, physiological and behavioural changes where the timing of this parr-smolt transition is cued by photoperiod and water temperature. Environmental conditions in the freshwater habitat control the downstream migration and contribute to within- and among-river variation in migratory timing. Moreover, the timing of the freshwater emigration has likely evolved to meet environmental conditions in the ocean as these affect growth and survival of the post-smolts. Using generalized additive mixed-effects modelling, we analysed spatio-temporal variations in the dates of downstream smolt migration in 67 rivers throughout the North Atlantic during the last five decades and found that migrations were earlier in populations in the east than the west. After accounting for this spatial effect, the initiation of the downstream migration among rivers was positively associated with freshwater temperatures, up to about 10 °C and levelling off at higher values, and with sea-surface temperatures. Earlier migration occurred when river discharge levels were low but increasing. On average, the initiation of the smolt seaward migration has occurred 2.5 days earlier per decade throughout the basin of the North Atlantic. This shift in phenology matches changes in air, river, and ocean temperatures, suggesting that Atlantic salmon emigration is responding to the current global climate changes.

Effects of temperature and food quality on age and size at maturity in ectotherms: an experimental test with Atlantic salmon.

The reaction norm between growth rate, age and size at maturity in ectotherms is widely debated in ecological literature. It has been proposed that the effect depends on whether growth is affected by food quality or temperature (called the Berrigan-Charnov puzzle). The present experiment tested this for Atlantic salmon (Salmo salar). We enhanced growth rates by increasing temperature and ratio of lipids to proteins in the food for groups of Atlantic salmon. Both treatments gave higher percentages of early mature and therefore smaller adults in contrast to the proposed Berrigan-Charnov puzzle. There was a difference between sexes in that males could attain maturity 1 year younger than females when reared under similar environmental conditions. Males that matured during the first year in sea water were smaller than similar aged immature males. The probability of that Atlantic salmon attained maturity for the first time during their second year in sea increased with growth rate during the preceding winter and if fed a high-lipid diet. Increased summer temperature exhibited no additional effect. Similar aged fish reared at elevated temperature and fed high-lipid diet attained maturity at a larger body mass and exhibited higher mass-length-ratios than those reared at natural temperature and fed a low-lipid diet, indicating that structural growth has priority over lipid deposits. Increased growth rate before the onset of maturation, whether this is owing to enhanced lipid content in food or increased water temperature, decreased age and therefore size at maturity. Enhanced lipid relative to protein content in food, but not temperature, had an additive positive effect on early maturation probability, likely due to increased amounts of reserve energy. These results may be general for ectotherm organisms.

Aichi virus infection in elderly people in Sweden.

Aichi virus (AiV), genus Kobuvirus, family Picornaviridae, is associated with gastroenteritis in humans. Previous studies have shown high seroprevalence but low incidence (0.9-4.1%) in clinical samples. We report here the first detection of AiV in Sweden. Two hundred twenty-one specimens from hospitalized patients with diarrhea, who were negative for other enteric viruses, were included in the study. AiV were detected in three specimens, all from elderly patients. Phylogenetic analysis revealed that the three Swedish isolates belonged to genotype A and were genetically closest to European and Asian strains of AiV.

Plus disease in retinopathy of prematurity: diagnostic impact of field of view.

PURPOSE: To examine the impact of retinal field of view and magnification on interexpert reliability of plus disease diagnosis in retinopathy of prematurity. METHODS: Fifteen wide-angle images from infants with retinopathy of prematurity were cropped and adjusted in magnification to create 2 additional image categories: medium angle (40°-50°) and narrow angle (20°-30°). These 45 images were uploaded to a Web-based system and interpreted independently by 13 experts of retinopathy of prematurity using a 3-level (plus, preplus, neither) and 2-level (plus, not plus) classification. Absolute agreement and kappa statistics were calculated to compare interexpert reliability. RESULTS: In the 3-level classification, ≥ 70% experts agreed on the same diagnosis in 8 of the 15 wide-angle images (53%), but only in 3 of the 15 medium-angle (20%) and 3 of the 15 narrow-angle (20%) images. In the 2-level classification, ≥ 80% experts agreed on the same diagnosis in 11 of the 15 wide-angle images (73%), but only in 9 of the 15 medium-angle (60%) and 3 of the 15 narrow-angle (20%) images. Mean kappa of each expert compared with all other experts was 0.40 to 0.59 in 8 of 13 experts (62%) using wide-angle images, was 0 to 0.19 in 7 of 13 experts (54%) using medium-angle images, and was 0.20 to 0.39 in 9 of 13 experts (69%) using narrow-angle images. CONCLUSION: Interexpert agreement in plus disease diagnosis in wide-angle images is higher than from medium-angle and narrow-angle images. Plus disease is defined using a narrow-angle standard published photograph, yet this study suggests that peripheral findings also contribute to diagnosis.

Computer-based image analysis for plus disease diagnosis in retinopathy of prematurity.

Presence of plus disease in retinopathy of prematurity (ROP) is an important criterion for identifying ROP requiring treatment. Plus disease is defined by a standard published photograph selected more than 20 years ago by expert consensus. However, diagnosis of plus disease has been shown to be subjective and qualitative. Computer-based image analysis using quantitative methods has potential to improve the objectivity of plus disease diagnosis. The objective was to review the published literature involving computer-based image analysis for ROP diagnosis. The PubMed and Cochrane library databases were searched for the keywords "retinopathy of prematurity" AND "image analysis" AND/OR "plus disease." Reference lists of retrieved articles were searched to identify additional relevant studies. All relevant English-language studies were reviewed. There are four main computer-based systems-ROPtool (area under the receiver operating characteristic curve [AUROC], plus tortuosity 0.95, plus dilation 0.87), RISA (AUROC, arteriolar TI 0.71, venular diameter 0.82), Vessel Map (AUROC, arteriolar dilation 0.75, venular dilation 0.96), and CAIAR (AUROC, arteriole tortuosity 0.92, venular dilation 0.91)-attempting to objectively analyze vessel tortuosity and dilation in plus disease in ROP. Some show promise for identification of plus disease using quantitative methods. This has potential to improve the diagnosis of plus disease and may contribute to the management of ROP using both traditional binocular indirect ophthalmoscopy and image-based telemedicine approaches.

Cytolytic replication of echoviruses in colon cancer cell lines.

BACKGROUND: Colorectal cancer is one of the most common cancers in the world, killing nearly 50% of patients afflicted. Though progress is being made within surgery and other complementary treatments, there is still need for new and more effective treatments. Oncolytic virotherapy, meaning that a cancer is cured by viral infection, is a promising field for finding new and improved treatments. We have investigated the oncolytic potential of several low-pathogenic echoviruses with rare clinical occurrence. Echoviruses are members of the enterovirus genus within the family Picornaviridae. METHODS: Six colon cancer cell lines (CaCo-2, HT29, LoVo, SW480, SW620 and T84) were infected by the human enterovirus B species echovirus 12, 15, 17, 26 and 29, and cytopathic effects as well as viral replication efficacy were investigated. Infectivity was also tested in spheroids grown from HT29 cells. RESULTS: Echovirus 12, 17, 26 and 29 replicated efficiently in almost all cell lines and were considered highly cytolytic. The infectivity of these four viruses was further evaluated in artificial tumors (spheroids), where it was found that echovirus 12, 17 and 26 easily infected the spheroids. CONCLUSIONS: We have found that echovirus 12, 17 and 26 have potential as oncolytic agents against colon cancer, by comparing the cytolytic capacity of five low-pathogenic echoviruses in six colon cancer cell lines and in artificial tumors.

Characterization of a putative ancestor of coxsackievirus B5.

Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein-encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major cocirculating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates by using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence dated back to 1854 (1807 to 1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates in terms of receptor preferences, plaque phenotypes, growth characteristics, and cell tropism. This is the first report describing the resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including a phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy for the development of vaccines.

Studies of Echovirus 5 interactions with the cell surface: heparan sulfate mediates attachment to the host cell.

Infections caused by Echovirus 5 (E5), an enterovirus of the Picornaviridae family, have been associated with fever, rashes and sporadic cases of aseptic meningitis. To elucidate the receptor usage of this virus, the significance of a previously proposed integrin binding arginine-glycine-aspartic acid (RGD) motif found in the VP3 capsid protein was investigated, as well as the capacity of E5 to interact with heparan sulfate on the cell surface. Using the prototype strain E5 Noyce (E5N), an E5N mutant where the aspartic acid of the RGD motif has been substituted to a glutamic acid and clinical E5 isolates, the RGD motif of VP3 was found to be non-essential and hence not involved in integrin receptor binding. However, E5N and clinical E5 isolates interact with heparan sulfate at the cell surface, as demonstrated by virus replication inhibition assays using heparin and heparinase III, and studies of E5 interactions at the cell surface measured by real-time PCR analysis. In conclusion, E5 utilizes heparan sulfate as a cellular receptor, but the RGD motif of VP3 is not essential for E5 infectivity.

A single coxsackievirus B2 capsid residue controls cytolysis and apoptosis in rhabdomyosarcoma cells.

Coxsackievirus B2 (CVB2), one of six human pathogens of the group B coxsackieviruses within the enterovirus genus of Picornaviridae, causes a wide spectrum of human diseases ranging from mild upper respiratory illnesses to myocarditis and meningitis. The CVB2 prototype strain Ohio-1 (CVB2O) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells. These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions.

A rapid and efficient method for studies of virus interaction at the host cell surface using enteroviruses and real-time PCR.

BACKGROUND: Measuring virus attachment to host cells is of great importance when trying to identify novel receptors. The presence of a usable receptor is a major determinant of viral host range and cell tropism. Furthermore, identification of appropriate receptors is central for the understanding of viral pathogenesis and gives possibilities to develop antiviral drugs. Attachment is presently measured using radiolabeled and subsequently gradient purified viruses. Traditional methods are expensive and time-consuming and not all viruses are stable during a purification procedure; hence there is room for improvement. Real-time PCR (RT-PCR) has become the standard method to detect and quantify virus infections, including enteroviruses, in clinical samples. For instance, primers directed to the highly conserved 5' untranslated region (5'UTR) of the enterovirus genome enable detection of a wide spectrum of enteroviruses. Here, we evaluate the capacity of the RT-PCR technology to study enterovirus host cell interactions at the cell surface and compare this novel implementation with an established assay using radiolabeled viruses. RESULTS: Both purified and crude viral extracts of CVB5 generated comparable results in attachment studies when analyzed with RT-PCR. In addition, receptor binding studies regarding viruses with coxsackie- and adenovirus receptor (CAR) and/or decay accelerating factor (DAF) affinity, further demonstrated the possibility to use RT-PCR to measure virus attachment to host cells. Furthermore, the RT-PCR technology and crude viral extracts was used to study attachment with low multiplicity of infection (0.05 x 10(-4)TCID50/cell) and low cell numbers (250), which implies the range of potential implementations of the presented technique. CONCLUSION: We have implemented the well-established RT-PCR technique to measure viral attachment to host cells with high accuracy and reproducibility, at low cost and with less effort than traditional methods. Furthermore, replacing traditional methods with RT-PCR offers the opportunity to use crude virus containing extracts to investigate attachment, which could be considered as a step towards viral attachment studies in a more natural state.