Neutrophils in Atlantic salmon (Salmo salar L.) are MHC class II+ and secret IL-12p40 upon bacterial exposure

Antigen-presentation via major histocompatibility complex (MHC) to T cells is the key event to initiate adaptive immune responses. In teleosts, as in mammals, the main types of professional antigen-presenting cells (APCs) are dendritic cells (DCs), monocytes/macrophages, and B cells. In the current study, flow cytometry, immunostaining and qPCR have been used to show that neutrophils in the teleost fish Atlantic salmon (Salmo salar L.) have antigen-presenting properties. The neutrophils were positive for MHC class II, CD83 and CD80/86, and upon in vitro bacterial exposure, gene expression analysis of purified neutrophils showed that IL-12p40, which is essential for proliferation of naïve T cells, was highly upregulated at both 6 and 24 h post bacterial exposure. Based on presence of MHC class II and upregulation of molecules involved in antigen presentation and T cell activation, we suggest that neutrophils in Atlantic salmon have potential to function as professional APCs. This work makes an important basis for further exploring the potential of using neutrophils to develop new, targeted immunoprophylactic measures.publishedVersio

Antibacterial treatment of lumpfish (Cyclopterus lumpus) experimentally challenged with Vibrio anguillarum, atypical Aeromonas salmonicida and Pasteurella atlantica

Lumpfish is a novel farmed species used as cleaner fish for the removal of lice from farmed salmon. As often with new, farmed species, there are challenges with bacterial infections. The frequency of prescription of antibiotic agents to lumpfish is increasing, despite the lack of knowledge about appropriate doses, duration of treatment and application protocols for the various antibacterial agents. In the current study, we have tested the effect of medicated feed with florfenicol (FFC), oxolinic acid (OA) and flumequine (FLU) on lumpfish experimentally challenged with Vibrio anguillarum, atypical Aeromonas salmonicida and Pasteurella atlantica. We found that all three antibacterial agents efficiently treated lumpfish with vibriosis using 10 and 20 mg kg−1 day−1 of FFC, 25 mg kg−1 day−1 of OA and 25 mg kg−1 day−1 FLU, whereas only FFC (20 mg kg−1 day−1) had good effect on lumpfish with pasteurellosis. None of the antibacterial agents were efficient to treat lumpfish with atypical furunculosis. FFC 20 mg kg−1 day−1 showed promising results in the beginning of the experiment, but the mortality increased rapidly 14 days post-medication. Efficient treatment is important for the welfare of lumpfish and for reducing the risk of development of antibiotic-resistant bacteria. To our knowledge, this is the first study to establish protocols for antibacterial treatment of lumpfish.publishedVersio

Exploring the Effect of Phage Therapy in Preventing <i>Vibrio anguillarum</i> Infections in Cod and Turbot Larvae

The aquaculture industry is suffering from losses associated with bacterial infections by opportunistic pathogens. Vibrio anguillarum is one of the most important pathogens, causing vibriosis in fish and shellfish cultures leading to high mortalities and economic losses. Bacterial resistance to antibiotics and inefficient vaccination at the larval stage of fish emphasizes the need for novel approaches, and phage therapy for controlling Vibrio pathogens has gained interest in the past few years. In this study, we examined the potential of the broad-host-range phage KVP40 to control four different V. anguillarum strains in Atlantic cod (Gadus morhua L.) and turbot (Scophthalmus maximus L.) larvae. We examined larval mortality and abundance of bacteria and phages. Phage KVP40 was able to reduce and/or delay the mortality of the cod and turbot larvae challenged with V. anguillarum. However, growth of other pathogenic bacteria naturally occurring on the fish eggs prior to our experiment caused mortality of the larvae in the unchallenged control groups. Interestingly, the broad-spectrum phage KVP40 was able to reduce mortality in these groups, compared to the nonchallenge control groups not treated with phage KVP40, demonstrating that the phage could also reduce mortality imposed by the background population of pathogens. Overall, phage-mediated reduction in mortality of cod and turbot larvae in experimental challenge assays with V. anguillarum pathogens suggested that application of broad-host-range phages can reduce Vibrio-induced mortality in turbot and cod larvae, emphasizing that phage therapy is a promising alternative to traditional treatment of vibriosis in marine aquaculture

Genomic analysis of pasteurella atlantica provides insight on its virulence factors and phylogeny and highlights the potential of reverse vaccinology in aquaculture

Pasteurellosis in farmed lumpsuckers, Cyclopterus lumpus, has emerged as a serious disease in Norwegian aquaculture in recent years. Genomic characterization of the causative agent is essential in understanding the biology of the bacteria involved and in devising an efficient preventive strategy. The genomes of two clinical Pasteurella atlantica isolates were sequenced (≈2.3 Mbp), and phylogenetic analysis confirmed their position as a novel species within the Pasteurellaceae. In silico analyses revealed 11 genomic islands and 5 prophages, highlighting the potential of mobile elements as driving forces in the evolution of this species. The previously documented pathogenicity of P. atlantica is strongly supported by the current study, and 17 target genes were recognized as putative primary drivers of pathogenicity. The expression level of a predicted vaccine target, an uncharacterized adhesin protein, was significantly increased in both broth culture and following the exposure of P. atlantica to lumpsucker head kidney leucocytes. Based on in silico and functional analyses, the strongest gene target candidates will be prioritized in future vaccine development efforts to prevent future pasteurellosis outbreaks.publishedVersio

Abrogation of Anthracycline and Ischemia-Reperfusion Induced Injury by Cell Signaling Modulators

Chemotherapy has a long history in cancer treatment, and the anthracycline used in Paper I and II are among the most effective anti-cancer drugs developed. Unfortunately, the use of anthracyclines is dose-restricted due to the risk of cumulative toxicity in healthy tissue, most notably in the heart. Targeted therapy using all-trans retinoic acid (ATRA) is used to differentiate, and hence, eliminate acute promyelocytic leukemia (APL) cells, and is combined with low dose anthracyclines to remove live or ATRA resistant cells. Recently, cAMP was recommended as adjuvant to standard APL therapy since it enhances ATRA induced differentiation of APL cells. In Paper I we demonstrate that cAMP in fact abrogate the anti-cancer effect of the anthracycline Daunorubicine (DNR) in blasts from APL patients and also in ATRA-sensitive and insensitive APL cell lines. The protection was dependent on the cytoplasmic PKA-type I rather than perinuclear PKA type-II, and was associated with (inactivating) phosphorylation of pro-apoptotic Bad and (activating) phosphorylation of the acute myeloid leukemia (AML) oncogene cAMPresponsive element binding protein (CREB). Mice with orthotopic NB4 cell leukemia showed a more rapid disease progression when given cAMP-increasing agents (prostaglandin E2 analog and aminophylline), both with and without DNR chemotherapy. Together this suggests that the beneficial pro-differentiating and nonbeneficial pro-survival APL cell effects of cAMP should be weighed against each other. Although the mechanism behind anthracycline mediated cardiotoxicity is highly contested, intramyocardial production of reactive oxygen species (ROS) is generally accepted as a strong candidate, and has increased the focus on antioxidants in cardioprotective strategies. In Paper II, we demonstrate that Red Palm Oil (RPO) supplemented diet during chemotherapy attenuate cardiotoxic side-effects of daunorubicin, by improving aortic output and coronary flow in the isolated working rat heart model. Improved hemodynamic was accompanied by stabilization of important antioxidant systems (SOD1 and NOS1) and reduction of stress-induced MAPK activation. While cancer is a consequence of restricted cell death, the opposite scenario, with increased cell death is an important component of ischemia-reperfusion induced injury. Since prolonged ischemia may lead to cardiac cell death, rapid and adequate reperfusion is a necessity to salvage the compromised cardiac tissue. Paradoxically, reperfusion per se also induces cell death (lethal reperfusion injury) a process involving opening of the mitochondrial permeability transition pore (mPTP). Different ways to limit or delay cardiomyocyte cell death have emerged in the laboratory setting, and are evaluated as clinical candidates to further improve the outcome of patients with acute myocardial infarction (AMI). In paper III and IV we utilize the Langendorff perfusion setup for the ex vivo rat heart, to evaluate different therapeutic strategies to reduce ischemia-reperfusion induced injury. In Paper III we show that corticotropin releasing factor (CRF) significantly reduce infarct size when applied to the heart prior to a lethal ischemic insult, and was cytoprotective in neonatal mouse cardiomyocytes when added prior to a lethal simulated ischemic event (hypoxia). CRF was however not protective when administered at the point of ischemic reperfusion or hypoxic reoxygenation. The cardioprotective effects of CRF was mediated via activation of PKA and PKC dependent signaling pathways downstream of CRF receptor type 2 (CRFR2). In Paper IV we evaluated the possible additive effects of combining known cardioprotective treatments. We found that combining insulin reperfusion therapy with direct Glycogen synthase kinase 3 β (GSKβ) inhibition at reperfusion did not confer any additive effect, but showed similar cardioprotection as seen when the treatments were administered separately. Surprisingly, we found that combining either of the two pharmacologic interventions with ischemic postconditioning (IPost) abrogated all cardioprotective effect. This loss of cardioprotection was accompanied with blunted Akt phosphorylation. To our knowledge, we are the first to demonstrate the loss of protection when combining two otherwise cardioprotective regimes

5-type HPV mRNA versus 14-type HPV DNA test: test performance, over-diagnosis and overtreatment in triage of women with minor cervical lesions

Publisher's version, source: http://doi.org/10.1186/s12907-016-0032-x

Reperfusion Therapy with Low-Dose Insulin or Insulin-Like Growth Factor 2; Myocardial Function and Infarct Size in a Porcine Model of Ischaemia and Reperfusion

In an open-chest porcine model, we examined whether myocardial pharmacological conditioning at the time of reperfusion with low-dose insulin or insulin-like growth factor 2 (IGF2), not affecting serum glucose levels, could reduce infarct size and improve functional recovery. Two groups of anaesthetized pigs with either 60 or 40 min. of left anterior descending artery occlusion (total n = 42) were randomized to receive either 0.9% saline, insulin or IGF2 infusion for 15 min., starting 5 min. before a 180-min. reperfusion period. Repeated fluorescent microsphere injections were used to confirm ischaemia and reperfusion. Area at risk and infarct size was determined with Evans blue and triphenyltetrazolium chloride staining. Local myocardial function was evaluated with multi-layer radial tissue Doppler strain and speckle-tracking strain from epicardial echocardiography. Western blotting and TUNEL staining were performed to explore apoptosis. Infarct size did not differ between treatment groups and was 56.7 ± 6.8%, 49.7 ± 9.6%, 56.2 ± 8.0% of area at risk for control, insulin and IGF2 group, respectively, in the 60-min. occlusion series. Corresponding values were 45.6 ± 6.0%, 48.4 ± 7.2% and 34.1 ± 5.8% after 40-min. occlusion. Global and local cardiac function did not differ between treatment groups. No differences related to treatment could be found in myocardial tissue cleaved caspase-3 content or the degree of TUNEL staining. Reperfusion therapy with low-dose insulin or with IGF2 neither reduced infarct size nor improved function in reperfused myocardium in this in vivo porcine model

Antibiotic uptake by cultured Atlantic cod leucocytes and effect on intracellular Francisella noatunensis subsp. noatunensis replication

The granuloma disease caused by Francisella noatunensis subsp. noatunensis in farmed Atlantic cod has not been successfully treated by use of antibacterials, even when antibacterial resistance testing indicates a sufficient effect. The reason for this treatment failure may be the intracellular existence of the bacteria within immune cells, mainly macrophages. To investigate the effect of antibacterials on intracellular Francisella replication, we established a protocol for the detection of drugs within Atlantic cod immune cells using high-performance liquid chromatography (HPLC). When the uptake and intracellular concentrations of oxolinic acid and flumequine were analysed in isolated adherent head kidney leucocytes (HKLs) by HPLC, we found that uptake was rapid and the intracellular concentrations reflected the extracellular exposure concentrations. To investigate the effect of the antibacterial compounds on intracellular bacterial replication, adherent HKLs experimentally infected with the bacteria were analysed using flow cytometry and intracellular labelling of bacteria by specific antibodies. We found that flumequine did not inhibit intracellular bacterial replication. Unexpectedly, the results indicated that the intracellularly effiacy of the drug was reduced. The HPLC method used proved to be highly applicable for accurate determination of intracellular drug concentrations. When combined with sensitive and specific flow cytometry analyses for identification and measurement of intracellular bacterial replication, we suggest that this approach can be very valuable for the design of antibacterial treatments of intracellular pathogens

Protection and antibody reactivity in lumpsucker (Cyclopterus lumpus L.) following vaccination against Pasteurella sp.

Two monovalent vaccines against pasteurellosis were developed and tested for efficacy using a previously established bath challenge model. High levels of specific antibodies were detected following vaccination. While the vaccine efficacy trial indicated that some level of protection was obtained, high mortality was still observed. qPCR analysis of head kidney tissues from surviving fish post challenge showed no difference in bacterial numbers in vaccinated and non-vaccinated fish. Clinical symptoms observed in moribund and diseased fish included white spots on the skin and around the eyes, frayed fins and redness around the mouth and fin bases. Despite production of specific antibodies, the protection against experimental challenge was relatively weak. A reason for this could potentially be that the specific antibodies produced are not alone enough to provide complete protection against pasteurellosis in lumpsuckers. Confocal and scanning electron microscopy of head kidney leucocytes exposed to Pasteurella sp. in vitro gave indications of the interactions between the pathogen and leucocytes. The results indicate that parts of the immune system other than humoral antibodies could be important for protection against pasteurellosis. Our combined results highlight the need for further work on host-pathogen interaction between Pasteurella sp. and lumpsuckers