Assessment of the Live Attenuated and Wild-Type Edwardsiella ictaluri-Induced Immune Gene Expression and Langerhans-Like Cell Profiles in the Immune-Related Organs of Catfish

Edwardsiella ictaluri is a Gram-negative intracellular pathogen that causes enteric septicemia of catfish (ESC). Successful vaccination against intracellular pathogens requires T cell priming by antigen presenting cells (APCs) that bridge innate and adaptive immunity. However, the evidence on immunological mechanisms that underscore E. ictaluri pathogenesis and the protective role of live attenuated vaccines (LAVs) is scarce. We assessed the expression of immune genes related to antigen presentation by real-time PCR and the distribution patterns of Langerhans-like (L/CD207+) cells by immunohistochemistry in the immune-related tissues of channel catfish challenged with two novel E. ictaluri LAVs, EiΔevpB, and ESC-NDKL1 and wild type (WT) strain. Our results indicated significantly elevated expression of IFN-γ gene in the anterior kidney (AK) and spleen of vaccinated catfish at the early stages of exposure, which correlated with increased numbers of L/CD207+ cells. In general, the ESC-NDKL1-induced IFN-γ gene expression patterns in the AK resembled that of the patterns induced by EiΔevpB. However the MHCII gene expression patterns differed between the strains with significant increases at 6 h post-challenge (pc) with the EiΔevpB and at 7 d pc with the ESC-NDKL1 strains, respectively. Significant increases in activity of T helper type polarization genes such as IFN-γ and T cell co-receptors after exposure to ESC-NDKL1, in combination with elevated numbers of L/CD207+ cells at 7 d pc with both LAVs compared to uninfected and the WT-exposed counterparts, were documented in the spleen. The dominant pro-inflammatory environment with dramatically overexpressed inflammatory genes in the AK and 7 d pc in the spleen in response to E. ictaluri was found in exposed catfish. In general, the pro-inflammatory gene expression profiles in the ESC-NDKL1 pc showed more similarities to the WT strain-induced gene profiles compared to the EiΔevpB counterpart. In addition, E. ictaluri WT significantly decreased the numbers of Langerhans-like L/CD207+ cells in the AK and spleen at 3 and 7 days pc. In conclusion, we report the differential framework of initiation of innate and adaptive immune responses between E. ictaluri strains with both LAVs having a potential of satisfying the stringent requirements for successful vaccines

Analysis of Bovine Viral Diarrhea Viruses-infected monocytes: identification of cytopathic and non-cytopathic biotype differences

<p>Abstract</p> <p>Background</p> <p>Bovine Viral Diarrhea Virus (BVDV) infection is widespread in cattle worldwide, causing important economic losses. Pathogenesis of the disease caused by BVDV is complex, as each BVDV strain has two biotypes: non-cytopathic (ncp) and cytopathic (cp). BVDV can cause a persistent latent infection and immune suppression if animals are infected with an ncp biotype during early gestation, followed by a subsequent infection of the cp biotype. The molecular mechanisms that underscore the complex disease etiology leading to immune suppression in cattle caused by BVDV are not well understood.</p> <p>Results</p> <p>Using proteomics, we evaluated the effect of cp and ncp BVDV infection of bovine monocytes to determine their role in viral immune suppression and uncontrolled inflammation. Proteins were isolated by differential detergent fractionation and identified by 2D-LC ESI MS/MS. We identified 137 and 228 significantly altered bovine proteins due to ncp and cp BVDV infection, respectively. Functional analysis of these proteins using the Gene Ontology (GO) showed multiple under- and over- represented GO functions in molecular function, biological process and cellular component between the two BVDV biotypes. Analysis of the top immunological pathways affected by BVDV infection revealed that pathways representing macropinocytosis signalling, virus entry via endocytic pathway, integrin signalling and primary immunodeficiency signalling were identified only in ncp BVDV-infected monocytes. In contrast, pathways like actin cytoskeleton signalling, RhoA signalling, clathrin-mediated endocytosis signalling and interferon signalling were identified only in cp BDVD-infected cells. Of the six common pathways involved in cp and ncp BVDV infection, acute phase response signalling was the most significant for both BVDV biotypes. Although, most shared altered host proteins between both BVDV biotypes showed the same type of change, integrin alpha 2b (ITGA2B) and integrin beta 3 (ITGB3) were down- regulated by ncp BVDV and up- regulated by cp BVDV infection.</p> <p>Conclusions</p> <p>This study shows that, as we expected, there are significant functional differences in the host proteins that respond to cp or ncp BVDV infection. The combined use of GO and systems biology network modelling facilitated a better understanding of host-pathogen interactions.</p

Zebrafish Kidney Phagocytes Utilize Macropinocytosis and Ca2+-Dependent Endocytic Mechanisms

Background: The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including active endocytosis by macrophages and granulocytes. Endocytosis can be used as a reliable measure of selective and non-selective mechanisms of antigen uptake in the early phase of an immune response. Numerous assays have been developed to measure this response in a variety of mammalian and fish species. The small size of the zebrafish has prevented the large-scale collection of monocytes/macrophages and granulocytes for these endocytic assays. Methodology/Principal Findings: Pooled zebrafish kidney hematopoietic tissues were used as a source of phagocytic cells for flow-cytometry based endocytic assays. FITC-Dextran, Lucifer Yellow and FITC-Edwardsiella ictaluri were used to evaluate selective and non-selective mechanisms of uptake in zebrafish phagocytes. Conclusions/Significance: Zebrafish kidney phagocytes characterized as monocytes/macrophages, neutrophils and lymphocytes utilize macropinocytosis and Ca 2+-dependant endocytosis mechanisms of antigen uptake. These cells do not appear to utilize a mannose receptor. Heat-killed Edwardsiella ictaluri induces cytoskeletal interactions for internalization in zebrafish kidney monocytes/macrophages and granulocytes. The proposed method is easy to implement and should prove especially useful in immunological, toxicological and epidemiological research

Національна доповідь про стан і перспективи розвитку освіти в Україні: монографія (До 30-річчя незалежності України)

The publication provides a comprehensive analysis of the state and development of national education over the 30 years of Ukraine’s independence, identifies current problems in education, ascertains the causes of their emergence, offers scientifically reasoned ways to modernise domestic education in the context of globalisation, European integration, innovative development, and national self-identification. Designed for legislators, state officials, education institutions leaders, teaching and academic staff, the general public, all those who seek to increase the competitiveness of Ukrainian education in the context of civilisation changes.У виданні здійснено всебічний аналіз стану і розвитку національної освіти за 30-річний період незалежності України, визначено актуальні проблеми освітньої сфери, виявлено причини їх виникнення, запропоновано науково обґрунтовані шляхи модернізації вітчизняної освіти в умовах глобалізації, європейської інтеграції, інноваційного розвитку та національної самоідентифікації. Розраховано на законодавців, державних управлінців, керівників закладів освіти, педагогічних і науково-педагогічних працівників, широку громадськість, усіх, хто прагне підвищення конкурентоспроможності української освіти в контексті цивілізаційних змін

Electronic separation of kidney leukocytes by flow cytometry and measurement of fluorescence in phagocytic cells.

<p>A) Forward Scatter (FSC) and Side Scatter (SSC) characteristics of kidney cell suspensions differentiate 3 distinct cell populations in zebrafish 1: macrophage/monocytes and granulocytes, 2: hematopoietic precursors, 3: lymphocytes and lymphocyte-like cells. Inset shows Wright stain of sorted cells from gate 1. B) Endocytosis was assessed by measuring green fluorescent intensity (FL1) in the gated macrophage/monocytes and granulocytes or lymphocytes. The fluorescent peaks in this example indicate active macropinocytosis of FITC-<i>E. ictaluri</i> at 30°C and 37°C compared to 4°C control in gate1. Since ingestion of bacteria could alter size and granularity of cells the analytical gating for phagocytes in gate 1 was expanded (square gate) to control a potential shift of cells. Note about inset: Sorted cells image was taken separately from endocytosis experiments. The described method does not rely on actual cell sorting but rather on electronic gating of cell populations.</p

Non-selective uptake via macropinocytosis of Lucifer Yellow (LY) in zebrafish kidney phagocytes.

<p>The <i>y</i>-axis represents fold increase in mean fluorescent intensity (MFI) compared to basal conditions (4°C treatment). The <i>x</i>-axis represents the experimental conditions during incubation. A) After an incubation time of 30 min a significant uptake of LY at 30°C and 37°C was observed. B) The inhibition affect of Cytochalasin D (CCD) (2.5 µg/ml) when added to kidney cells prior to incubation for 1 hour with LY. Addition of the inhibitor EDTA (1 mM) had no significant effect on LY uptake in granulocytes and macrophages. C) Macropinocytosis in zebrafish lymphocyte population. Non-selective uptake of LY was also observed in cells of the lymphocyte gate. This uptake was significantly inhibited by CCD (2.5 µg/ml) but not by EDTA (1 mM). Same letters indicate no significant difference in MFI. Average fold change in MFI±s.e.m. from 3 replicates is shown (<i>p</i><0.05).</p

Uptake of heat-killed FITC-<i>E. ictaluri</i> in zebrafish kidney phagocytes.

<p>The <i>y</i>-axis represents fold increase in mean fluorescent intensity (MFI) compared to basal conditions (4°C treatment). The <i>x</i>-axis represents the experimental conditions during incubation. A) Significant uptake of FITC-<i>E. ictaluri</i> at 30°C and 37°C when compared to 4°C control treatment was demonstrated. B) Significant inhibition after incubation of kidney cells with CCD is shown indicating non-selective uptake via macropinocytosis. Adding EDTA also significantly inhibited FITC-<i>E. ictaluri</i> uptake. EDTA is known to inhibit receptor-mediated endocytosis. Same letters indicate no significant difference in MFI. Average fold change in MFI±s.e.m. from 3 replicates is shown (<i>p</i><0.05).</p