Neuroendocrine-immune interactions in carp: a role for cortisol and interleukin-1

Maintaining a dynamic internal equilibrium, homeostasis, is crucial for survival of an organism. Disturbances in the environment may threaten the homeostasis and this will subsequently evoke an adaptive response in order to restore homeostasis. In vertebrates the adaptive response is mediated via the neuroendocrine system by adrenocortical and adrenergic activation. Glucocorticoids (GC) and catecholamines are the main actors in the response and can affect a whole range of processes, including those in the immune system. In response to pathogenic challenges the immune system is triggered, resulting in activation of components of innate and acquired immunity. Bi-directional communication between the Hypothalamus-Pituitary-Adrenal (HPA)-axis, sympathetic nervous system and the immune system is crucial to ensure homeostasis in mammals. Shared use of ligands and especially receptors forms a key component of this mutual interaction. The Hypothalamus-Pituitary-Interrenal (HPI)-axis is the teleost equivalent of the HPA-axis. Stress induced immuno-suppression in fish is mostly attributed to actions of cortisol, major GC in fish and end-product of the HPI-axis. Stress in aquaculture is one of the potential factors causing increased susceptibility of fish to pathogens and subsequently considerable losses in production. As part of a programme investigating adaptive strategies of carp ( Cyprinus carpio L.) after temperature stress, this study focuses on the possible neuroendocrine modulation of immune functioning during acute stress. We studied the effects of in vitro cortisol and in vivo acute temperature stress on carp leucocytes and functioning of these leucocytes. Moreover, the cortisol influence on gene expression of the cytokine interleukin-1b(IL-1b) was studied. IL-1bin mammals is part of the reciprocal signalling between neuroendocrine and immune system, therefore it may be an important candidate for modulating hormone secretion in carp. Cortisol acts upon lymphocytes differentially; in previous research it was demonstrated that in carp, in particular the B lymphocytes are affected. In vertebrates B lymphocytes play an important role in acquired immunity as precursors of antibody producing cells. Maturation and activation state of B lymphocytes may have consequences for the influence cortisol has on these cells. Therefore, carp B lymphocytes were isolated from different tissues and compared with regard to their proliferation, apoptosis and the effects of cortisol on these processes. Head kidney and spleen B lymphocytes were characterised by high basal proliferation. Peripheral blood B lymphocytes showed a low basal proliferation which could be up-regulated by stimulation with lipopolysaccharide (LPS), a major constituent of the cell wall of gram-negative bacteria. LPS could not alter proliferation of head kidney B lymphocytes. In addition, Ig-crosslinking induced higher intracellular calcium responses in circulating B lymphocytes compared with B lymphocytes from head kidney or spleen origin. With respect to apoptosis, stimulation could enhance cell viability in all organs. However, in combination with cortisol high levels of apoptosis were induced. Especially activated peripheral blood B lymphocytes were sensitive to cortisol-induced apoptosis. Also head kidney and to a lesser extent spleen B lymphocytes, although less sensitive than their equivalent in circulation, underwent cortisol-induced apoptosis irrespective of extra stimulation. Proliferation was suppressed by cortisol in blood and spleen B lymphocytes and to a more limited extent in head kidney, regardless of LPS stimulation. It is suggested that cortisol may be important for immunoregulation in both stress and non-stress conditions, because the relatively modest concentration of cortisol used (compared to plasma values measured during stress conditions) could induce a significant increase in apoptosis in all three populations of B lymphocytes. This implies an impact of stress on B lymphocyte development and activity. Stress-induced immunological changes that may contribute to a decreased disease resistance in carp were investigated. A 3 h drop in ambient water temperature was used as model for a relative mild and acute stressor for carp. After single or multiple temperature shocks, the relative number of circulating B lymphocytes decreased significantly within 4 h after the onset of the stressor, which was even more pronounced than after challenging the immune system. After a single temperature shock the relative number of B lymphocytes returned to control levels within 24 hours. In head kidney, an increase was measured in the relative number of B lymphocytes. Migration of B lymphocytes resulting in a redistribution of these cells to other body compartments may contribute to the relative drop in B lymphocytes in the circulation. Granulocyte numbers showed opposite reactions, doubling in circulation and decreasing significantly in head kidney. This demonstrates differential modulation of immune cells in vivo by a relative mild stressor. Freshly isolated blood lymphocytes from stressed carp showed a considerable higher number of apoptotic cells than lymphocytes from unstressed animals. Besides B lymphocytes, Ig -lymphocytes contributed significantly to this stress-induced apoptosis. Glucocorticoid receptors could be detected in the vast majority of the B lymphocytes and also part of the Ig -lymphocytes. As distribution of B lymphocytes was substantially affected by temperature stress, the effects of multiple temperature shocks on humoral antibody responses were determined. The kinetics of the antibody response to both, T lymphocyte independent (TI) antigens and T lymphocyte dependent (TD) antigens consistently showed a trend to decreased antibody response in stressed carp. In carp immunised with the TI-antigen TNP-LPS the antibody response was significantly slower in the stressed carp. These observations confirm the effect of temperature stress on the B lymphocyte population. These results show that even a mild stressor can affect distribution of B lymphocyte and granulocyte cell populations reversibly with differential effects and thus can have implications for a subsequent immune response. However, during acute stress, the role of cortisol is most probably not purely immunosuppressive but more immunomodulatory. A stress-induced enhancement of an innate type of response could facilitate a fast and effective reaction of the immune system. Cytokines, like IL-1b, play a pivotal role in the regulation of the immune system. Macrophages and a whole range of other cells release IL-1bas a response to infection or tissue damage. IL-1bhas pleiotropic effects as an immune and inflammatory mediator. Furthermore, IL-1bis an important candidate able to affect the HPI-axis by altering the release of corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH). In fish, most interleukin molecules await identification but the IL-1bsequences of several teleost fishes were recently elucidated. In the tetraploid carp we describe gene organisation and expression of two IL-1bgenes: IL-1b1 and IL-1b2. The two carp mRNA sequences share about 74% amino acid identity. The existence of two IL-1bcopies in the carp genome probably originates from the tetraploid nature of the species. In contrast to carp IL-1b1, the IL-1b2 locus is represented by multiple sequences with 95-99% identity. Detection of up to 6 distinct IL-1b2 sequences within single homozygous fish suggests the presence of multiple copies of the IL-1b2 gene in the carp genome. Both IL-1b1 and IL-1b2 comprise seven exons with typical IL-1 characteristics as an IL-1 family motif and instability motifs in the 3'-untranslated region. A general discrepancy of teleost IL-1bsequences described thus far with mammalian IL-1b, is the lack of a clear caspase-1 (interleukin- 1b-converting enzyme; ICE) cleavage site. Three IL-1b1 RNA transcripts could be detected in carp: (1) a fully spliced product, (2) exon 1-7 with introns 5 and 6 and (3) exon 1-7 with intron 5 only. Intron-containing products were also detected for IL-1b2. These intron-containing products probably represent partially spliced transcripts. IL-1bmRNA expression in carp was semi-quantitatively analysed by RT-PCR in multiple organs, including brain and pituitary. In vivo , mRNA of both IL-1bsequences were constitutively expressed in healthy carp, for IL-1b1 this was predominantly in the immune organs head kidney and spleen. Furthermore, a scattered distribution of IL-1b1 producing cells was shown by in situ hybridisations of head kidney tissue. Administration of phorbol-myristate-acetate (PMA) or LPS to phagocytes isolated from the head kidney, resulted in up-regulation of IL-1b1 expression. Also IL-1b2 transcripts could be up-regulated by in vitro LPS stimulation of head kidney phagocytes. Interestingly, by determining the ratio of expression it was demonstrated that IL-1b2 is expressed at a maximum of one tenth of the amount of the IL-1b1 sequence. Together with the high number of amino acid substitutions in the IL-1b2 sequences this suggests either that IL-1b2 is approaching a pseudogene status or IL-1b2 is part of a complex receptor - ligand interaction network. The involvement of nuclear factor (NF)-kB in carp IL-1b1 expression was shown with suppression of the LPS-induced IL-1bexpression by the NF-kB inhibitor, pyrrolidine dithiocarbamate (PDTC). Data suggests also that carp IL-1b2 is regulated via NF-kB and consequently both IL-1bsequences appear to have similar promoter regions. Cortisol, as endocrine-derived factor potentially mediating carp IL-1bexpression, was able to inhibit constitutive expression of IL-1b1 as well as IL-1b2 transcripts in vitro. However, when cortisol was added in combination with LPS at a physiological dose, cortisol could not inhibit LPS-induced expression. Moreover, it appears that cortisol synergistically enhances LPS-induced IL-1bexpression in carp. Probably LPS overrules the glucocorticoid receptor mediated inhibition via the NF-kB pathway. This might imply that cortisol can not suppress IL-1bactivation during infection. At a tenfold higher cortisol dose, however, the expression is inhibited. In conclusion, data presented in this thesis show that carp leucocytes are differentially sensitive to cortisol and in vivo stress, with regard to cell type, location and maturation or activation state. This affects cell viability, replication and migration with subsequent consequences for the immune status of carp. Also interaction of the neuroendocrine system with immune regulating factors was demonstrated: cortisol affects carp IL-1bmRNA expression. IL-1bin carp consists of multiple forms and is part of an immune regulating mechanism which probably matches that of mammals in complexity

Eerste detectie van oesterherpesvirus OsHv-1 in Nederland

In het late voorjaar en zomer van 2008 en 2009 werd verhoogde sterfte geconstateerd onder Japanse oesters (Crassostrea gigas) in verscheidene kweekgebieden in Frankrijk, Ierland en de Kanaaleilanden. Het oesterherpesvirus, Ostreid herpesvirus 1 µvar (OsHV-1 µvar), lijkt een belangrijke rol te hebben gespeeld bij de sterfte. Gezien de mogelijkheid op herhaling van de problemen in het voorjaar en zomer van 2010 en de mogelijke verspreiding van OsHV-1 µvar zijn EU maatregelen opgesteld ter bestrijding van de verhoogde mortaliteit bij Japanse oesters in samenhang met de aanwezigheid van het OsHB-1 µvar. In Nederland is in 2010 een programma gestart voor de vroegtijdige detectie van OsHV-1 µvar. In dit artikel zijn de resultaten van de monitoring naar OsHV-1 in de zomer van 2010 in Nederland en de verspreiding van OsHV-1 in 2010 in Europa beschreve

Progressive slip after removal of screw fixation in slipped capital femoral epiphysis: two case reports

<p>Abstract</p> <p>Introduction</p> <p>In slipped capital femoral epiphysis the femoral neck displaces relative to the head due to weakening of the epiphysis. Early recognition and adequate surgical fixation is essential for a good functional outcome. The fixation should be secured until the closure of the epiphysis to prevent further slippage. A slipped capital femoral epiphysis should not be confused with a femoral neck fracture.</p> <p>Case presentation</p> <p>Case 1 concerns a 15-year-old boy with an adequate initial screw fixation of his slipped capital femoral epiphysis. Unfortunately, it was thought that the epiphysis had healed and the screw was removed after 11 weeks. This caused new instability with a progressive slip of the femoral epiphysis and subsequently re-fixation and a subtrochanteric correction osteotomy was obligatory. Case 2 concerns a 13-year-old girl with persistent hip pain after screw fixation for slipped capital femoral epiphysis. The screw was removed as lysis was seen around the screw on the hip X-ray. This operation created a new unstable situation and the slip progressed resulting in poor hip function. A correction osteotomy with re-screw fixation was performed with a good functional result.</p> <p>Conclusion</p> <p>A slipped epiphysis of the hip is not considered ‘healed’ after a few months. Given the risk of progression of the slip the fixation material cannot be removed before closure of the growth plate.</p

Phylogeny of the Viral Hemorrhagic Septicemia Virus in European Aquaculture

<p>One of the most valuable aquaculture fish in Europe is the rainbow trout, Oncorhynchus mykiss, but the profitability of trout production is threatened by a highly lethal infectious disease, viral hemorrhagic septicemia (VHS), caused by the VHS virus (VHSV). For the past few decades, the subgenogroup Ia of VHSV has been the main cause of VHS outbreaks in European freshwater-farmed rainbow trout. Little is currently known, however, about the phylogenetic radiation of this Ia lineage into subordinate Ia clades and their subsequent geographical spread routes. We investigated this topic using the largest Ia-isolate dataset ever compiled, comprising 651 complete G gene sequences: 209 GenBank Ia isolates and 442 Ia isolates from this study. The sequences come from 11 European countries and cover the period 1971-2015. Based on this dataset, we documented the extensive spread of the Ia population and the strong mixing of Ia isolates, assumed to be the result of the Europe-wide trout trade. For example, the Ia lineage underwent a radiation into nine Ia clades, most of which are difficult to allocate to a specific geographic distribution. Furthermore, we found indications for two rapid, large-scale population growth events, and identified three polytomies among the Ia clades, both of which possibly indicate a rapid radiation. However, only about 4% of Ia haplotypes (out of 398) occur in more than one European country. This apparently conflicting finding regarding the Europe-wide spread and mixing of Ia isolates can be explained by the high mutation rate of VHSV. Accordingly, the mean period of occurrence of a single Ia haplotype was less than a full year, and we found a substitution rate of up to 7.813 × 10^-4 nucleotides per site per year. Finally, we documented significant differences between Germany and Denmark regarding their VHS epidemiology, apparently due to those countries' individual handling of VHS.</p

Insights into the Function of the CRM1 Cofactor RanBP3 from the Structure of Its Ran-Binding Domain

Proteins bearing a leucine-rich nuclear export signal (NES) are exported from the nucleus by the transport factor CRM1, which forms a cooperative ternary complex with the NES-bearing cargo and with the small GTPase Ran. CRM1-mediated export is regulated by RanBP3, a Ran-interacting nuclear protein. Unlike the related proteins RanBP1 and RanBP2, which promote disassembly of the export complex in the cytosol, RanBP3 acts as a CRM1 cofactor, enhancing NES export by stabilizing the export complex in the nucleus. RanBP3 also alters the cargo selectivity of CRM1, promoting recognition of the NES of HIV-1 Rev and of other cargos while deterring recognition of the import adaptor protein Snurportin1. Here we report the crystal structure of the Ran-binding domain (RBD) from RanBP3 and compare it to RBD structures from RanBP1 and RanBP2 in complex with Ran and CRM1. Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex. The comparison of RBD structures thus provides an insight into the functional diversity of Ran-binding proteins

Estimating genetic diversity across the neutral genome with the use of dense marker maps

<p>Abstract</p> <p>Background</p> <p>With the advent of high throughput DNA typing, dense marker maps have become available to investigate genetic diversity on specific regions of the genome. The aim of this paper was to compare two marker based estimates of the genetic diversity in specific genomic regions lying in between markers: IBD-based genetic diversity and heterozygosity.</p> <p>Methods</p> <p>A computer simulated population was set up with individuals containing a single 1-Morgan chromosome and 1665 SNP markers and from this one, an additional population was produced with a lower marker density i.e. 166 SNP markers. For each marker interval based on adjacent markers, the genetic diversity was estimated either by IBD probabilities or heterozygosity. Estimates were compared to each other and to the true genetic diversity. The latter was calculated for a marker in the middle of each marker interval that was not used to estimate genetic diversity.</p> <p>Results</p> <p>The simulated population had an average minor allele frequency of 0.28 and an LD (r²) of 0.26, comparable to those of real livestock populations. Genetic diversities estimated by IBD probabilities and by heterozygosity were positively correlated, and correlations with the true genetic diversity were quite similar for the simulated population with a high marker density, both for specific regions (r = 0.19-0.20) and large regions (r = 0.61-0.64) over the genome. For the population with a lower marker density, the correlation with the true genetic diversity turned out to be higher for the IBD-based genetic diversity.</p> <p>Conclusions</p> <p>Genetic diversities of ungenotyped regions of the genome (i.e. between markers) estimated by IBD-based methods and heterozygosity give similar results for the simulated population with a high marker density. However, for a population with a lower marker density, the IBD-based method gives a better prediction, since variation and recombination between markers are missed with heterozygosity.</p