Paraptosis-like cell death induced by yessotoxin.

This study shows that BC3H1 myoblast cell lines exposed to 100 nM yessotoxin (YTX) undergo a form of programmed cell death distinct from apoptosis and with features resembling paraptosis. Morphologically, cells treated with YTX reveal extensive cytoplasmic vacuolation, mitochondrial and endoplasmic reticulum swelling, uncondensed chromatin and cytoskeletal alterations. DNA electrophoresis evidences lack of DNA fragmentation and Western blotting analysis demonstrates activation of the mitogen-activated protein kinase JNK/SAPK1. Further characterisation of this form of programmed cell death may have interest within medicine and cancer therapy.

Depletion of CD8 alpha cells from tissues of Atlantic salmon during the early stages of infection with high or low virulent strains of infectious salmon anaemia virus (ISAV).

The virulence of an infectious salmon anaemia virus (ISAV) isolate is influenced by the response of the host's immune system to virus infection. Here we report the fate of immune responsive cells in head kidney, spleen and gills of Atlantic salmon during infection with high and low virulent strains of ISAV. A comparison of real-time PCR detection of virus and immunohistochemical detection of immune responsive cells revealed that peak viral load was coincident with both an elevated presence of MHC class I cells and a marked depletion of CD8 alpha cells. There was a larger CD8 alpha population in tissues from salmon infected with the low virulent strain compared with tissues from salmon infected with the high virulent strain at early stages of infection. These findings suggest a protective role for the CD8 alpha cell population in immune defences against ISAV.

In situ localisation of major histocompatibility complex class I and class II and CD8 positive cells in infectious salmon anaemia virus (ISAV)-infected Atlantic salmon.

It is assumed that the mobilisation of a strong cellular immune response is important for the survival of Atlantic salmon infected with infectious salmon anaemia virus (ISAV). In this study, the characterisation of immune cell populations in tissues of non-ISAV infected Atlantic salmon and during the early viraemia of ISAV was undertaken. Immunohistochemical investigations of spleen, head kidney and gills using monoclonal antibodies against recombinant proteins from MHC I, II and CD8 were performed on tissues from Atlantic salmon collected day 17 post-challenge in a cohabitant infection model. The localisations of MHC I and II in control salmon were consistent with previous reports but this study presents novel observations on the distribution of CD8 labelled cell populations in Atlantic salmon including the description of significant mucosal populations in the gills. The distribution of MHC I, MHC II and CD8 positive cell populations differed between control salmon and cohabitant salmon in the early stages of ISAV infection. The changes in MHC I labelled cells differed between organs in ISAV cohabitants but all investigated organs showed a decreased presence of MHC II labelled cells. Together with a clustering of CD8 labelled cells in the head kidney and a reduced presence of CD8 labelled cells in the gills, these observations support the early mobilisation of cellular immunity in the response of Atlantic salmon to ISAV infection. However, differences between the present study and the findings from studies investigating immune gene mRNA expression during ISAV infection suggest that viral strategies to interfere with protein expression and circumvent the host immune response could be operative in the early response to ISAV infection.

Effect of early infectious salmon anaemia virus (ISAV) infection on expression of MHC pathway genes and type I and II interferon in Atlantic salmon (Salmo salar L.) tissues.

A number of viral diseases affecting teleost fish are characterized but few studies have addressed the effects of viral infection on gene expression in vivo. In this study, we investigated the effect of the early stages of infectious salmon anaemia virus (ISAV) infection on important components of the innate and adaptive immune response by monitoring expression of five genes in the MHC class I pathway, MHC class IIbeta, type I IFN-alpha, Mx, and type II IFN-gamma from cohabitant-infected Atlantic salmon tissues using quantitative real-time PCR. There was an increased expression of type I IFN-alpha in all tissues analyzed in response to infection that was proportional to viral load (relative to virus RNA levels) in gills and head kidney. Basal expression of IFN-gamma was modest or absent in all tissues, but expression was strongly induced and proportional to ISAV RNA levels in heart, spleen and head kidney. A 10-fold or higher level of virally induced IFN-alpha, in addition to significantly elevated levels of IFN-gamma, enhanced transcription of MHC class I pathway genes in heart, spleen and head kidney. In gills, the main entry site for ISAV, there was no induction of MHC class I pathway genes. MHC IIbeta and PSMB9 were not significantly induced in any tissue. Thus, by analysing various immune genes in a range of tissues from early cohabitant ISAV-infected salmon, we demonstrate that ISAV infection induced a rapid type I and II IFN response in the major infected lymphoid tissues, which was concurrent with induced expression of MHC class I pathway genes but not MHC IIbeta. This may suggest that CD8(+) T cell responses are more important than CD4(+) T cell responses during early ISAV viraemia.

Cleavage of tensin during cytoskeleton disruption in YTX-induced apoptosis.

Yessotoxin (YTX) is a marine algal toxin previously shown to induce apoptosis in L6 and BC3H1 myoblast cell lines. Disassembly of the F-actin cytoskeleton and cleavage of tensin, a cytoskeletal protein localised at the focal adhesion contacts, appear during this apoptotic process. Tensin binds to actin filaments at the focal adhesion contacts and it links the actin cytoskeleton to the extracellular matrix (ECM). This binding occurs via integrin receptors and it makes tensin a potential link between the actin cytoskeleton and signal transduction. This study evaluates disruption in the F-actin cytoskeleton and change of tensin in myoblast cell lines exposed to 100 nM YTX up to 72 h. YTX treatment cleaves tensin and makes it translocate to the cell centre. Tensin has normally a role in the maintenance of cell shape and YTX-treatment may therefore alter the shape of the cells. YTX exposure also induces formation of lamellas associated with pseudopodia. Alternative linkages and cytoskeletal proteins anchoring the actin filaments to focal contacts remain to be identified.

Induction of apoptosis by YTX in myoblast cell lines via mitochondrial signalling transduction pathway.

Yessotoxin (YTX) can induce apoptotic events in myoblast L6 and BC3H1 cell lines from rat and mouse, respectively. The present study indicates that apoptosis induced by YTX in these cell lines can occur through activation of the mitochondrial pathway indicating an intracellular response. Terminal events during mitochondrial-mediated apoptosis involve perturbations to mitochondria resulting in loss of mitochondrial membrane potential (DeltaPsi(m)), permeability transition pore (PTP) opening and the release of proapoptotic factors cytochrome c, smac/DIABLO into the cytosol. Results from western blotting, electron and fluorescent microscopy of YTX-treated myoblast cells provided experimental data for evaluation of cytochrome c, smac/DIABLO release and caspase-9 activation. Loss of mitochondrial membrane potential and swelling of mitochondria indicated an active role of mitochondria during the early phase of apoptosis in L6 and BC3H1 cells after YTX exposure. These observations show that YTX targets mitochondria and involve activation of a cascade of events through mitochondrial regulation.

Apoptotic events induced by yessotoxin in myoblast cell lines from rat and mouse.

This study reports apoptotic events after yessotoxin (YTX) exposure in L6 (rat) and BC3H1 (mouse) skeletal muscle myoblast cell lines. These cell lines are relevant targets to study the cytotoxic effect since this toxin has been reported as cardiotoxic. Mechanisms of action of YTX in multicellular organisms are not fully elucidated. Cell culture studies can contribute to find some of these mechanisms and trace the molecular pathways involved. The present work shows results from exposing cells to 100 nM purified YTX for 72 h. Morphological and biochemical changes characteristic of apoptotic cell death were evaluated in the two cell lines. Immunofluorescence and western blot techniques showed caspase-3 and caspase-9 activation. Western blot analysis of poly(ADP-ribose)-polymerase (PARP) confirmed caspase-3 activation in both cell lines. DNA fragmentation was not detected in these cell lines. This evidence reflect that oligonucleosomal DNA fragmentation is not a biochemical event that can be used as a definitive apoptotic marker in L6 and BC3H1 myoblast cell lines. The results indicate that the time-course and degree of apoptotic events induced by YTX depend on cell line sensitivity.