Neutrophils and B-cells in Atlantic cod (Gadus morhua L.)

Proportions of leucocytes from head kidney, blood and spleen were identified as B-cells and neutrophils using a polyclonal antibody to cod IgM and a monoclonal antibody which previously has been shown to bind specifically to salmon and trout neutrophils. The cell specific binding of the antibodies was supported by double immunostaining. The morphology of isolated leucocytes was examined on Diff Quick stained slide preparations, and myeloperoxidase positive neutrophils were identified by diaminobenzidine staining. The antibodies clearly identified distinct cell populations. Using flow cytometry, high proportions of neutrophils were observed in peripheral blood leucocytes and high proportions of B-cells were found in head kidney leucocytes when compared to proportions of these cells in Atlantic salmon (Salmo salar L.). The spleen contained the highest proportion of B-cells. Cytoplasmic staining of immunoglobulin positive cells in slide preparations indicated that plasma cells were present, but not strikingly abundant, in head kidney, spleen and peripheral blood. Staining for myeloperoxidase identified, in accordance with the flow cytometry results, a large number of neutrophils, especially in peripheral blood leucocytes. The neutrophil nucleus was not clearly segmented, but appeared more irregular than rounded. The findings of high proportions of neutrophils in peripheral blood suggest that these cells of the innate immune system might have a central role in defence and protection against infections in cod.     

A recombinant CHSE-214 cell line expressing an Mx1 promoter-reporter system responds to both interferon type I and type II from salmonids and represents a versatile tool to study the IFN-system in teleost fish

A transgenic cell line for the detection of salmon interferons (IFNs) has been established. It is based on a CHSE-214 cell line containing a reporter construct expressing firefly luciferase under the control of the rainbow trout promoter for the IFN-induced Mx1 gene. This cell line, named CHSE-Mx10, showed IFN-induced luciferase expression after more than 80 passages, confirming the stability of this cell line. Interestingly, the Mx promoter was shown to respond to both salmon IFN-alpha/beta and trout IFN-gamma in a dose-dependent manner, while there was no response to TNF-alpha and IL-1beta. IFN-alpha/beta activity could be measured at a range of 9-150 U/ml, and IFN-gamma showed activity between 10 and 100 ng/ml. The reproducibility of both responses was good. The CHSE-Mx10 reporter system constitutes a versatile tool to study the induction and regulation of IFN signaling in teleost fish. A preliminary study presented herein suggests that both infectious pancreas necrosis virus (IPNV) and salmon pancreas disease virus (SPDV) may block activation of the Mx promoter in CHSE-Mx10 stimulated with IFN-alpha/beta.     

Biogeographical and phylogenetic origins of African fig species (Ficus section Galoglychia)

Ficus section Galoglychia (subgenus Urostigma; Moraceae) includes 72 species restricted to the African floristic region (a few extending to the Arabian Peninsula and Socotra). We present the first molecular phylogenetic analysis of the section including 56 ingroup (representing 44 species) and three outgroup taxa, to investigate its monophyly, classification and evolution. We used sequence data from the nuclear ribosomal internal and external transcribed spacers (ITS and ETS). Our results suggest that section Galoglychia is paraphyletic to the neotropical section Americana, although this is not supported by bootstrap analysis and only weakly supported by Bayesian posterior probabilities. Maximum parsimony analysis conflict with maximum likelihood and Bayesian analyses with respect to the closest relatives of section Americana in Africa. The subsections of section Galoglychia proposed by Berg [Berg, C.C., 1986. Subdivision of Ficus subg. Urostigma sect. Galoglychia (Moraceae). Proc. Kon. Ned. Akad. Wetensch., Ser. C, 89, 121-127] are generally supported. We find two major clades of section Galoglychia within Africa possibly corresponding to two main centres of diversity. One clade comprises members of subsections Platyphyllae and Chlamydodorae, which are more concentrated in Eastern Africa, and extend to Madagascar and neighbouring archipelagos (Comores, Mascarenes, Aldabra Islands and Seychelles). The other main clade includes members of subsections Caulocarpae, Cyathistipulae, Crassicostae and Galoglychia, which are concentrated in West and Central Africa.     

mRNA expression patterns of the BPI/LBP molecule in the Atlantic cod (Gadus morhua L.)

Bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) are important components of the mammalian innate defence system against Gram-negative infections. cDNA encoding a protein related to mammalian BPI and LBP have been cloned from several teleosts including the Atlantic cod (Gadus morhua L.). Using real-time PCR an increase in cod BPI/LBP expression in whole blood and peritoneal cells was demonstrated one, two and four days after intraperitoneal injection of inactivated Vibrio anguillarum. Although constitutively produced in the head kidney, a moderate rise of BPI/LBP mRNA production was seen on day two in this organ. After seven days the BPI/LBP mRNA levels at the three locations examined were almost back to normal. In situ hybridisation demonstrated a leucocytic localisation and morphology of the BPI/LBP expressing cells in various tissues. A combination of in situ hybridisation and peroxidase staining of head kidney leucocytes showed that the BPI/LBP producing cells are peroxidase positive and possible neutrophil like cells. The results suggest that the cod BPI/LBP is important in the first-line defence against bacterial infections and has a function more related to the mammalian BPI molecule than the LBP counterpart.     

Repeated inhalations of diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase (OGG1) deficient mice

DNA repair may prevent increased levels of oxidatively damaged DNA from prolonged oxidative stress induced by, e.g. exposure to diesel exhaust particles (DEP). We studied oxidative damage to DNA in broncho-alveolar lavage cells, lungs, and liver after 4 × 1.5 h inhalations of DEP (20 mg/m³) in Ogg1^- / -  and wild type (WT) mice with similar extent of inflammation. DEP exposure increased lung levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in Ogg1^- / -  mice, whereas no effect on 8-oxodG or oxidized purines in terms of formamidopyrimidine DNA glycosylase (FPG) sites was observed in WT mice. In both unexposed and exposed Ogg1^- / -  mice the level of FPG sites in the lungs was 3-fold higher than in WT mice. The high basal level of FPG sites in Ogg1^- / -  mice probably saturated the assay and prevented detection of DEP-generated damage. In conclusion, Ogg1^- / -  mice have elevated pulmonary levels of FPG sites and accumulate genomic 8-oxodG after repeated inhalations of DEP.     

V beta profiles in African children with acute cerebral or uncomplicated malaria: very focused changes among a remarkable global stability

T cells are thought to play a critical role in cerebral malaria pathogenesis. However, available evidences are restricted to rodent models in which V beta specific T cell expansion has been associated with neurological syndrome suggesting involvement of superantigens or dominant antigens. Using flow cytometry, we studied the peripheral V beta T cell repertoire of Ghanaian children with cerebral malaria, uncomplicated malaria and asymptomatic control children, to look for either expansion or deletion of specific V beta associated with cerebral malaria. At admission, the general pattern of the repertoire of the patients was very similar, with no major distortion compared to the control group a part a significant increase of the frequency of the V beta 21.3 subset correlating with disease severity and attributed to the CD4 subset. During convalescence very limited fluctuations were observed including a significant decrease of the V beta 21.3 subset and increase of the V beta 20 subset, a subset not detected at admission. The remarkable stability of the V beta repertoire observed in acute malaria either cerebral or uncomplicated argues against the idea that cerebral malaria would result from a T cell-mediated inflammatory shock syndrome driven by some dominant super-antigenic activity(ies). The significance of the reproducible increase of the CD4+V beta 21.3T cell subset deserves further investigations.     

Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium

Eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) either by unfolded protein response that leads to an increase in the capacity of the ER to fold its client proteins or by apoptosis when the function of ER cannot be restored. Emerging data now indicate that ER stress is also a potent inducer of macroautophagy, a process whereby eukaryotic cells recycle their macromolecules and organelles. Depending on the context, autophagy counterbalances ER stress-induced ER expansion, enhances cell survival or commits the cell to non-apoptotic death. Here, we discuss the signaling pathways linking ER stress to autophagy and possibilities for their clinical exploitation.     

Emodin negatively affects the phosphoinositide 3-kinase/AKT signalling pathway: a study on its mechanism of action

The development of selective cell-permeable inhibitors of protein kinases whose aberrant activation contributes to cell transformation is a promising approach in cancer treatment. Emodin is a natural anthraquinone derivative that exhibits anti-proliferative effects in various cancer cell lines by efficient induction of apoptosis. The phosphoinositide 3-kinase (PI3K)/AKT pathway has been shown to be central in the promotion of cell survival since the alteration of this signalling cascade is a frequent event in human malignancies. Previous published results indicated that treatment of cells with inhibitors of protein kinase CK2, such as emodin, induces apoptosis and that the anti-apoptotic effect of CK2 is partially mediated by target phosphorylation and up-regulation of AKT by CK2. In the present study, a screening with selected CK2 inhibitors induced a variable response with respect to AKT down-regulation, emodin being the most effective, suggesting that other mechanisms other than the inhibition of CK2 were responsible for the emodin-mediated modulation of AKT. We found that emodin does not directly affect AKT kinase. Furthermore, we show that the down-regulation of AKT is due to the emodin-mediated target inhibition of components of the PI3K pathway, which directly or indirectly affect AKT activity, i.e. the mammalian target of rapamycin and the phosphatase and tensin homolog deleted on chromosome 10, but not the phosphoinositide-dependent kinase 1. Taken together, our results highlight a new mechanism by which emodin exerts anti-cancer activity and suggest the further investigation of plant polyphenols, such as emodin, as therapeutic and preventive agents for cancer therapy.     

Growth inhibitory factors in bovine faeces impairs detection of Salmonella Dublin by conventional culture procedure

AIMS: To analyse the relative importance of different biological and technical factors on the analytical sensitivity of conventional culture methods for detection of Salmonella Dublin in cattle faeces. METHODS AND RESULTS: Faeces samples collected from six adult bovines from different salmonella-negative herds were split into subpools and spiked with three strains of S. Dublin at a concentration level of c. 10 CFU g(-1) faeces. Each of the 18 strain-pools was divided into two sets of triplicates of four volumes of faecal matter (1, 5, 10 and 25 g). The two sets were pre-enriched with and without novobiocin, followed by combinations of culture media (three types) and selective media (two types). The sensitivity of each combination and sources of variation in detection were determined by a generalized linear mixed model using a split-plot design. CONCLUSIONS: Biological factors, such as faecal origin and S. Dublin strain influenced the sensitivity more than technical factors. Overall, the modified semi-solid Rappaport Vassiliadis (MSRV)-culture medium had the most reliable detection capability, whereas detection with selenite cystine broth and Mueller Kauffman tetrathionate broth combinations varied more in sensitivity and rarely reached the same level of detection as MSRV in this experiment. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that for MSRV-culture medium and xylose lysine decarboxylase agar as the indicative medium, the sensitivity of the faecal culture method may be improved by focusing on the strain variations and the ecology of the faecal sample. Detailed investigation of the faecal flora (pathogens and normal flora) and the interaction with chemical factors may result in developing an improved method for detection of S. Dublin.     

Predicting survival from microarray data--a comparative study

MOTIVATION: Survival prediction from gene expression data and other high-dimensional genomic data has been subject to much research during the last years. These kinds of data are associated with the methodological problem of having many more gene expression values than individuals. In addition, the responses are censored survival times. Most of the proposed methods handle this by using Cox's proportional hazards model and obtain parameter estimates by some dimension reduction or parameter shrinkage estimation technique. Using three well-known microarray gene expression data sets, we compare the prediction performance of seven such methods: univariate selection, forward stepwise selection, principal components regression (PCR), supervised principal components regression, partial least squares regression (PLS), ridge regression and the lasso. RESULTS: Statistical learning from subsets should be repeated several times in order to get a fair comparison between methods. Methods using coefficient shrinkage or linear combinations of the gene expression values have much better performance than the simple variable selection methods. For our data sets, ridge regression has the overall best performance. AVAILABILITY: Matlab and R code for the prediction methods are available at http://www.med.uio.no/imb/stat/bmms/software/microsurv/.