Teleost intestinal immunology

Teleosts clearly have a more diffuse gut associated lymphoid system, which is morphological and functional clearly different from the mammalian GALT. All immune cells necessary for a local immune response are abundantly present in the gut mucosa of the species studied and local immune responses can be monitored after intestinal immunization. Fish do not produce IgA, but a special mucosal IgM isotype seems to be secreted and may (partly) be the recently described IgZ/IgT. Fish produce a pIgR in their mucosal tissues but it is smaller (2 ILD) than the 4–5 ILD pIgR of higher vertebrates. Whether teleost pIgR is transcytosed and cleaved off in the same way needs further investigation, especially because a secretory component (SC) is only reported in one species. Teleosts also have high numbers of IEL, most of them are CD3-?⁺/CD8-α⁺ and have cytotoxic and/or regulatory function. Possibly many of these cells are TCRγδ cells and they may be involved in the oral tolerance induction observed in fish. Innate immune cells can be observed in the teleost gut from first feeding onwards, but B cells appear much later in mucosal compartments compared to systemic sites. Conspicuous is the very early presence of putative T cells or their precursors in the fish gut, which together with the rag-1 expression of intestinal lymphoid cells may be an indication for an extra-thymic development of certain T cells. Teleosts can develop enteritis in their antigen transporting second gut segment and epithelial cells, IEL and eosinophils/basophils seem to play a crucial role in this intestinal inflammation model. Teleost intestine can be exploited for oral vaccination strategies and probiotic immune stimulation. A variety of encapsulation methods, to protect vaccines against degradation in the foregut, are reported with promising results but in most cases they appear not to be cost effective yet. Microbiota in fish are clearly different from terrestrial animals. In the past decade a fast increasing number of papers is dedicated to the oral administration of a variety of probiotics that can have a strong health beneficial effect, but much more attention has to be paid to the immune mechanisms behind these effects. The recent development of gnotobiotic fish models may be very helpful to study the immune effects of microbiota and probiotics in teleosts.     

Constraints in the identification and interpretation of ozone as a significant predictor of effects on the supersensitive indicator Nicotiana tabacum Bel-W3 in biomonitoring studies

Biomonitoring by means of the supersensitive cultivar Nicotiana tabacum Bel-W3 is frequently used to obtain information on ozone effects on plants and estimates of ozone exposure. However, most of biomonitoring surveys do not account for other environmental variables (predictors in a statistical model) and their inherent multicollinearity with ozone. We tested the relative role of different predictors (fixed: time and site; random: ozone, temperature and humidity) on height growth and on the development of visible foliar symptoms of N. tabacum Bel-W3 plants. To do this, we investigated a relatively small area (256 km²), used a random design at every stage of the survey, controlled watering and protected plants from direct solar radiation and wind. QA/QC procedures were adopted at every stage of the investigation. Linear correlation shows that Leaf Injury Index (LII) and height increment (H.I.) positively related to ozone concentration, elevation and temperature, and negatively to relative humidity. All the predictors correlate to each other. However, relationships between response and ozone vary with the site and the monitoring week. The effect of the random factor “ozone” in combination with fixed factors “site” and “time” on the response variables was therefore formally investigated using the ANCOVA model. Besides ozone, the interactions “ozone × site” and “ozone × time” resulted always significant (0.001 < P < 0.05). While the factor “time” emphasize the inherent development of injury and growth through time, the interaction “ozone × site” pointed out the importance of local conditions. When watering, solar radiation, wind and plant characteristics were controlled, the remaining site-specific covariates of interest were temperature (T) and humidity (RH). When T and RH were accounted for by means of partial correlation analysis, no significant relationship was found between ozone and LII. On the other side, O₃ and RH resulted significant for both absolute and relative height increment. In short, LII seemed to be not solely dependent on ozone, T and RH, but showed to integrate their combined effect. On the other side, H.I. seemed to be favoured by high RH and T, and depressed by high ozone. Based on the above results, we recommend caution when handling bioindicator data: if the purpose is to infer ozone concentrations by leaf injury data, results may be affected by a serious bias, as the frequently reported correlations may be partly an artefact due to co-variation between predictors.     

Mapping phosphoproteins in Neisseria meningitidis serogroup A

To investigate the phosphorylation capability of serogroup A Neisseria meningitidis (MenA) and to implement our knowledge in meningococcal biology and in bacterial post-translational modifications, cell extracts were separated by 2-DE and 51 novel phosphoproteins were revealed by the use of the highly specific Ser/Thr/Tyr-phosphorylated proteins staining by Pro-Q Diamond and identified by MALDI-ToF/MS. Our results indicate that phosphorylation in MenA is comparable to that of other bacterial species. A first functional characterization of the identified modified proteins was also given, in order to understand their role in meningococcal physiopathology.     

Redox proteomics of fat globules unveils broad protein lactosylation and compositional changes in milk samples subjected to various technological procedures

The Maillard reaction between lactose and proteins occurs during thermal treatment of milk and lactosylated β-lactoglobulin, α-lactalbumin and caseins have widely been used to monitor the quality of dairy products. We recently demonstrated that a number of other whey milk proteins essential for nutrient delivery, defense against bacteria/virus and cellular proliferation become lactosylated during milk processing. The extent of their modification is associated with the harshness of product manufacturing. Since fat globule proteins are also highly important for the health-beneficial properties of milk, an evaluation of their lactosylation is crucial for a complete understanding of aliment nutritional characteristics. This is more important when milk is the unique dietary source, as in the infant diet. To this purpose, a sequential proteomic procedure involving an optimized milk fat globule (MFG) preparation/electrophoretic resolution, shot-gun analysis of gel portions for protein identification, selective trapping of lactosylated peptides by phenylboronate chromatography and their analysis by nanoLC-ESI-electron transfer dissociation (ETD) tandem MS was used for systematic characterization of fat globule proteins in milk samples subjected to various manufacturing procedures. Significant MFG protein compositional changes were observed between samples, highlighting the progressive adsorption of caseins and whey proteins on the fat globule surface as result of the technological process used. A significant lactosylation of MFG proteins was observed in ultra-high temperature sterilized and powdered for infant nutrition milk preparations, which well paralleled with the harshness of thermal treatment. Globally, this study allowed the identification of novel 157 non-redundant modification sites and 35 MFG proteins never reported so far as being lactosylated, in addition to the 153 ones ascertained here as present on other 21 MFG-adsorbed proteins whose nature was already characterized. Novel MFG proteins include components involved in nutrient delivery, defense response against pathogens and cellular proliferation/differentiation. Nutritional, biological and toxicological consequences of these findings are here discussed, highlighting their possible impact on children's diet.     

Copper exposure effects on yeast mitochondrial proteome

Mitochondria play an important role on the entire cellular copper homeostatic mechanisms. Alteration of cellular copper levels may thus influence mitochondrial proteome and its investigation represents an important contribution to the general understanding of copper-related cellular effects. In these study we have performed an organelle targeted proteomic investigation focusing our attention on the effect of non-lethal 1mM copper concentration on Saccharomyces cerevisiae mitochondrial proteome. Functional copper effects on yeast mitochondrial proteome were evaluated by using both 2D electrophoresis (2-DE) and liquid chromatography coupled with tandem mass spectrometry. Proteomic data have been then analyzed by different unsupervised meta-analysis approaches that highlight the impairment of mitochondrial functions and the activation of oxidative stress response. Interestingly, our data have shown that stress response generated by 1mM copper treatment determines the activation of S. cerevisiae survival pathway. To investigate these findings we have treated yeast cells responsiveness to copper with hydrogen peroxide and observed a protective role of this metal. These results are suggestive of a copper role in the protection from oxidative stress possibly due to the activation of mechanisms involved in cellular survival and growth.