Development and Screening of a Marker to Detect Activated Rainbow Trout Leukocytes

Monoclonal antibodies (mAbs) have been essential tools in the elucidation of the immune system of mammals, and their application to identify surface molecules on leukocytes have allowed important functions of these cell to be identified (such as receptors that bind antigens, ligands involved in cell to cell signaling or in initiating immune response activity). Not only have mAbs been used to discriminate cells during different stages of cell development, but have also assisted in understanding the dynamics of molecules expressed during functional processes. Such molecules detected on human leukocytes are called human leukocyte differentiation antigens or HLDA. In order to group the antibodies that detect similar molecules and have similar patterns of reaction, immunologists have organised the mAbs that bind to these antigens into Clusters of Differentiation (CD). So far, there are about 350 leukocyte surface molecules detected by mAbs with a CD nomenclature for human leukocytes (www.hcdm.org). In fish immunology there is a great need to produce mAbs that are able to differentiate the various components of the fish immune system to assist in the elucidation of the fish immune system. The present study was an endeavour to develop and characterise mAbs that could be accredited to such scheme. A better understanding of the fish immune system is urgently required so that effective strategies of control can be developed for significant diseases during fish farming. Monoclonal antibodies were prepared by immunizing mice with thymic leukocytes from rainbow trout. The leukocytes were activated with the lectin Concanavalin A to promote the activation and proliferation of the target T-cell population. The selection of clones producing antibodies during screening was performed on the basis of the response of the supernatant from hybridomas using three consecutive assays. First, selection was determined by the positive staining of cells from the thymus in a Dot blot assay. Secondary screening was performed by means of flow cytometry (FCM) and the criterion for selection was the preferential detection of leukocytes gated in the lymphocyte region. Finally, the positive supernatants from hybridomes were evaluated to determine their effectiveness in the detection of modifications in the labelled cells during a multiple way activation by detection of foreign histocompatibility complex enhanced with mitogens. Monoclonal antibody TcOm15 was selected from 564 hybridomas produced and then used to stain cells from various Rainbow Trout tissues. It was clear from FCM, microscopy and Western blot analysis that mAb TcOm15 not only reacted with thymic cells but also with cells from other tissues. Differential staining of cells with mAb TcOm15 was observed with 27.1 ±1.4 % of leukocytes from peripheral blood leukocytes (PBL) stained in comparison to 2.0 ±0.2 % from the thymus, 13.8 ±0.4 % from the spleen, and 5.6 ±0.6 % cells stained from head kidney. The labeled cells showed characteristics of lymphocytes and monocytes, presenting a distinctive staining in immunohistochemistry and confocal microscopy. Western blot analysis, using electrophoresed proteins under denaturing conditions with leukocytes from several different tissues, showed that mAb TcOm15 did not detect a single protein. At least three proteins appeared to be identified by the mAb at 105, 160 and 200 kDa. The proteins were identified as α Actinin-4, non-erythroid Spectrin αII chain or Ig-like protein and non-muscle Myosin (MYH10) by MALDI-TOF analysis. Three of these identities are for compositional molecules for the cytoskeleton of different types of cells, and one it is associated to immunoglobulin superfamily. The identification of these proteins by mAb TcOm15 suggests an ability of this mAb to detect a specific function, possibly related with the synchronicity of expression or interaction of cytoskeleton-membrane proteins forming a multiprotein complex. Another possibility is as a carrier role for a protein during interactions. Colocalization of the mAb with F actin from the cytoskeleton was also observed suggesting the possibility that mAb TcOm15 detects a specific site in a multi-protein complex from the cytoskeleton. The molecule detected showed down-regulation in a dose dependant way with Concanavalin A and the expression was almost lost following stimulation of cells with phorbol 12-myristate 13-acetate stimulation. Leukocytes from the PBL and thymus up-regulated the expression of the TcOm15 molecule under mitogenic conditions in vitro, and results from in vivo experiments suggested the possibility of up-regulation on thymic cells. In conclusion, the results obtained in the present study provide information on a potentially useful marker (mAb TcOm15) for a cytoskeleton-membrane antigen that is modulated during stimulation of teleost lymphocytes. Additionally, this may enable insights into the relationship between cytoskeletal proteins and membrane associated immunoglobulin. Future research is necessary in order to explain this relationship and to determine the functional participation of the TcOm15 molecule during the activation of rainbow trout cells

Applicability of flow cytometry γH2AX assay in population studies: suitability of fresh and frozen whole blood samples

Phosphorylation of H2AX histone (γH2AX) represents an early event in the DNA damage response against double-strand breaks (DSB); hence, its measurement provides a surrogate biomarker of DSB. Recently, we reported initial steps in the standardization of γH2AX assay in peripheral blood leukocytes (PBL), addressing the possibility of using cryopreserved samples, and the need of phytohaemagglutinin (PHA) stimulation prior analysis (Toxicol Sci 2015, 144:406-13). Validating the use of whole blood samples as cell specimen for this assay would be particularly useful for human population studies. Hence, in the current study we determined for the first time the feasibility of whole blood samples, both fresh and frozen, to be used in the γH2AX assay, evaluated by flow cytometry, and the convenience of PHA stimulation. Freshly collected and cryopreserved whole blood samples were treated with bleomycin (BLM), actinomycin-D (Act-D) and mitomycin C (MMC); half of the samples were previously incubated with PHA. Results were compared with those from PBL. Negative responses in MMC treatments were probably due to the quiescence of unstimulated cells, or to the short treatment time in PHA stimulated cells. Fresh whole blood samples exhibited a more intense response to BLM and Act-D treatments in stimulated cells, probably due to DSB indirectly produced from other less relevant types of DNA damage. Results obtained in frozen whole blood samples indicate that PHA stimulation is not advisable. In conclusion, this study demonstrates that whole blood samples can be used to assess DSB-related genotoxicity by the flow cytometry γH2AX assay.This work was supported by Xunta de Galicia [ED431B 2019/02], Ministerio de Educación, Cultura y Deporte [BEAGAL18/00142 to V.V], and Deputación Provincial de A Coruña [to M.S.-F. and N.F.-B.]

Development and screening of a marker to detect activated rainbow trout leukocytes

Monoclonal antibodies (mAbs) have been essential tools in the elucidation of the immune system of mammals, and their application to identify surface molecules on leukocytes have allowed important functions of these cell to be identified (such as receptors that bind antigens, ligands involved in cell to cell signaling or in initiating immune response activity). Not only have mAbs been used to discriminate cells during different stages of cell development, but have also assisted in understanding the dynamics of molecules expressed during functional processes. Such molecules detected on human leukocytes are called human leukocyte differentiation antigens or HLDA. In order to group the antibodies that detect similar molecules and have similar patterns of reaction, immunologists have organised the mAbs that bind to these antigens into Clusters of Differentiation (CD). So far, there are about 350 leukocyte surface molecules detected by mAbs with a CD nomenclature for human leukocytes (www.hcdm.org). In fish immunology there is a great need to produce mAbs that are able to differentiate the various components of the fish immune system to assist in the elucidation of the fish immune system. The present study was an endeavour to develop and characterise mAbs that could be accredited to such scheme. A better understanding of the fish immune system is urgently required so that effective strategies of control can be developed for significant diseases during fish farming. Monoclonal antibodies were prepared by immunizing mice with thymic leukocytes from rainbow trout. The leukocytes were activated with the lectin Concanavalin A to promote the activation and proliferation of the target T-cell population. The selection of clones producing antibodies during screening was performed on the basis of the response of the supernatant from hybridomas using three consecutive assays. First, selection was determined by the positive staining of cells from the thymus in a Dot blot assay. Secondary screening was performed by means of flow cytometry (FCM) and the criterion for selection was the preferential detection of leukocytes gated in the lymphocyte region. Finally, the positive supernatants from hybridomes were evaluated to determine their effectiveness in the detection of modifications in the labelled cells during a multiple way activation by detection of foreign histocompatibility complex enhanced with mitogens. Monoclonal antibody TcOm15 was selected from 564 hybridomas produced and then used to stain cells from various Rainbow Trout tissues. It was clear from FCM, microscopy and Western blot analysis that mAb TcOm15 not only reacted with thymic cells but also with cells from other tissues. Differential staining of cells with mAb TcOm15 was observed with 27.1 ±1.4 % of leukocytes from peripheral blood leukocytes (PBL) stained in comparison to 2.0 ±0.2 % from the thymus, 13.8 ±0.4 % from the spleen, and 5.6 ±0.6 % cells stained from head kidney. The labeled cells showed characteristics of lymphocytes and monocytes, presenting a distinctive staining in immunohistochemistry and confocal microscopy. Western blot analysis, using electrophoresed proteins under denaturing conditions with leukocytes from several different tissues, showed that mAb TcOm15 did not detect a single protein. At least three proteins appeared to be identified by the mAb at 105, 160 and 200 kDa. The proteins were identified as α Actinin-4, non-erythroid Spectrin αII chain or Ig-like protein and non-muscle Myosin (MYH10) by MALDI-TOF analysis. Three of these identities are for compositional molecules for the cytoskeleton of different types of cells, and one it is associated to immunoglobulin superfamily. The identification of these proteins by mAb TcOm15 suggests an ability of this mAb to detect a specific function, possibly related with the synchronicity of expression or interaction of cytoskeleton-membrane proteins forming a multiprotein complex. Another possibility is as a carrier role for a protein during interactions. Colocalization of the mAb with F actin from the cytoskeleton was also observed suggesting the possibility that mAb TcOm15 detects a specific site in a multi-protein complex from the cytoskeleton. The molecule detected showed down-regulation in a dose dependant way with Concanavalin A and the expression was almost lost following stimulation of cells with phorbol 12-myristate 13-acetate stimulation. Leukocytes from the PBL and thymus up-regulated the expression of the TcOm15 molecule under mitogenic conditions in vitro, and results from in vivo experiments suggested the possibility of up-regulation on thymic cells. In conclusion, the results obtained in the present study provide information on a potentially useful marker (mAb TcOm15) for a cytoskeleton-membrane antigen that is modulated during stimulation of teleost lymphocytes. Additionally, this may enable insights into the relationship between cytoskeletal proteins and membrane associated immunoglobulin. Future research is necessary in order to explain this relationship and to determine the functional participation of the TcOm15 molecule during the activation of rainbow trout cells.EThOS - Electronic Theses Online ServiceCONACYT, MexicoGBUnited Kingdo

Exploring frailty-related biomarkers and potential influence of environmental factors

Recent evidence advocates that healthy ageing may be possible, with morbidity compressed to later years. One area of concern is the burden of environmentally induced disease in susceptible populations. Older adults are a well-recognized susceptible population due to the decline of immune defences and the burden of multiple chronic diseases. As a susceptible population, the burden of environmentally induced disease and lifestyle factors is an increasing concern. Frailty is an age-related syndrome expected to increase over the next decades given the observed demographic shift. This syndrome has been identified to be the most common condition leading to disability, institutionalization and death in the older adults and the risk factors associated with its development are yet to be clarified. The main aim of the present study is to investigate a relation between frailty status, biomarkers and environmental exposures

Effects of Zinc Oxide Nanoparticle Exposure on Human Glial Cells and Zebrafish Embryos

Zinc oxide nanoparticles (ZnO NPs) are among the most widely used nanomaterials. They have multiple applications in cosmetics, textiles, paints, electronics and, recently, also in biomedicine. This extensive use of ZnO NPs notably increases the probability that both humans and wildlife are subjected to undesirable effects. Despite being among the most studied NPs from a toxicological point of view, much remains unknown about their ecotoxicological effects or how they may affect specific cell types, such as cells of the central nervous system. The main objective of this work was to investigate the effects of ZnO NPs on human glial cells and zebrafish embryo development and to explore the role of the released Zn2+ ions in these effects. The effects on cell viability on human A172 glial cells were assessed with an MTT assay and morphological analysis. The potential acute and developmental toxicity was assessed employing zebrafish (Danio rerio) embryos. To determine the role of Zn2+ ions in the in vitro and in vivo observed effects, we measured their release from ZnO NPs with flame atomic absorption spectrometry. Then, cells and zebrafish embryos were treated with a water-soluble salt (zinc sulfate) at concentrations that equal the number of Zn2+ ions released by the tested concentrations of ZnO NPs. Exposure to ZnO NPs induced morphological alterations and a significant decrease in cell viability depending on the concentration and duration of treatment, even after removing the overestimation due to NP interference. Although there were no signs of acute toxicity in zebrafish embryos, a decrease in hatching was detected after exposure to the highest ZnO NP concentrations tested. The ability of ZnO NPs to release Zn2+ ions into the medium in a concentration-dependent manner was confirmed. Zn2+ ions did not seem entirely responsible for the effects observed in the glial cells, but they were likely responsible for the decrease in zebrafish hatching rate. The results obtained in this work contribute to the knowledge of the toxicological potential of ZnO NPs.This research was funded by the Ministry of Science and Innovation: MCIN/AEI/10.13039/501100011033 (grant PID2020-114908GA-I00), Xunta de Galicia (ED431B 2022/16 and ED481A 2019/003 to A.A-G.), CICA-Disrupting Project 2021SEM-B2, and Ministry of Education, Culture and Sport (BEAGAL18/00142 to V.V.)

Neural response to the observable self in social anxiety disorder

Background: Distorted images of the observable self are considered crucial in the development and maintenance of social anxiety. We generated an experimental situation in which participants viewed themselves from an observer's perspective when exposed to scrutiny and evaluation by others. Method: Twenty patients with social anxiety disorder (SAD) and 20 control subjects were assessed using functional magnetic resonance imaging (fMRI) during the public exposure of pre-recorded videos in which they were each shown performing a verbal task. The examiners acted as the audience in the experiment and rated performance. Whole-brain functional maps were computed using Statistical Parametric Mapping. Results: Robust activation was observed in regions related to self-face recognition, emotional response and general arousal in both study groups. Patients showed significantly greater activation only in the primary visual cortex. By contrast, they showed significant deactivation or smaller activation in dorsal frontoparietal and anterior cingulate cortices relevant to the cognitive control of negative emotion. Task-related anxiety ratings revealed a pattern of negative correlation with activation in this frontoparietal/cingulate network. Importantly, the relationship between social anxiety scores and neural response showed an inverted-U function with positive correlations in the lower score range and negative correlations in the higher range. Conclusions: Our findings suggest that exposure to scrutiny and evaluation in SAD may be associated with changes in cortical systems mediating the cognitive components of anxiety. Disorder severity seems to be relevant in shaping the neural response pattern, which is distinctively characterized by a reduced cortical response in the most severe cases

Genotoxic effects of occupational exposure to lead and influence of polymorphisms in genes involved in lead toxicokinetics and in DNA repair

This work was partly supported by the Spanish Ministry of Science and Innovation (PSI2010-15115) and Portuguese Fundação para a Ciência e a Tecnologia (grants PDCT/SAU-OBS/59821/2004, PTDC/QUI/ 67522/2006 and PTDC/SAU-OSM/105572/2008, and fellowship SFRH/ BD/22612/2005 to M. Pingarilho).publishersversionpublishe