Denis Flipo

Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia.

Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin‐1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/ syncytin‐2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta‐derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum‐derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead‐associated flow cytometry approach, and a syncytin‐2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome–cell interactions were compared between cells incubated in the presence of control exosomes, syncytin‐1 or syncytin‐2‐deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin‐1‐ and syncytin‐2‐dependent manner and that syncytin‐2 is reduced in serum‐derived exosomes from women with PE when compared to exosomes from normal pregnant women.—Vargas, A., Zhou, S., Éthier‐Chiasson, M., Flipo, D., Lafond, J., Gilbert, C., Barbeau, B. Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia. FASEB J. 28, 3703–3719 (2014). www.fasebj.org

Immune functions in beluga whales (Delphinapterus leucas) : Evaluation of phagocytosis and respiratory burst with peripheral blood leukocytes using flow cytometry

Flow cytometric assays using peripheral blood were developed to study phagocytosis and respiratory burst, the two major functions of neutrophils and among the most important non-specific defense mechanisms, in beluga whales. The use of flow cytometry avoids the problems associated with the isolation and purification of different cell types, and allows the measurement of a large number of cells (10,000) in a very short period of time. The methods described will be used to compare these functions in blood samples from highly contaminated beluga whales from the St. Lawrence and from relatively clean arctic beluga whales.

Combined effects of selected insecticides on humoral immune response in mice

Biological effects data with single insecticides are far more abundant than with mixtures. These data cannot be used directly to predict the effects of insecticide mixtures. Three insecticides of different chemical classes: organochlorine; dieldrin, organophosphate; malathion, and carbamate; carbofuran, previously evaluated for their immunotoxic potential, were selected for studies of combined acute exposure in C57B1/6 inbred mice. The humoral response to sheep red blood cells (SRBC) and the functional activities of peritoneal macrophages, such as phagocytosis of fluorescent beads and presentation of a single protein antigen, avidin, were examined after in vivo exposure of mice to different combinations of the selected pesticides and compared with the vehicle controls. Regarding exposure to single substances, the data confirmed the immunosuppressive potential of dieldrin and carbofuran and the immunopotentiating effect of malathion. Following the acute concomitant exposure to dieldrin/carbofuran mixture, however, values for the parameters of antigen presentation, primary IgM antibody response to SRBC antigen, and macrophage phagocytosis, returned to control or above-control values, indicating a lack of any synergistic or additive effects of the chemicals on the immune response. Thus, it was concluded the dieldrin/carbofuran mixture had an antagonistic effect on the humoral response to SRBC and the macrophage phagocytic activity, in comparison with the action of administration of each of the insecticides alone.

Increased susceptibility to mouse hepatitis virus 3 of peritoneal macrophages exposed to dieldrin

Abstract Interaction of a single dose (36 mg/kg body wt) of the organochlorine pesticide dieldrin with mouse peritoneal macrophages was examined in C57Bl 6 , ( C57Bl 6 × A J )F1 , and A J strains of different genetic resistance to mouse hepatitis virus 3 (MHV3) infection. In vivo studies showed increased susceptibility to MHV3 acute disease of C57Bl 6 and ( C57Bl 6 × A J )F1 animals challenged with the pesticide. Significant decrease of mean time of death in dieldrin-exposed, MHV3-infected susceptible C57Bl 6 mice was observed similarly upon po or ip administration of a single, sublethal dose of dieldrin. In addition, decrease of humoral response to the virus was quantified by determination of anti-MHV3 IgG antibodies in spleen cell supernatant fractions and in blood sera of dieldrin-exposed C57Bl 6 mice. A single dose of dieldrin did not alter the in vivo resistance of A J animals to acute MHV3 disease. The resistant A J mice, however, showed increased mortality upon two subsequent exposures to dieldrin followed by infection with high lethal doses of MHV3. Phagocytic activity, cell adherence capacity, and attachment and uptake of 3H-radiolabeled MHV3 by C57Bl 6 peritoneal macrophages were determined by in vitro studies. These affector activities of peritoneal macrophages were slightly decreased or unchanged in cells originating from animals exposed to the pesticide. However, the intrinsic activity of MHV3 restriction appeared to be affected in macrophages derived from dieldrin-treated animals: (i) peritoneal C57Bl 6 macrophages collected from the early phase of acute MHV3 disease contained increased MHV3 antigen and (ii) increased cytolysis was observed after in vitro MHV3 infection of macrophages originating from dieldrin-exposed C57Bl 6 mice.

Suppression of avidin processing and presentation by mouse macrophages after sublethal exposure to dieldrin

The molecular events in macrophage antigen processing and presentation were examined to determine the possible site(s) of cell-xenobiotic interaction. Antigenic processing by mouse peritoneal macrophages of a single protein antigen, avidin, was significantly suppressed following sublethal exposure of animals to an organochlorine pesticide, dieldrin. Exposure of C57B1/6 female mice to dieldrin affected the in vitro uptake of [methyl-14C]avidin by peritoneal macrophages and markedly decreased phagocytosis of fluorescein-labelled microspheres and Salmonella typhimurium. Release of the processed avidin, determined by immunochemical quantification of immunogenic avidin and by bioassay of immunogenicity of the released antigen, was also markedly affected. Dieldrin markedly affected presentation of avidin on the macrophage surface, observed by cytoimmunochemical staining of the antigen with fluorescent antibody and flow cytometry. Inhibition of the release of processed avidin was dieldrin dose- and time-dependent, following single sublethal intraperitoneal (ip) exposure to the pesticide. The antigenic properties of processed avidin, determined by biological assay using lymphocyte cultures of normal C57B1/6 mice primed with avidin, were proportional to the antigen concentration in supernatants of macrophage cultures, for both vehicle controls and dieldrin-exposed animals. This observation and analysis of the kinetics of release of processed avidin by macrophages from control and dieldrin-exposed animals suggested that the release of processed avidin, but not the immunogenicity of the antigen itself, was affected by the pesticide exposure. Generally, impairment of avidin processing and presentation appeared to be more dramatic than other pesticide-related injuries to macrophages, such as the uptake of the antigen. In conclusion, antigen processing could be a sensitive target for dieldrin-related injury of macrophage functional activities, which, in consequence, could produce suppression of the humoral immune response.

Cytometric profiles of bone marrow and spleen lymphoid cells after mercury exposure in mice

The potential immunotoxic effects of mercury chloride on murine bone marrow (bm) cell subpopulations, including analysis of maturation patterns for B-cells, were evaluated by flow cytometric analysis. CD-1 outbred mice were exposed for 28 days to relatively low doses of 25-100 ppm HgCl2 in drinking water and the mercury-related functional cellular changes were validated in a macrophage phagocytosis assay. Lymphocyte subsets from the bone marrow population were stained with PNA lectin and a panel of monoclonal antibodies against cell surface antigens. The incidence of subset-specific staining was also monitored in spleens and thymuses. A dose-effect correlation was noted for the mercury-related activation of macrophage phagocytosis. Subchronic exposure to mercuric chloride resulted in a transient (7-14 day) decrease of the lymphoid/total bm cell ratio and affected the incidence of splenic T-cell subsets, however, without a clear dose-response correlation. The B-cell population in spleen and maturation patterns of B-cells in bm appeared to be unaffected by the mercury exposure. Overall, cytometric analysis of lymphoid cell subsets in murine bone marrow revealed transient and subset-non-specific cell fluctuations after subchronic exposure to inorganic mercury.

Evaluation of pesticide effects on humoral response to sheep erythrocytes and mouse hepatitis virus 3 by immunosorbent analysis

Abstract Effect of selected organochlorine, organophosphorus, and carbamate pesticides on the humoral immune IgM response was examined upon immunization of inbred C57B1/6 mice with neutral, polyvalent, T-dependent sheep erythrocytes (SRBC) and T-independent lipopolysaccharide (LPS). In addition, a pathogenic antigen, mouse hepatitis virus 3 (MHV3) was used for determination for the interaction of selected pesticides on the primary IgG immune response in a model of viral infection, of the genetically resistant A/J mouse strain. Single, sublethal doses of dieldrin, carbofuran, and matacil induced a marked immunosuppression of the humoral responses to both neutral and pathogenic antigens. The data showed that single, sublethal doses (0.4 ≤ LD 50 ≤ 0.6) of dieldrin, carbofuran, and matacil inhibited the number of SRBC-primed cells without any direct cytotoxic effect on the activated plasmocyte, as the titer of specific antibody measured by an enzyme-linked immunosorbent assay (ELISA) per activated cell, was constant. In contrast, exposure to malathion at 10–14 days prior to the assay increased the number of plaque-forming cells (PFC) and the anti-SRBC IgM and anti-MHV3 IgG antibody titer, suggesting therefore an increase in the humoral response to neutral and pathogenic antigens in C57B1/6 and A/J mice. Immunomodulation of the humoral IgM response by selected pesticides was shown to take place at a stage prior to antibody secretion from the activated cell, as the ELISA/PFC index was similar to the control value. The data obtained for dieldrin-induced inhibition of the humoral response to SRBC and LPS antigens suggest a mechanism of immunosuppression common for both T-dependent and T-independent antigens. Good correlations were obtained for the immunomodulatory effects of selected pesticides, as measured by PFC and ELISA, which encourages support for the latter technique in the immunotoxicological screening of pesticides.

Immunotoxicity of subchronic versus chronic exposure to aldicarb in mice

In this study we compared the immunotoxicity of subchronic vs chronic exposure to the aldicarb insecticide at a relatively low, 0.1-10 ppb, level in drinking water. The immunotoxicity of aldicarb was evaluated in 28- and 90-day studies by determination of the humoral, cellular and nonspecific immunity in inbred C57BL/6 mice. Quantification of splenic plaque-forming cells (PFC) to sheep erythrocytes (SRBC), mitogen activation of spleen lymphocytes, mixed lymphocyte reaction (MLR) and the cytofluorometric assay of the phagocytic uptake of fluorescent beads were among the parameters studied. Neither the cell viability nor the splenic cell count was affected by the insecticide exposure. Immunophenotyping and cytometric determination of L3T4+, Lyt2+ and Ig+ cells revealed no effect of the insecticide exposure on the total count of cell subsets in the ungated splenocyte population. However, a marked shift in the percentages of L3T4+ and Lyt2+ cells was noted after subchronic exposure to 1 and 10 ppb aldicarb, possibly indicating activation of these splenic T-cell subsets. Subchronic aldicarb exposure significantly suppressed the splenic PFC response to SRBC at 1 ppb dose, however, no dose-effect correlation could be concluded. Similarly, no dose-effect correlation was observed for subchronic aldicarb-related changes in mitogen responses. Subchronic exposure to aldicarb had no statistically significant effect on the mixed lymphocyte reaction (MLR) or on the macrophage phagocytosis. Chronic exposure to 0.1-10 ppb aldicarb did not affect any of the parameters measured, including the cell subsets. Thus, aldicarb-related changes in immune parameters, noted after a 28-day exposure, were compensated over chronic exposure to the insecticide.

Ploidy level stability of callus tissue, axillary and adventitious shoots of Larix × eurolepis Henry regenerated in vitro

Abstract Studies on the ploidy level of conifers in tissue culture are comparatively rare; however, the confirmation of genetic stability is of particular importance when considering the relatively long generation time of most coniferous species. This study focuses on hybrid larch ( Larix × eurolepis Henry) in vitro systems established from vegetative tissue from a mature tree. These systems involve the regeneration of: (1) multiple shoots without a callus phase (both from axillary buds and from adventitious shoot development from in vitro propagated shoots) and (2) callus tissue and shoots regenerated from callus tissue. Both systems have been in culture for over a year. The objective was to test whether one or both of these systems differ in DNA ploidy level from foliar tissue of mother-plant material, using flow cytometric analysis. There was no evidence of polyploidy in any of the samples and only 2C G0/GI peaks were evident. Our results show that the hybrid larch belongs to the group of conifers that remain genetically stable with respect to ploidy levels under in vitro conditions. Thus, over an extensive culture period, both adventitious shoots and shoots regenerated from callus levels under in vitro conditions. Thus, over an extensive culture period, both adventitious shoots and shoots regenerated from callus tissue of hybrid larch, as well as callus tissue itself retain the diploid DNA level of the mother-plant material.

Immune response of earthworms (Lumbricus terrestris, Eisenia andrei and Aporrectodea tuberculata) following in situ soil exposure to atmospheric deposition from a cement factory

In order to reduce their energy costs, many cement plants use fuel product substitutes (old tyres and used oil). The combustion of these products generates a metal increase (e.g. Cu, Cd, Pb and Zn) in the atmospheric emissions. After their release, these elements are deposited into the environment and could eventually accumulate up to concentrations of concern. At the Saint-Laurent cement factory (Joliette, QC, Canada), maximum deposition of these elements occurs in the direction of prevailing winds (North-East). We evaluated the potential impact of these depositions upon the immune system of three earthworm species (Lumbricus terrestris, Eisenia andrei and Aporrectodea tuberculata) exposed in a natural environment. The exposure sites were 0.5, 1.0, and 2.0 km downwind from the cement factory, along with an upwind reference site. The immune parameters studied were the cell viability and phagocytic potential of the immune cells (coelomocytes). For both L. terrestris and E. andrei, after 7 d exposure, none of the measured parameters showed significant differences among the sites. On the other hand, for the indigenous worm A. tuberculata, in the most exposed zone (at 0.5 km), we observed an increase in cell viability and phagocytic potential. This increase could possibly be attributed to physicochemical effects such as the alkaline pH of the soil, or alternatively, it could result from beneficial effects induced by an increased calcium supply.