Kjell-Olov Grönvik

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters intrathymic T-cell development in mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was administered to 2-4-week-old mice (5, 25, and 50 micrograms/kg body wt.) and to in vitro cultures (10(-9) M) of fetal thymi. By monitoring thymocyte populations with respect to the differentiation antigens CD4 and CD8, it was found that the cell number in all thymocyte populations except for CD8+ decreased significantly compared with controls. In vivo the most marked decrease occurred among double negative (DN) and double positive (DP) cells, whereas in vitro, the DP cells were most severely affected. The cell number had already decreased to some extent by day 1 after a dose of 50 micrograms/kg body wt. of TCDD, although a severe reduction did not become apparent until day 4. There was a clear dose/response relationship between 5 and 50 micrograms/kg body wt. Autoradiography and liquid scintillation counting studies showed that incorporation of [3H]thymidine in the thymus had already decreased 24 h after TCDD treatment, with the decrease being even more pronounced at 48 h. By 96 h, the rate of cell proliferation had returned to approximately normal values. The results show that TCDD has a long-lasting effect on thymocyte abundance together with a transient effect on cell proliferation. This indicates that in addition to the initial effects of TCDD on cell proliferation, it may also more permanently disturb the normal process of elimination by means of selection.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits the activation of antigen-specific T-cells in mice

There are conflicting data in the literature regarding target cells in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity. In the present study, adult male C57BL/6 mice were exposed to TCDD (50 micrograms/kg) 4 days prior to immunization with ovalbumin (OVA). The effect of TCDD on the specific immune response in vivo was determined by T-cell proliferation and IL-2 production in response to either OVA or anti-mouse-CD3 antibodies plus PMA in vitro. The antigen-specific T-cell proliferation and IL-2 production in response to OVA were significantly suppressed by TCDD, while the polyclonal response to anti-CD3 antibodies plus PMA was not affected. This indicates that even at a high dose of TCDD the intra T-cell signalling pathways in resting cells are not disturbed, but TCDD selectively impairs the antigen-specific activation of T-cells. Since activated T-cells are required in antibody responses to T-dependent antigens, the low number of such cells observed in the present study, may well explain the suppressive effects of TCDD on humoral immunity reported previously.

Shb deficient mice display an augmented TH2 response in peripheral CD4+ T cells

BackgroundShb, a ubiquitously expressed Src homology 2 domain-containing adaptor protein has previously been implicated in the signaling of various tyrosine kinase receptors including the TCR. Shb associates with SLP76, LAT and Vav, all important components in the signaling cascade governing T cell function and development. A Shb knockout mouse was recently generated and the aim of the current study was to address the importance of Shb deficiency on T cell development and function.ResultsShb knockout mice did not display any major changes in thymocyte development despite an aberrant TCR signaling pattern, including increased basal activation and reduced stimulation-induced phosphorylation. The loss of Shb expression did however affect peripheral CD4+ TH cells resulting in an increased proliferative response to TCR stimulation and an elevated IL-4 production of naïve TH cells. This suggests a TH2 skewing of the Shb knockout immune system, seemingly caused by an altered TCR signaling pattern.ConclusionOur results indicate that Shb appears to play an important modulating role on TCR signaling, thus regulating the peripheral CD4+ TH2 cell response.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment in vivo on thymocyte functions in mice after activation in vitro

Thymocytes from 15-day old C57BL/6 mice, pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 4 days before sacrifice, showed an earlier response and a higher maximal cell proliferation than thymocytes from control mice upon stimulation by concanavalin A in vitro. This is partly in contrast to the conclusions from earlier published studies. IL-2 content--as measured by growth of CTLL cells--was equally high in TCDD and in control cultures at day 1. At day 2, TCDD cultures had decreased dramatically in IL-2 content, possibly due to a high rate of consumption. At this point in time, the controls still contained a high concentration of IL-2, although less than at day 1. In contrast to the increased sensitivity to mitogen stimulation, thymocytes from TCDD-treated mice induced B-cells less avidly with respect to antibody production, and could also inhibit the T-cell help of thymocytes from untreated animals, a phenomenon which could be reversed if TCDD-treated thymocytes were irradiated before culture.

2,3,7,8-tetrachlorodibenzo-P-dioxin (TCDD) induced suppression of the local immune response

TCDD suppressed the normal immune response in popliteal and inguinal lymph nodes, when administered i.p. (50 micrograms/kg) to C57BL/6 mice, 4 days before immunization with the T-dependent antigen ovalbumin (10 micrograms/pad) in the hind foot pads. A hampered increase in lymph node cell number and a reduced frequency of antigen-specific B-cells were observed, despite the fact that cell proliferation in vivo was normal. While the restimulation of lymph node cells in vitro with ConA or LPS was normal, suggesting that the APC function was largely unaffected, the OVA-induced proliferation was greatly reduced. The anti-OVA antibody (ab) concentration both in serum and in supernatants of cultured lymph node cells was lower than in controls. In contrast, the production of anti-BSA ab upon LPS stimulation was normal. This indicates that the ability of the B-cells to produce ab and to proliferate was not disturbed. The DTH assay clearly showed an impaired T-cell function in TCDD-treated animals. Since APC or B-cells have appeared normal in their functions tested in this study, we propose that TCDD disturbed T-cell functions, leading to an impaired activation of B-cells.

Mouse blastocyst surface expression of galactose-containing epitopes coinciding with trophoblast differentiation

A galactose-containing cell surface epitope of mouse blastocysts was identified and partially characterized by means of immuno- and lectincytochemistry, using a mouse IgM anti-blastocyst monoclonal antibody (mAb N63) and four different galactose-binding lectins (BSL-1, DBA, PNA and SBA) as molecular probes. The mAb was produced by syngeneic intrasplenic immunization with adhesive mouse blastocysts, obtained 18 h after estrogen reactivation from facultative delay of implantation. Labelling of different mouse embryonic stages collected by uterine flushings revealed that the labelling of the epitope by monoclonal antibodies was restricted to the blastocyst stage. A peak labelling intensity was observed on late blastocysts. When examining blastocyst outgrowths, both trophoblast and embryoblast were weakly stained by mAb N63. Direct antigen characterization performed on blastocysts indicated that the mAb N63 recognized a galactose-containing glycolipid antigen. Immunochemistry of cryosectioned, unfixed mouse tissues including ovary, testis, uterus in delay and at implantation, Day 12 and term placenta, liver, kidney, brain, intestine, heart, striated muscle, and skin was negative. In addition, labelling of rat and hamster blastocysts was negative. In vitro experiments demonstrated that the galactose-containing blastocyst surface epitope was not involved in blastocyst attachment to plastic culture dishes. The appearance of the epitope at the embryonic surface in vivo coincides with the time of trophoblast differentiation and implantation in the mouse.

Experiments with Immunization of Mice with Blastocysts by an Intrasplenic Route

A few attaching blastocysts from CBA/H mice were irradiated from a Cesium source and transferred into the spleen of male DBA2 mice. A booster immunization was performed after four weeks. Blood samples for preparation of antiserum to test for the presence of immunoglobulins directed against blastocyst surface determinants were obtained by a retro-orbital puncture. Specific antibodies were detected with a protein A-gold method, modified for transmission electron microscopy of air-dried blastocysts. The results showed that CBA/H blastocyst incubated in DBA2 immune serum were positive for protein A-gold labelling, while control blastocysts only possessed a few irregularly scattered gold particles. Thus, it seems as a deposition of antigens in the spleen tissue with persistence of the antigens at this site will result in detectable antibodies in the peripheral blood.

Identification of in vivo released products of Onchocerca with diagnostic potential, and characterization of a dominant member, the OV1CF intermediate filament

A sensitive and specific test for the routine diagnosis of active Onchocerca infection is currently lacking. A major drawback in the development of such a test has been the paucity of knowledge of suitable parasite antigens that can serve as targets in antigen-detection assays. In the present investigation, we employed mass spectrometry, bioinformatics and molecular techniques to identify and characterize several potentially diagnostic Onchocerca antigens in the in vivo nodular fluid, which is being investigated for the first time. The majority of the 27 identified antigens lacked a secretory signal. One of them, also identified and characterized in greater detail with the aid of previously developed monoclonal antibodies (Mabs), was a dominant circulating cytoplasmic intermediate filament protein, previously identified and named, OV1CF. Although OV1CF lacks a secretory signal in its amino acid sequence and is not detected in the pure 42 h in vitro released products, it is easily detected in the in vivo nodular fluid. We conclude that these in vivo released products offer promise as diagnostics markers in onchocerciasis.

PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST BOVINE MYELOPEROXIDASE

We established eight cloned B-cell hybridomas producing monoclonal antibodies (Mo abs) against bovine myeloperoxidase (MPO). These anti-MPO (AM) Mo abs, designated AM1-AM8, all reacted similarly to three chromatographic forms of MPO, isolated from a single donor, in an enzyme linked immunosorbent assay. According to immunoblot analysis and ELISA the AM Mo abs are specific to bovine MPO and show no cross reactivity with other neutrophil granule proteins such as lactoferrin, lactoperoxidase and serum albumin. In immunoblot analyses IgG1 class AM1, AM2, AM3 and AM4 Mo abs immunostained the heavy subunit of the MPO (57 kDa). Additionally, the AM Mo abs seem to bind either the reactive site or epitopes on bovine MPO that affect the peroxidase activity of this enzyme. AM Mo abs reacted specifically with neutrophils but did not react with lymphocytes or epithelial cells. The present study shows that these AM Mo abs could be used for developing immunoassays to measure bovine MPO from biological fluids and for localizing neutrophils at sites of infections. They could also be useful in studies assessing the involvement of MPO in inflammatory processes in bovine species.

Methods for Production and Detection of Monoclonal Antibodies against Surface Components of Adhesive Implanting Mouse Blastocysts

Monoclonal antibodies against mouse blastocysts in the adhesion stage of implantation were obtained by intrasplenic immunization with blastocysts either injected into the spleen after irradiation or transferred after drying on a piece of nitrocellulose paper. About 10 blastocysts were deposited at each of 4 immunizations before the spleen cells were used for hybridoma production. The supernatants obtained were examined for anti-blastocyst antibodies in indirect immunofluorescence labelling of native blastocysts and with ABC-immunoperoxidase staining either of blastocysts attached to nitrocellulose paper or of methanol-fixed blastocysts. Further, sections of paraffin-embedded blastocysts were used for detection of antigens.