Tamoxifen induces apoptotic neutrophil efferocytosis in horses.

Macrophages and neutrophils are important cellular components in the process of acute inflammation and its subsequent resolution, and evidence increasingly suggests that they play important functions during the resolution of chronic, adaptive inflammatory processes. Exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including allergic asthma and chronic obstructive pulmonary disease in humans, and equine asthma (also known as recurrent airway obstruction (RAO). Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with effects on cell growth and survival. Previous studies showed that TX treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction of BALF neutrophils, and improvement in animals' clinical status. The aim of this study was to describe if TX induces in vitro efferocytosis of neutrophils by alveolar macrophages. Efferocytosis assay, myeloperoxidase (MPO) detection and translocation phosphatidylserine (PS) were performed on neutrophils isolated from peripheral blood samples from five healthy horses. In in vitro samples from heathy horses, TX treatment increases the phenomenon of efferocytosis of peripheral neutrophils by alveolar macrophages. Similar increases in supernatant MPO concentration and PS translocation were observed in TX-treated neutrophils, compared to control cells. In conclusion, these results confirm that tamoxifen has a direct effect on equine peripheral blood neutrophils, through stimulation of the engulfment of apoptotic neutrophils by alveolar macrophages.

Aspergillus fumigatus-sensitive IgE is associated with bronchial hypersensitivity in a murine model of neutrophilic airway inflammation.

Neutrophils are the predominant inflammatory cells that infiltrate airways during acute exacerbation of asthma. The importance of A. fumigatus sensitization, and IgE response in the airways in patients with acute asthma is unclear. Rockefeller (RK) mice were sensitized with A. fumigatus extract protein. The animals were subsequently challenged with different degrees of A. fumigatus contamination in the cage bedding. All groups of mice were euthanized to obtain bronchoalveolar lavage fluid (BALF) for cytological and Elisa assays, and lung tissue for histological analysis. Moreover, several bioassays were conducted to determine whether BALF IgE antibodies can activate mast cells. In this study, we demonstrated that exposure of sensitized mice to a known concentration of A. fumigatus conidia produces bronchial hyperreactivity with marked neutrophilic bronchial infiltration and increased BALF IgE, capable of triggering mast cell degranulation. This study suggests that IgE may play a role in bronchial hyperreactivity associated to A. fumigatus exposure in mice. Mice sensitized and challenged with this fungus showed characteristics of severe asthma, with an increase of BALF neutrophils, histological changes consistent with severe asthma and an increase of IgE capable of triggering type I hypersensitivity.

In Vitro effects of tamoxifen on equine neutrophils.

Neutrophils participate in innate immunity as the first line of host defense against microorganisms. However, exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including allergic asthma and chronic obstructive pulmonary disease in humans, and equine asthma (also known as recurrent airway obstruction (RAO). Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with effects on cell growth and survival. Previous preliminary studies showed that TX treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction of BALF neutrophils, and improvement in animals' clinical status. The aim of this study was to evaluate the in vitro effect of TX on functional tests in equine peripheral blood neutrophils. Chemotaxis, respiratory burst production and phagocytosis assays were performed on neutrophils isolated from peripheral blood samples from 10 healthy horses. Results showed that IL-8 stimulated cells decrease their chemotactic index when treated with TX (1 and 10µM). Respiratory burst production was also dampened after treatment with TX. In conclusion, these results confirm that tamoxifen has a direct action on equine peripheral blood neutrophils. However, more in vivo and in vitro studies are required to fully understand the mechanisms of action of TX on neutrophils, in order to elucidate if it can be used as treatment in disorders such as allergic asthma in humans and horses.

Apoptotic effects of tamoxifen on leukocytes from horse peripheral blood and bronchoalveolar lavage fluid.

A reduction in inflammatory cell apoptosis is an important concept in the maintenance of inflammation and a potential target for the resolution of inflammation in many inflammatory diseases. Dysregulation of apoptosis has been implicated in a range of diseases, including tumors, neurodegenerative disorders and autoimmunity, and may also be implicated in allergic asthma. In horses, recurrent airway obstruction (RAO) is an asthma-like condition that is characterized increased survival neutrophil bronchial. Tamoxifen is a synthetic, non-steroidal, anti-estrogen agent that is widely used for treating all stages of breast cancer and has been approved for the prevention of breast cancer in high-risk women. The observed efficacy of tamoxifen has been attributed to both growth arrest and the induction of apoptosis. Therefore, the aim of our study was to evaluate the ability of tamoxifen to induce apoptosis in vitro in granulocytic cells from peripheral blood and in mononuclear cells from bronchoalveolar lavage fluid (BALF) in horses. Flow cytometry using commercial AnnexinV-FITC and propidium iodide was used to quantify early and late apoptotic leukocytes, respectively. The results showed a significant increase in early apoptosis in peripheral blood and bronchial granulocytic cells treated with tamoxifen. The rate of early apoptosis of mononuclear cells from blood and BALF when incubated with tamoxifen was significantly lower compared with granulocytic cells. We did not observe a direct effect of tamoxifen on late apoptosis in any of the in vitro assays in the cell types used here. These results indicate that the apoptotic mechanisms under these experimental conditions would affect only blood and BALF granulocytic cells, particularly in early apoptosis. Finally, further in vitro and in vivo studies are needed to better understand apoptotic mechanisms because tamoxifen could be used to treat chronic, inflammatory pathologies associated with granulocytes and allergic diseases, such as asthma or equine RAO.

Reaginic antibodies from horses with recurrent airway obstruction produce mast cell stimulation.

Reaginic antibodies (IgE and some IgG subclasses) and mast cells play important roles in the induction of type I immediate hypersensitivity reactions. These antibodies bind through their Fc fragment to high affinity receptors (FcεRI) present in the membrane of mast cells and basophils. The cross-linking of the receptor initiates a coordinated sequence of biochemical and morphological events that results in exocytosis of secretory granules containing pre-formed inflammatory mediators, secretion of newly formed lipid mediators, and secretion of cytokines. Previously, several studies have investigated the role of reaginic antibodies in the pathogenesis of Recurrent Airway Obstruction (RAO). However, whereas the immunological aspects of RAO have been extensively studied, the precise sequence of events involved in the pathogenesis remains not completely understood, and the role of IgE in this disease remains controversial. Therefore, in this study, several bioassays were conducted to determine whether reaginic antibodies from RAO-affected horses have the ability to activate mast cells. These bioassays involved measuring degranulation of rat peritoneal mast cells, activation of NF-κB and morphological changes in basophilic leukemia cells (RBL-2H3) following incubation with horse serum from RAO-affected horses that were sensitive and insensitive to Aspergillus fumigatus (A. fumigatus) or from unaffected horses. Our results show that reaginic antibodies from horses sensitive to A. fumigatus were able to degranulate rat peritoneal mast cells. In additon, there was an increase in the activity of the transcription factor NF-κB in RBL-2H3 cells, and morphological changes were observed in these cells once cross-linking was produced. These findings were not found in horses not sensitive to A. fumigatus and healthy horses. These bioassays demonstrate the ability of reaginic antibodies to stimulate mast cells and indicate that these antibodies could be involved in the immunological mechanisms leading to RAO.

Membranoproliferative glomerulonephritis possibly associated with over-vaccination in a cocker spaniel.

Membranoproliferative glomerulonephritis was observed in a seven-month-old male cocker spaniel dog. The clinical, microbiological, biochemical, radiographic and ultrasonographic examinations ruled out neoplasia, congenital disease and infectious disease. The anamnesis revealed that the owner had vaccinated the dog seven times, one vaccination per month, without veterinarian supervision. In both kidneys, severe thickening of the glomerular capillary walls was observed. Electron microscope examination revealed a large number of electron-dense deposits that were primarily in the glomerular subendothelial spaces and the basal membrane, which is compatible with antigen-antibody complexes. The immunohistochemical examination revealed that the antigen present in the glomeruli corresponded with the antigen present in the vaccine. We report a type III hypersensitivity nephropathy in a young dog, which was possibly caused by over-vaccination.

Ultraviolet exposure of thymocytes: selective inhibition of apoptosis.

PURPOSE: To evaluate selective effects of ultraviolet (UV) irradiation on spontaneous and induced apoptosis in freshly extracted mice thymocytes. MATERIALS AND METHODS: Cells were exposed to UV radiation with emission peaks of 365 nm (UVA) exposures of 1620-10200 J m(-2), of 312 nm (UVB) exposures of 34-1620 J m(-2) or of 254 nm (UVC) exposures of 1.5-1620 J m(-2), and incubated for 5.5 h with or without hydrocortisone, phorbol-12-myristate-13-acetate or anti-Fas antibody. Additionally, cells were irradiated with gamma-rays (5 Gy) before UVB exposure (408 J m(-2)) at different times. Apoptosis was quantified by DNA fragmentation. RESULTS: Up to an irradiation of 5000 J m(-2), UVA exposure did not show any effect on thymocyte apoptosis, while at 10200 J m(-2) irradiation, considerable DNA fragmentation was observed. In contrast, UVB and UVC irradiation clearly inhibited natural and cortisone-induced apoptosis. Moreover, UVB inhibited apoptosis triggered by phorbol-12-myristate-13-acetate and gamma-irradiation, but not by anti-Fas antibody. CONCLUSIONS: The response of mouse thymocytes in culture to UV irradiation strongly depends on the wavelength used. It is suggested that either a survival or an apoptotic pathway occurs depending on the physiological state of the cell, spectral composition of the UV light and cell type. The possible involvement of extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase in the apoptotic pathway is discussed.

Frequent exposure of mice to crude Brucella abortus proteins down-regulates immune response.

Mice repeatedly immunized via the intraperitoneal route with a Brucella abortus antigen lost their ability to develop a strong in vitro lymphoproliferative response. This result correlates with a decreased tendency of the lymphoid population to produce interferon-gamma when stimulated in culture with the immunizing antigen. With respect to the humoral response, as the number of immunizations increased, the animals produced more specific immunoglobulin M and immunoglobulin G1 antibodies. It is postulated that the long-term exposure of an animal to Brucella antigen changes the nature of the immune response from a T-cell-mediated response to a humoral response favouring the establishment of the disease.

Vaccination with live Escherichia coli expressing Brucella abortus Cu/Zn superoxide dismutase protects mice against virulent B. abortus.

Vaccination of mice with Escherichia coli expressing Brucella Cu/Zn superoxide dismutase (SOD) [E. coli(pBSSOD)] induced a significant level of protection against virulent Brucella abortus challenge, although this level was not as high as the one reached with B. abortus vaccine strain RB51. In addition, vaccination with E. coli(pBSSOD) induced antibodies to Cu/Zn SOD and a strong proliferative response of splenocytes when stimulated in vitro with a thioredoxin-Cu/Zn SOD fusion protein.

Production and characterization of monoclonal antibodies to the major protein of boar outer dense fibers.

Outer dense fibers (ODF) are structural elements in the mammalian sperm tail which surround the axoneme in the midpiece and principal piece and probably may help to maintain the elastic structures and elastic recoil of the sperm tail. In the present study, we have generated and characterized and describe a series of monoclonal antibodies (mAbs) against the 30 kDa major protein from boar ODF. For antibody screening an ELISA was developed using a newly developed method to fix the ODF proteins to the solid phase. A total of seven mAbs were selected and characterized by ELISA, Western blot and immunofluorescence. The mAbs recognize the major protein component of boar ODF on preparative Western blot and mark the mid- and principal piece of demembranated flagella. These mAbs also recognize the mid- and principal piece of demembranated human spermatozoa from normozoospermic patients, but not from those with asthenozoospermia. For the first time, we succeeded in obtaining hybridoma cell lines that secrete mAbs of class IgM, which react with the 30 kDa protein of boar ODF.

Proliferation and apoptosis in infection with infectious bursal disease virus: a flow cytometric study.

Programmed cell death, or apoptosis, is involved in the normal physiology of many immunocompetent organs, including lymphocytes of the bursa of Fabricius in chickens. Involvement of apoptosis has also been described in some viral diseases such as AIDS. The purpose of this work was to study the potential role of apoptosis in the pathogenesis of Gumboro disease in the bursa of Fabricius. Our results show that 1-3 days after infection of young chickens with infectious bursal disease virus, the number of apoptotic cells increases and cellularity and proliferation decrease. Because of the dynamic nature of bursal lymphocyte populations and the involvement of apoptosis in lymphocyte cell physiology, the increased level of cells undergoing apoptosis may be due to an impairment in the withdrawal of apoptotic cells. A concomitant increase in macrophages in infected bursae and a dramatic decrease in cellularity suggest that an increase in apoptosis may be an important cause of cell depletion.

[Intervention of Campylobacter jejuni subsp. jejuni flagella in the adhesion to cellular cultures: bacteriological and immunological evidence]. / Intervención del flagelo de Campylobacter jejuni subsp. jejuni en la adherencia a cultivos celulares: evidencias bacteriológicas e inmunológicas.

The participation of the flagella of a virulent strain (O52) of Campylobacter jejuni subsp. jejuni in the adhesion to HEp-2 cells and their inhibition by means of homologous polyclonal antibodies, monoclonal anti-flagella antibodies and colostral natural antibodies (IgA) was studied. An aflagellated strain (T1) was used as negative control. Adhesion was observed in higher rates with O52 strain (72%) than with T1 strain (27.5%). Polyclonal, monoclonal and colostral antibodies inhibited O52 strain adhesion in more than 70% (p < 0.001). T1 strain adhesion was inhibited only by polyclonal and colostral natural antibodies. Our results suggest that the flagella of C. jejuni subsp. jejuni could participate effectively in the adhesion process. However, the inhibition of T1 strain by polyclonal and colostral antibodies suggests the existence of other binds of adhesins in the bacterial surface.

Radiation-induced apoptosis in thymocytes: pH sensitization.

Thymocytes were used as a model system to study the effect of microenvironmental pH changes on the radiation-induced apoptosis. We found that the sensitivity of thymocytes toward radiation induced apoptosis is increased by increasing the pH of the incubation medium. The major sensitivity change occurs between pH 7 and 8. In a given cell suspension the results obtained where similar when the apoptosis evaluation was carried out either by counting the picnotic nuclei, or monitoring the fraction of apoptotic nuclei by flow cytometry; both methods show a radiosensitization when the pH value of incubation media rises from 7 to 8. These results may be important when "in vitro" experiments are performed with lymphoid cells, since changes in pH of the media may determine important changes in the results.

[Participation of Campylobacter jejuni flagellar epitopes in cellular adhesion]. / Participación de epitopes flagelares de Campylobacter jejuni en la adhesion celular.

Using a flagellated (052) and an aflagellated (T-1) strains we studied the participation of flagellar epitopes of C. jejuni in the adhesion to HEp-2 cells in vitro. Strain 052 was significantly more adherent than strain T-1. When adhesion assays were carried out with antiflagella monoclonal antibodies, strain 052 showed inhibition of their adhesive capacity that varied between 64.3 and 92.9%. With an ELISA test it was demonstrated that those monoclonal antibodies were specific and directed exclusively against flagellar epitopes of strain 052 being unreactive with strain T-1. Our results show that flagellar epitopes could participate in the adhesion process suggesting that flagella could be involved in the installation of the infectious process.

Induction of apoptosis in thymocytes: new evidence for an interaction of PKC and PKA pathways.

The second messenger cascades connected to PKC and PKA are involved in the induction of apoptosis. Here we study the interaction of those two second messenger pathways with respect to the induction of apoptosis by stimulation or inhibition. The stimulators used were phorbol dibutyrate for PKC and one of the cAMP agonists Sp-5,6 DCl-cBIMPS or Sp-cAMP for PKA. The inhibitors used were staurosporin for PKC and the cAMP antagonist Rp-cAMPS for PKA. We found a synergism between both second messenger systems with regard to the induction of apoptosis in thymus lymphocytes.

Radiation induced membrane changes and programmed cell death: possible interrelationships.

A short review of the evidence that lymphocyte membranes are a target for the initiation of irradiation induced programmed cell death (PCD) is given. It is assumed that for lymphocytes PCD represents an essential physiological mechanism in order to prevent degeneration of the biological system involved. Initiation of PCD can be obtained by a pharmacological activation as well as with irradiation. In both cases, protein kinase-C (PKC) is involved in the signal transduction from the cellular membrane to the nucleus where, by means of a metabolically active process, DNA fragmentation is induced. It is hypothesized that processes connected to lipid peroxidation in the cell membrane constitute a primary effect of irradiation induced PCD, where membrane fluidization or a compensatory process aimed to the maintenance of membrane fluidity (membrane homeoviscosity hypothesis) are likely to be involved.

A flow-cytometric method to study DNA fragmentation in lymphocytes.

A method to measure DNA fragmentation cell by cell in a cell population was implemented based on acridine orange procedure to determine DNA content of single cells by flow cytometry. Using this method it can be observed that the fragmentation process induced by irradiation in thymic cells occurs in a fraction of the population, thus indicating that this process is not evenly distributed over the total population, and that it corresponds to a fast phenomenon in which the cells suddenly lose DNA material.

Role of protein kinase-C in thymocyte apoptosis induced by irradiation.

The role of protein kinase C in radiation-induced death of thymocytes was studied. For this purpose murine thymocytes were irradiated and incubated for 6 h at 37 degrees C and afterwards the fraction of fragmented DNA was measured. Results indicate that radiation-induced DNA fragmentation can be prevented by adding the protein kinase C inhibitor H-7 or staurosporine to the thymocytes during incubation time. Incubation of irradiated cells with HA-1004, an inhibitor of cAMP-dependent protein kinase, with a minor effect on protein kinase C did not affect the DNA fragmentation induced by irradiation. Incubation of cells with phorboldibutyrate gave a dose-dependent induction of DNA fragmentation. This effect can be inhibited by staurosporine. These results suggest that radiation-induced DNA fragmentation is an active cellular process in which protein kinase C plays an important role.

Age-related capacity of neuroendocrine factors to differentiate T cells in vitro.

The present report shows the capacity of hypothalamic extract (HE) to differentiate bone marrow cells to Thy-1+ cells in vitro. A two-step short-term culture was used. In the first step thymus and pituitary were co-cultured in the presence of HE. Supernatant was then transferred to a bone marrow cell suspension and following a period of culture, the percentage of Thy-1+ cells was determined by a microcytotoxicity assay. Results indicated that: (a) HE from young mice show a very efficient differentiating capacity; (b) HE from young mice is equally efficient when old pituitary, thymus and marrow are used; (c) HE from old donors has no capacity to differentiate T cells; (d) there is a progressive age-related decline of this capacity; and (e) there is a feed-back mechanism involved in this process. It is concluded that hypothalamic factors can regulate the differentiation of T cells and that this effect operates through a mechanism involving pituitary and thymus.