RESUMO
Lymphocyte stimulation tests (LST) were performed in five dogs sensitised with ovalbumin (OVA) and seven healthy dogs. In addition, all five OVA-sensitised and two control dogs were tested after two in vivo provocations with OVA-containing eye drops. The isolated cells were suspended in culture media containing OVA and were cultured for up to 12 days. Proliferation was measured as reduction in 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) intensity by flow cytometry on days 0, 3, 6, 9 and 12. A cell proliferation index (CPI) for each day and the area under the curve (AUC) of the CPI was calculated for each dog. All OVA-sensitised dogs demonstrated increased erythema after conjunctival OVA application. The presence of OVA-specific lymphocytes was demonstrated in 2/5 OVA-sensitised dogs before and 4/5 after in vivo provocation. Using the AUC, the difference between OVA-sensitised and control dogs was significant in all three LST before in vivo provocation (P<0.05) and borderline significant (P=0.053) in 2/3 LST after provocation. The most significant difference in CPI was observed after 9 days of culture (P=0.001). This pilot study indicates that the LST allows detection of rare antigen specific memory T-cells in dogs previously sensitised to, but not concurrently undergoing challenge by a specific antigen.
Assuntos
Doenças do Cão/imunologia , Hipersensibilidade/veterinária , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Ovalbumina/efeitos adversos , Animais , Divisão Celular/efeitos dos fármacos , Doenças do Cão/sangue , Cães , Eritema/etiologia , Eritema/veterinária , Hipersensibilidade/sangue , Hipersensibilidade/imunologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Valores de ReferênciaRESUMO
Photodynamic therapy (PDT) is widely used to treat preneoplastic skin lesions and non-melanoma skin tumours. Studies analyzing the effects of PDT on malignant melanoma have yielded conflicting results. On the one hand, melanoma cell lines in culture as well as cell lines transplanted into experimental animals were sensitive to PDT. On the other hand, spontaneous melanomas of human patients responded poorly to most PDT regimens tested so far. Here, we analyzed effects of 5-aminolaevulinic acid (5-ALA)-based PDT on melanoma cell lines and on experimental melanomas. To mimic the clinical situation as closely as possible, metallothionein-I/ret (MT-ret) mice, a transgenic model of skin melanoma development, were used. Optimal doses of 5-ALA as well as energy doses and power densities were determined in vitro using a cell line (Mel25) established by us from a melanoma of an MT-ret transgenic mouse as well as commercially available human and mouse melanoma cell lines. Treatment with light irradiation alone had no effect. In combination with 5-ALA, however, this illumination readily induced the death of all mouse and human melanoma cell lines examined. Still, 5-ALA PDT caused only minor focal regressive changes including haemorrhages and fibrosis of MT-ret melanomas in vivo and did not significantly delay tumour growth. These results show that, even though MT-ret melanoma cells are vulnerable to 5-ALA PDT in vitro, malignant MT-ret melanomas in vivo are quite resistant to this type of therapy at doses which are highly effective in vitro.
Assuntos
Ácido Aminolevulínico/uso terapêutico , Melanoma Experimental/terapia , Melanoma/terapia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/uso terapêutico , Neoplasias Cutâneas/terapia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Modelos Animais de Doenças , Fibrose , Citometria de Fluxo , Humanos , Luz , Melanoma/genética , Melanoma Experimental/genética , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Metástase Neoplásica , Neoplasias Experimentais , Neoplasias Cutâneas/genética , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Azul Tripano/farmacologiaRESUMO
Fas (CD95/APO-1) ligand (FasL)-mediated cytotoxicity has been implicated in tissue destruction in a variety of diseases, including acute graft-vs-host disease (GVHD). In this study, we have analyzed FasL expression and regulation during the course of experimental murine acute GVHD. Although activation-induced FasL-mediated cytotoxicity in control T cells was sensitive to the immunosuppressant cyclosporin A, we observed that functional FasL expression of GVHD T cells became increasingly cyclosporin A unresponsive. This was found to be the result of a massive in vivo accumulation and intracellular storage of FasL protein and its release in a transcription- and protein synthesis-independent manner. Immunohistochemistry analysis of FasL expression in situ revealed accumulation of FasL-expressing cells in the spleen, the liver, and small intestine, with a typical cytoplasmic and granular expression pattern. Thus, we conclude that the release of preformed FasL by infiltrating donor T cells may contribute to recipient tissue damage during the pathogenesis of acute GVHD.
Assuntos
Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Linfócitos T/imunologia , Receptor fas/metabolismo , Doença Aguda , Animais , Ciclosporina/farmacologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Imunossupressores/farmacologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/imunologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Especificidade de Órgãos , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologiaRESUMO
Interleukin-6 (IL-6), a major cytokine with diverse effects on cells mainly of the immune and hematopoietic systems, has been linked to several neurological disorders such as acquired immune deficiency syndrome dementia, multiple sclerosis, and Alzheimer's disease. Central nervous system (CNS)-specific expression of IL-6 caused neurodegeneration, massive gliosis, and vascular proliferation in transgenic mice. However, the effects of systemically circulating IL-6 and its receptor IL-6Ralpha on the CNS are unknown. IL-6Ralpha is the specific component of the IL-6 receptor system and hence an important co-factor of IL-6. IL-6Ralpha is bioactive in a membrane-bound and in a soluble (s) form. We investigated the effects of systemically elevated levels of either human IL-6 or human sIL-6Ralpha or both on the CNS of transgenic mice. Although IL-6 and sIL-6Ralpha single transgenic mice were free of neurological disease, IL-6/sIL-6Ralpha double-transgenic mice showed neurological signs, such as tremor, gait abnormalities, and paresis. However, these mice also frequently showed prominent general weakness probably because of the systemic effects of IL-6/IL-6Ralpha such as liver damage and plasmacytomas. IL-6/sIL-6Ralpha transgenic mice exhibited massive reactive gliosis. Lack of signs of neuronal breakdown versus ample astrogliosis suggested that astrocytes were selectively affected in these mice. There was neither vascular proliferation nor inflammatory infiltration. Ultrastructural analysis revealed blood-brain barrier (BBB) changes manifested by hydropic astrocytic end-feet. However, albumin immunohistochemistry did not reveal major BBB leakage. Our results indicate that increased and constitutive systemic expression of IL-6 together with its soluble receptor sIL-6Ralpha is less harmful to the brain than to other organs. The BBB remains primarily intact. IL-6/IL-6Ralpha, however, might be directly responsible for the selective activation of astrocytes.
Assuntos
Astrócitos/patologia , Sistema Nervoso Central/patologia , Interleucina-6/metabolismo , Receptores de Interleucina-6/metabolismo , Animais , Encéfalo/patologia , Sistema Nervoso Central/ultraestrutura , Expressão Gênica , Humanos , Interleucina-6/genética , Camundongos , Camundongos Transgênicos/genética , Fenótipo , Receptores de Interleucina-6/genética , Solubilidade , Transgenes/genéticaRESUMO
Acute graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality following bone marrow transplantation, and early detection is important to allow effective therapy. Since the presence of apoptotic keratinocytes (dyskeratotic bodies) has been suggested as a useful diagnostic criterion for GVHD, attention has focused on the use of the TUNEL assay to detect apoptosis in clinical specimens. We reviewed clinical specimens upon which TUNEL had been performed for possible artifacts that might interfere with accurate evaluation for GVHD. Several distinct types of artifact were found and could be re-created in experimental systems. Artifacts in TUNEL staining generally resulted from the lack of specificity of this reaction for apoptotic cell death. Artifacts were found resulting from inadequate fixation, over-exposure of the TUNEL reaction, and proximity to the section edge. In addition, a novel artifact, apparently resulting from DNA shearing during the sectioning process, was noted and confirmed using confocal microscopy of experimental specimens. The TUNEL assay must therefore must be interpreted with caution in the clinical setting. In our laboratory, we consider TUNEL-positive cells as apoptotic only when accompanied by apoptotic morphology. Although these criteria clearly miss some cells in the early stages of apoptosis, they provide the highest specificity for apoptotic cell death.
Assuntos
Doença Enxerto-Hospedeiro/diagnóstico , Marcação In Situ das Extremidades Cortadas , Animais , Apoptose/fisiologia , Artefatos , Transplante de Medula Óssea/patologia , Mama/patologia , Mama/fisiologia , Feminino , Técnicas de Preparação Histocitológica , Humanos , Queratinócitos/patologia , Camundongos , Microscopia Confocal , Sensibilidade e EspecificidadeRESUMO
Physiological apoptosis in mammals is a type of programmed cell death, an important element in the developmental repertoire ensuring tissue homeostasis and proper disposal of cells that are no longer needed, such as milk-producing epithelial cells in the mammary gland after lactation, luteal cells in the post partum Corpus luteum or secretory cells in the prostate after castration. Although incompletely described, apoptosis in hormone-dependent tissues is apparently initiated and executed using common biochemical strategies. These include survival pathways governed by local and systemic factors and hormones, diverse regulatory pathways and caspase-dependent execution pathways. Using an antibody that recognizes processed effector caspases or a fluorogenic caspase substrate, we present for the first time evidence that caspases are activated in the mammary gland, in the prostate and in the ovary at the time when apoptosis occurs. Most likely phagocytosis of apoptotic cells by neighboring cells may represent an important step, since only a modest involvement of professional phagocytes is apparent. Here, we will summarize and discuss recent data and will attempt to draw a generalized picture of how physiological apoptosis may occur in these organs.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Hormônios/metabolismo , Glândulas Mamárias Animais/fisiologia , Ovário/fisiologia , Próstata/fisiologia , Animais , Feminino , Regulação da Expressão Gênica , Masculino , Ocitocina , ProlactinaRESUMO
Apoptotic cell death was shown to be accompanied or preceded by an elevated expression of the c-fos protooncogene and DNA binding activity of transcription factor AP-1. We used Fos-deficient mice to study the role of c-Fos during programmed cell death in the prostate. In normal mice apoptosis is induced in the prostate within 2-4 days after castration. Histological features of reduced secretory activity and morphological signs of programmed cell death become obvious. No apparent decrease in secretory activity and no epithelial cell death were observed in Fos-deficient animals after castration. Fragmentation of nuclear DNA was measured by in situ terminal transferase reaction. DNA fragmentation was observed in the prostate epithelium of control mice after castration whereas no similar fragmentation was found in Fos-deficient animals. After castration an AP-1 complex accumulated in the prostate of Fos deficient mice which mainly consists of FosB, Fra-2 and JunD whereas in control animals the AP-1 complex in addition contained c-Fos. Our data strongly suggest that c-Fos is required for programmed cell death of prostate epithelial cells.
Assuntos
Apoptose/fisiologia , Genes fos , Orquiectomia , Próstata/patologia , Proteínas Proto-Oncogênicas c-fos/fisiologia , Fator de Transcrição AP-1/fisiologia , Animais , Atrofia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Ligação a DNA/isolamento & purificação , Antígeno 2 Relacionado a Fos , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-fos/isolamento & purificação , Proteínas Proto-Oncogênicas c-jun/isolamento & purificação , Espermatogênese , Testículo/patologia , Fator de Transcrição AP-1/isolamento & purificação , Fatores de Transcrição/isolamento & purificaçãoRESUMO
At birth, the mammalian lung is still immature. The alveoli are not yet formed and the interairspace walls contain two capillary layers which are separated by an interstitial core. After alveolarization (first 2 postnatal weeks in rats) the alveolar septa mature: their capillary layers merge, the amount of connective tissue decreases, and the mature lung parenchyma is formed (second and third week). During the first 3 wk of life the role of tissue transglutaminase (tTG) was studied in rat lung by immunostaining of cryostat and paraffin sections, by Northern and Western blotting, and by a quantitative determination of gamma-glutamyl-epsilon-lysine. While enzyme activity and intracellular tTG were already present before term, the enzyme product (gamma-glutamyl-epsilon-lysine-crosslink) and extracellular tTG appeared between postnatal days 10 and 19 in the lung parenchyma. In large blood vessels and large airways, which mature earlier than the parenchyma, both the enzyme product and extracellular tTG had already appeared at the end of the first postnatal week. We conclude that tTG is expressed and externalized into the extracellular matrix of lung shortly before maturation of an organ area. Because tTG covalently and irreversibly crosslinks extracellular matrix proteins, we hypothesize that it may prevent or delay further remodeling of basement membranes and may stabilize other extracellular components, such as microfibrils.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Matriz Extracelular/enzimologia , Pulmão/embriologia , Transglutaminases/genética , Transglutaminases/metabolismo , Animais , Especificidade de Anticorpos , Reagentes de Ligações Cruzadas/química , Dipeptídeos/análise , Dipeptídeos/metabolismo , Matriz Extracelular/química , Feminino , Feto/química , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Pulmão/química , Pulmão/enzimologia , Gravidez , RNA Mensageiro/análise , Ratos , Ratos Wistar , Transglutaminases/imunologiaRESUMO
In an effort to develop a suitable bioassay for testing lung growth factors that might be operative during compensatory lung growth following partial pneumonectomy, a simple and inexpensive lung organ culture system was characterized. The culture employs lung tissue slices obtained by means of a device allowing thicknesses of 500 microns to be cut reproducibly. To avoid the collapse of the organ, the alveolar spaces were filled prior to culture with Noble agar-containing Eagle's-Dulbecco's modified medium. Lung tissue sections could be maintained ultrastructurally intact for at least one week. The results showed that upon culture, a part of the type II pneumocytes undergo differentiation into type I pneumocytes, thus demonstrating that the culture system may be suited for differentiation studies. One surprising feature of this culture system was the mitogenic impulse associated with culture. Radioactively labeled thymidine incorporation was strongly stimulated in the culture, mainly affecting the epithelial cells, as could be established by "back-to-back" autoradiography. With a reconstruction experiment, it was possible to demonstrate the local release of a mitogenic factor following slicing, mincing, or dissection of the lung tissue, which could be assayed by its ability to induce serum-starved Balb/c 3T3 cells to synthesize DNA in culture.
Assuntos
Pulmão/metabolismo , Mitógenos/metabolismo , Células 3T3 , Animais , Diferenciação Celular , Divisão Celular , Sobrevivência Celular , DNA/biossíntese , Pulmão/citologia , Masculino , Camundongos , Técnicas de Cultura de Órgãos , RatosRESUMO
We have analysed the temperature dependence of the transport of Semliki Forest virus (SFV) envelope proteins in mosquito cells, the natural host cells of alphaviruses. These cells are cultivated at a lower temperature (28 degrees C) and have a different lipid composition as compared to mammalian cells. When the incubation temperature was reduced at early times after infection, the onset of virus shedding was delayed and the maximal titers decreased correspondingly to the temperature. No virus was shed at 12 degrees C. No evidence was observed for a block of virus release due to a shift of the sites of virus maturation. When the incubation temperature was reduced at later times after infection a critical temperature of 12 degrees C was again observed. At this temperature no transport of viral proteins took place, p62 remained uncleaved, the glycan processing of E1 did not occur and the envelope proteins accumulated in a pre-Golgi compartment. We suggest a mathematical formula which allows the extrapolation of transport data to the temperature at which intracellular protein transport becomes blocked.