RESUMO
In immature thymocytes, T cell receptor for antigen (TCR) mobilization leads to an active T cell suicide process, apoptosis, which is involved in the selection of the T cell repertoire. We have proposed that inappropriate induction of such a cell death program in the mature CD4+ T cell population could account for both early qualitative and late quantitative CD4+ T lymphocyte defects of human immunodeficiency virus (HIV)-infected individuals (Ameisen, J.C., and A. Capron. 1991. Immunol. Today. 4:102). Here, we report that the selective failure of CD4+ T cells from 59 clinically asymptomatic HIV-infected individuals to proliferate in vitro to TCR mobilization by major histocompatibility complex class II-dependent superantigens and to pokeweed mitogen (PWM) is due to an active CD4+ T cell death process, with the biochemical and ultrastructural features of apoptosis. Activation-induced cell death occurred only in the CD4+ T cell population from HIV-infected asymptomatic individuals and was not observed in T cells from any of 58 HIV-seronegative controls, including nine patients with other acute or chronic infectious diseases. Activation-induced CD4+ T cell death was prevented by cycloheximide, cyclosporin A, and a CD28 monoclonal antibody (mAb). The CD28 mAb not only prevented apoptosis but also restored T cell proliferation to stimuli, including PWM, superantigens, and the tetanus and influenza recall antigens. These findings may have implications for the understanding of the pathogenesis of acquired immune deficiency syndrome and for the design of specific therapeutic strategies.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Ativação Linfocitária/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos CD28 , Linfócitos T CD4-Positivos/ultraestrutura , Morte Celular/imunologia , Cicloeximida/farmacologia , Ciclosporina/farmacologia , Eletroforese em Gel de Ágar , Enterotoxinas/imunologia , Feminino , Soropositividade para HIV/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Mitógenos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
Interleukin 5 (IL-5), the major factor involved in eosinophil differentiation, is produced by T cells or mast cells. In the present study, we found that eosinophils infiltrating the mucosa of four patients with active coeliac disease also express the IL-5 mRNA. No positive signal was obtained in normal duodenum tissues and in the cell infiltrate from patients submitted to gluten restriction. The identification of labeled mucosal cells as eosinophils relied on their typical morphology. Moreover, highly purified blood eosinophils from three out of four patients with eosinophilia were also strongly labeled with the IL-5 antisense but not with the corresponding sense probe. Together, these results suggest that eosinophils have the capacity to synthesize IL-5, which could contribute to paracrine interactions with T and B cells and, in autocrine fashion, locally participate, through binding to the IL-5 receptor, to eosinophil differentiation and activation. These data might have implications not only in the pathology of coeliac disease but also in other diseases associated with eosinophil infiltration.
Assuntos
Doença Celíaca/imunologia , Eosinófilos/imunologia , Interleucina-5/genética , Mucosa Intestinal/imunologia , RNA Mensageiro/genética , Doença Celíaca/patologia , Sondas de DNA , Duodeno/patologia , Eosinófilos/patologia , Humanos , Mucosa Intestinal/patologia , Jejuno/patologia , Hibridização de Ácido Nucleico , RNA Mensageiro/análiseRESUMO
A role for immunoglobulin E and its high affinity receptor (Fc epsilon RI) in the control of bacterial pathogenicity and intestinal inflammation has been suggested, but relevant animal models are lacking. Here we compare transgenic mice expressing a humanized Fc epsilon RI (hFc epsilon RI), with a cell distribution similar to that in humans, to Fc epsilon RI-deficient animals. In hFc epsilon RI transgenic mice, levels of colonic interleukin 4 were higher, the composition of fecal flora was greatly modified, and bacterial translocation towards mesenteric lymph nodes was increased. In hFc epsilon RI transgenic mice, 2,4,6-tri-nitrobenzenesulfonic acid (TNBS)-induced colitis was also more pronounced, whereas Fc epsilon RI-deficient animals were protected from colitis, demonstrating that Fc epsilon RI can affect the onset of intestinal inflammation.
Assuntos
Colite/imunologia , Colite/microbiologia , Receptores de IgE/metabolismo , Animais , Bactérias/isolamento & purificação , Bactérias/patogenicidade , Sequência de Bases , Colite/patologia , Primers do DNA/genética , Modelos Animais de Doenças , Humanos , Imunoglobulina E/biossíntese , Imunoglobulina E/genética , Interleucina-4/genética , Interleucina-4/metabolismo , Linfonodos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Permeabilidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de IgE/genética , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
Lipoprotein transport across the blood-brain barrier (BBB) is of critical importance for the delivery of essential lipids to the brain cells. The occurrence of a low density lipoprotein (LDL) receptor on the BBB has recently been demonstrated. To examine further the function of this receptor, we have shown using an in vitro model of the BBB, that in contrast to acetylated LDL, which does not cross the BBB, LDL is specifically transcytosed across the monolayer. The C7 monoclonal antibody, known to interact with the LDL receptor-binding domain, totally blocked the transcytosis of LDL, suggesting that the transcytosis is mediated by the receptor. Furthermore, we have shown that cholesterol-depleted astrocytes upregulate the expression of the LDL receptor at the BBB. Under these conditions, we observed that the LDL transcytosis parallels the increase in the LDL receptor, indicating once more that the LDL is transcytosed by a receptor-mediated mechanism. The nondegradation of the LDL during the transcytosis indicates that the transcytotic pathway in brain capillary endothelial cells is different from the LDL receptor classical pathway. The switch between a recycling receptor to a transcytotic receptor cannot be explained by a modification of the internalization signals of the cytoplasmic domain of the receptor, since we have shown that LDL receptor messengers in growing brain capillary ECs (recycling LDL receptor) or differentiated cells (transcytotic receptor) are 100% identical, but we cannot exclude posttranslational modifications of the cytoplasmic domain, as demonstrated for the polymeric immunoglobulin receptor. Preliminary studies suggest that caveolae are likely to be involved in the potential transport of LDL from the blood to the brain.
Assuntos
Barreira Hematoencefálica , Receptores de LDL/metabolismo , Receptores de LDL/fisiologia , Animais , Astrócitos , Transporte Biológico , Encéfalo/irrigação sanguínea , Capilares/metabolismo , Capilares/fisiologia , Bovinos , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Humanos , Modelos Biológicos , RatosRESUMO
Alveolar macrophages from nonatopic donors were passively sensitized with allergen-specific IgE antibody from the serum of asthmatic patients. A selective release of 4-8% of the lysosomal beta-glucuronidase of these cells occurred within 30 min of contact with the related allergen or with anti-human IgE antibody, in the absence of any mast or basophil cells. The cell reactivity was dependent on the interaction of macrophages with IgE, as shown by the disappearance of the allergen-induced enzyme release after heating or IgE-immune adsorption of the sensitizing serum, but not after IgG-adsorption. Alveolar macrophages from asthmatic patients behaved similarly to passively sensitized normal macrophages. Contact with the related allergen or with anti-IgE antibody induced the same percentage of enzyme release, demonstrating that these cells possess allergen-specific IgE bound on their surface. 18% of them formed rosettes with anti-IgE-coated sheep erythrocytes, and 15-22% with allergen-coated erythrocytes, but lost this property after preincubation with the specific allergen. The presence of IgE-specific receptors on the macrophage surface was demonstrated both at the ultrastructural level with immunoperoxidase labeling, and at low magnification by the formation of 15-18% rosettes with human IgE-coated erythrocytes. The formation of such rosettes was inhibited after incubation of alveolar phagocytes with aggregated myeloma IgE. On the basis of these observations, the participation of the alveolar macrophages in IgE-mediated pulmonary hypersensitivity must be considered. Its precise involvement requires, however, further investigations.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Asma/patologia , Imunoglobulina E/imunologia , Macrófagos/imunologia , Adulto , Alérgenos/imunologia , Basófilos/metabolismo , Feminino , Liberação de Histamina , Humanos , Macrófagos/enzimologia , Masculino , Mastócitos/metabolismo , Microscopia Eletrônica , Alvéolos Pulmonares/citologia , Receptores de Antígenos de Linfócitos B , Formação de RosetaRESUMO
A sequential immunoaffinity chromatography procedure was developed to isolate from whole normolipidemic human plasma a subpopulation of apoB containing particles (Lp-B) which is virtually free of non apoB protein. The absence of non apoB protein in Lp-B was assessed by enzyme immunoassay against apolipoproteins A-I, A-II, A-IV, E, C-III and (a). Electron microscopy and fractionation of the isolated particles by gel filtration demonstrated that these particles were heterogeneous in size. However, most of them had diameters between 18 and 26 nm. These particles were found to be rich in cholesterol (molar ratio cholesterol/apoB = 2246 +/- 995) poor in triacylglycerol (molar ratio triacylglycerol/apoB = 555 +/- 518) and had a phospholipids/apoB molar ratio of 713 +/- 348. Most of the cholesterol was esterified (66% +/- 5%). Lp-B particles bound to the apoB, E receptor of HeLa cells with a lower affinity than LDL prepared by ultracentrifugation (1.030 kg/l less than d less than 1.053 kg/l). (KD = 18.9 vs 10.5 nmol/l).
Assuntos
Apolipoproteínas B/química , Apolipoproteínas B/isolamento & purificação , Apolipoproteínas B/análise , Células Cultivadas , Fracionamento Químico , Colesterol/análise , Cromatografia de Afinidade , Cromatografia em Gel , Humanos , Técnicas Imunoenzimáticas , Fosfolipídeos/análise , Triglicerídeos/análiseRESUMO
Endothelial cells and pericytes are closely associated in brain capillaries. Together with astrocytic foot processes, they form the blood-brain barrier. Capillaries were isolated from bovine brain cortex. Pure populations of endothelial cells and pericytes were isolated and cultured in vitro. Polarized monolayers of endothelial cells preferentially secreted immunoreactive endothelin-1 (Et-1) at their abluminal (brain-facing) membrane. They did not express receptors for Et-1. Pericytes expressed BQ-123-sensitive ETA receptors for endothelins as evidenced by 125I-Et-1 binding experiments. These receptors were coupled to phospholipase C as demonstrated by intracellular calcium measurements using indo-1-loaded cells. Addition of Et-1 to pericytes induced marked changes in the cell morphology that were associated with a reorganization of F-actin and intermediate filaments. It is concluded that Et-1 is a paracrine mediator at the bovine blood-brain barrier and that capillary pericytes are target cells for endothelium-derived Et-1.
Assuntos
Comunicação Celular , Circulação Cerebrovascular , Endotelina-1/fisiologia , Endotélio Vascular/citologia , Animais , Barreira Hematoencefálica , Capilares/citologia , Bovinos , Polaridade Celular , Técnicas de Cocultura , Antagonistas dos Receptores de Endotelina , Endotelina-1/biossíntese , Endotélio Vascular/metabolismo , Peptídeos Cíclicos/farmacologia , Receptores de Endotelina/metabolismoRESUMO
The insect moulting hormones, ecdysone and 20 hydroxyecdysone, were detected by the combined use of radioimmunoassay and high performance liquid chromatography in the human parasite Schistosoma mansoni. On day 11 after infection only the ecdysone form is present, but, on day 40 after infection the ratio between ecdysone and 20 hydroxyecdysone changes with anatomic localization of the adult worms in mammalian host. In the eggs, the ratio of these two hormones is identical to the ratio found in sexually mature worms located in mesenteric veins. These data demonstrate for the first time that S. mansoni synthesizes the steroid hormones ecdysone and 20 OH ecdysone which are potent molecules in stimulating growth and vitello-genesis of this gonochoric trematode.
Assuntos
Ecdisona/análise , Ecdisterona/análise , Schistosoma mansoni/crescimento & desenvolvimento , Envelhecimento , Animais , Cromatografia Líquida de Alta Pressão , Cricetinae , Humanos , Mesocricetus , RadioimunoensaioRESUMO
Ecdysteroids produced by schistosomes are released in biological fluids of infected hosts. In the sera, the concentration of ecdysteroids correlates with the permissiveness of the host to schistosome infection and its detection is available in the absence of positive parasitological tests. In the urine, ecdysteroid concentration decreases markedly after chemotherapy. 20-Hydroxyecdysone and its epimer were identified in the urine of infected patients using mass spectrometry. These data demonstrate for the first time that ecdysteroids are released by organisms. Moreover, they are potent molecules of parasite infection and can be used for parasite diagnosis.
Assuntos
Hormônios de Invertebrado/metabolismo , Esquistossomose/metabolismo , Adolescente , Animais , Criança , Cricetinae , Ecdisteroides , Ecdisterona/urina , Haplorrinos , Humanos , Hormônios de Invertebrado/sangue , Hormônios de Invertebrado/urina , Cinética , Espectrometria de Massas , Ratos , Schistosoma haematobium/metabolismo , Schistosoma mansoni/metabolismo , EstereoisomerismoRESUMO
Cholesterol efflux was studied in cultured Ob1771 adipose cells after preloading with LDL cholesterol. Exposure to particles containing apo AII (LpAI) and particles containing apo AI and apo AII (LpAI:AII) isolated from native human plasma, and from HDL2 or HDL3, showed that only LpAI were able to promote cholesterol efflux, despite the fact that both kinds of particles were able to bind to receptor sites within the same range of concentrations (apparent Kd values between 10 and 25 micrograms/ml). During this long-term exposure, LpAI:AII demonstrated a concentration-dependent inhibition (10-60 micrograms/ml) of LpAI-mediated cholesterol efflux from adipose cells under conditions where LpAI:AII did not deliver cholesterol to the cells and where no net change in the distribution of apo AI between LpAI and LpAI:AII was observed. The antagonizing and modulating role of LpAI:AII in preventing cholesterol efflux mediated by LpAI appears not to be related to the lipid composition and cholesterol content of the particles but, rather, appears dependent upon the presence of apo AI in LpAI particles and apo AII in LpAI:AII particles. The actual concentrations of these particles in the interstitial fluid bathing peripheral cells might be critical for the in vivo occurrence of cholesterol efflux.
Assuntos
Tecido Adiposo/metabolismo , Apolipoproteínas A/farmacologia , Colesterol/metabolismo , Animais , Apolipoproteína A-I , Apolipoproteínas A/química , Apolipoproteínas A/fisiologia , Ligação Competitiva , Células Cultivadas , Humanos , Lipídeos/análise , Camundongos , Proteínas/análiseRESUMO
Abetalipoproteinemia (ABL) and homozygous hypobetalipoproteinemia (HBL) are inherited disorders which are classically characterized by progressive retinal and spinocerebellar disease, fat-soluble vitamin deficiency, and absence of apolipoprotein (apo) B from the plasma. Using immunoaffinity chromatography with an anti-apo B antiserum, we isolated apo B-containing lipoprotein (LpB) particles from the plasma of 4 ABL and 2 HBL patients. The LpB particles were characterized and compared with low density lipoprotein (LDL) and LpB isolated from normal plasma. The ABL/HBL LpB particles were similar in size and charge to normal LpB particles but were relatively enriched in several other apolipoproteins. They contained alpha-tocopherol in a ratio to cholesterol that was proportionately much higher than the very low ratio of alpha-tocopherol to cholesterol in plasma. They bound saturably to fibroblasts and were internalized and degraded similarly to LDL. Hence, the molecular defects in ABL and HBL permit the secretion of a very small number of apo B-containing lipoproteins which may be important for transport of alpha-tocopherol to peripheral tissues.
Assuntos
Abetalipoproteinemia/classificação , Apolipoproteínas B/análise , Hipobetalipoproteinemias/sangue , Lipoproteínas/isolamento & purificação , Vitamina E/sangue , Abetalipoproteinemia/complicações , Abetalipoproteinemia/genética , Adulto , Apolipoproteína B-100 , Apolipoproteínas B/sangue , Apolipoproteínas B/genética , Colesterol/sangue , Feminino , Fibroblastos/metabolismo , Homozigoto , Humanos , Hipobetalipoproteinemias/complicações , Hipobetalipoproteinemias/genética , Lipoproteínas/sangue , Lipoproteínas/classificação , Lipoproteínas LDL/sangue , Masculino , Pessoa de Meia-Idade , Tamanho da Partícula , Deficiência de Vitamina E/sangue , Deficiência de Vitamina E/etiologiaRESUMO
A cell membrane permeabilizing treatment is described which involves the use of lysolecithin at low concentration in acidic acetate buffer and paraformaldehyde fixation. It preserved well-separated scatter cytograms of small and large lymphocytes. The accuracy of the immunochemical detection of internal antigens by flow cytofluorography was demonstrated by the linear relationship between the percentage of fluorescent cells detected and the proportion of intracellular antigen-containing cells in mixtures with antigen-negative cell lines. Cell cycle analysis by dual nuclear staining with propidium iodide and FITC-conjugated Ki-67 antibody recognising in vitro stimulated human T lymphocytes verified that the proliferating lymphocytes retained their increased light scatter properties after permeabilization. Enumeration of interleukin-2 (IL-2) producing cells by their cytoplasmic immunofluorescence showed that enlarged lymphocytes were the main IL-2 producing cells. This improved permeabilization procedure, by gating small and enlarged lymphocytes separately, makes it possible to determine by two color fluorescence the immunophenotype of activated T cells committed to interleukin production.
Assuntos
Permeabilidade da Membrana Celular , Citometria de Fluxo , Interleucina-2/biossíntese , Linfócitos T/metabolismo , Ciclo Celular , Linhagem Celular , Citometria de Fluxo/métodos , Fluoresceína-5-Isotiocianato , Fluoresceínas , Corantes Fluorescentes , Humanos , Ativação Linfocitária , Lisofosfatidilcolinas , Propídio , Linfócitos T/imunologia , Linfócitos T/ultraestrutura , Tiocianatos , Células Tumorais CultivadasRESUMO
The haem enzyme myeloperoxidase (MPO) (EC 1.11.1.7) with a spectral A430/A280 ratio greater than 0.7 and a specific activity of 125 U/mg was purified from isolated human neutrophils. To obtain a radioimmunoassay (RIA) for this enzyme, a specific antiserum against human neutrophil MPO was raised in rabbits and used at an initial dilution of 1/10,000. MPO labelled with 125iodine by a technique of self-labelling in the presence of H2O2, had a specific activity of 24 mCi/mg. After incubation at room temperature (2 h) and separation by double antibody precipitation in the presence of polyethylene glycol, the sensitivity of the RIA was 21 ng/ml. The RIA showed good precision and accuracy with intra- and interassay coefficients of variation of less than 7% for MPO concentrations ranging from 100 to 800 ng/ml, and satisfactory recoveries of known amounts of exogenous MPO in plasma. For the measurement of MPO in blood, the best sampling technique was to collect blood into EDTA. Rapid centrifugation (within 20 min) was necessary for blood collected into heparin. Mean MPO values in normal individuals were 340 +/- 98 ng/ml in EDTA plasma (n = 152) and 332 +/- 82 ng/ml in heparinized plasma (n = 34). When MPO was measured 12-6 h after injury in critically ill patients high values (above 1000 ng/ml) were found in 6/15 patients with multiple injuries. In patients with sepsis (n = 22), MPO values were always above 1000 ng/ml.
Assuntos
Neutrófilos/enzimologia , Peroxidase/sangue , Radioimunoensaio/métodos , Animais , Formação de Anticorpos/imunologia , Infecções Bacterianas/sangue , Coleta de Amostras Sanguíneas , Feminino , Humanos , Imunização , Leucemia Linfoide/sangue , Masculino , Traumatismo Múltiplo/sangue , Neutrófilos/ultraestrutura , Peroxidase/isolamento & purificação , Coelhos , Reprodutibilidade dos TestesRESUMO
Metabolic pathways leading to lipid biosynthesis in four different developmental stages of Schistosoma mansoni were explored and quantified by incubation in the presence of labeled precursors in a chemically defined medium. At the schistosomulum stage and in male, female, or paired worms, glycerol and oleate incorporation into neutral lipids, mainly in the form of triacylglycerols, was greater than into phospholipids, whereas in 11-and 15-day-old worms, synthesis mainly led to phospholipids. Incorporation into phospholipids was recovered largely in phosphatidylcholine, and distribution into other phospholipids depended on the developmental stage. Incorporation of choline and ethanolamine into their respective phospholipids represented up to 15% of the parasitic phospholipid content. The formation of phosphatidylcholine by phosphatidylethanolamine methylation occurred mainly in the immature parasitic stages. Inositol incorporation was also measurable, whereas [14C]serine incorporation was low or undetectable. Addition of 1-palmitoyl-2-[14C]oleyl phosphatidylcholine revealed a very high uptake of this phospholipid by the immature stages but further metabolism was not detectable. In contrast, adult S. mansoni were completely unable to take up or absorb this exogenous phospholipid. The most striking aspect of this study was the relatively high metabolic activity in 11-day-old worms and the lower but sustained activity on day 15 and at the schistosomulum stage. By comparison, biosynthetic activity in adult S. mansoni, on which research studies have been focused until now, was very low. We also discuss the participation of lipid metabolism in the constant renewal of the membrane complex which is essential to parasitism by S. mansoni.
Assuntos
Lipídeos de Membrana/metabolismo , Schistosoma mansoni/metabolismo , Envelhecimento , Animais , Biomphalaria , Radioisótopos de Carbono , Colina/metabolismo , Etanolamina , Etanolaminas/metabolismo , Feminino , Glicerol/metabolismo , Inositol/metabolismo , Masculino , Fosfolipídeos/metabolismo , Surfactantes Pulmonares/metabolismo , Schistosoma mansoni/crescimento & desenvolvimento , Serina/metabolismo , Fatores Sexuais , TrítioRESUMO
Molecular mimicry has been considered as a possible way for parasites to escape host immune responses. This work concerns the characterization of protein determinants shared by Schistosoma mansoni and its intermediate host Biomphalaria glabrata. Parasite (Sm39) and mollusc (Bg 39) cross-reactive proteins were identified and shown to induce in rabbit and mouse, antibodies specific for invertebrate determinants. Ultrastructural studies demonstrated that antibodies to Sm39 specifically bound to muscular structures of parasite and mollusc. Molecular cloning and sequencing indicated that Bg39 corresponded to a muscular isoform of tropomyosin. The mollusc sequence showed a 51-65% homology with seven different muscular tropomyosins from vertebrate and invertebrate species. The highest score of homology was observed with S. mansoni tropomyosin, suggesting that cross-reactive determinants could be specific for the trematode and its intermediate host. In miracidia, Sm39 epitopes were also shown to be contained in the vesicles present in epidermal ridges and cellular bodies. Such vesicles are involved in the formation of a protective tegument around sporocysts, suggesting a possible role of cross-reactive tropomyosins in miracidia and/or sporocyst-snail interactions.
Assuntos
Biomphalaria/análise , Schistosoma mansoni/análise , Tropomiosina/química , Sequência de Aminoácidos , Animais , Biomphalaria/imunologia , Biomphalaria/parasitologia , Northern Blotting , Clonagem Molecular , Reações Cruzadas , Interações Hospedeiro-Parasita , Imuno-Histoquímica , Dados de Sequência Molecular , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/imunologia , Homologia de Sequência do Ácido Nucleico , Tropomiosina/imunologiaRESUMO
A Schistosoma mansoni cDNA library was constructed from the mRNA of adult worms in the expression vector lambda gt11 and screened with a rabbit antiserum raised against the 26-kDa S. mansoni glutathione S-transferase isoforms (Sm GST 26). Two clones were selected and the nucleotide sequences deduced. The predicted amino acid sequence, specified by these cDNAs, shows strong homology with a Schistosoma japonicum 26 kDa glutathione S-transferase and a lower level of homology with mammalian glutathione S-transferase class mu isoenzymes (EC 2.5.1.18). No significant homology score was found with a 28-kDa S. mansoni glutathione S-transferase (Sm GST 28). A study of the tissue distribution of the cloned Sm GST 26 by immunoelectron microscopy shows similarities to Sm GST 28 in that they are present in the tegument and in subtegumentary parenchymal cells. However, a major difference exists in the protonephridial region in which Sm GST 26 is present in the cytoplasmic digitations localized in the apical chamber delineated by the flame cell body, suggesting that Sm GST 26 may be actively excreted by adult worms.
Assuntos
Glutationa Transferase/análise , Schistosoma mansoni/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , Eletroforese em Gel Bidimensional , Glutationa Transferase/genética , Soros Imunes , Imuno-Histoquímica , Microscopia Eletrônica , Dados de Sequência Molecular , Schistosoma mansoni/genética , Homologia de Sequência do Ácido NucleicoRESUMO
The P21 antigen of Toxoplasma gondii, defined by the monoclonal antibody TG17-113, has been described as a dense granule component, secreted in the parasitophorous vacuole during host cell invasion. The present work reports the cloning of the gene encoding the P21 antigen, for which we propose the name GRA 5. A cDNA library was screened with a rat antiserum raised against an HPLC fraction enriched in the P21 antigen. cDNA clones encoding GRA 5 were selected by antibody selection on the recombinant proteins. All these clones were incomplete at the 5' end. The 5' fragment of the longest cDNA clone isolated by this first screening was used as a probe in secondary screenings of cDNA and genomic DNA libraries. A genomic fragment containing the P21 gene and nearly full-length cDNAs have been isolated and sequenced. The gene encoding GRA 5 is 834 bp long and does not contain any intron. The deduced amino acid sequence of an open reading frame encoding 133 amino acids perfectly matched that of 5 peptides microsequenced from the native antigen. A N-terminal hydrophobic region was found to possess the characteristics of a signal peptide of 25 amino acids. A second hydrophobic domain, bordered by two hydrophilic regions strongly suggests a transmembrane region. This molecular structure is supported by ultrastructural studies showing the association of the P21 antigen with the parasitophorous vacuole membrane.
Assuntos
Antígenos de Protozoários/genética , Toxoplasma/genética , Toxoplasma/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/metabolismo , Sequência de Bases , Clonagem Molecular , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/ultraestrutura , DNA de Protozoário/genética , Genes de Protozoários , Membranas Intracelulares/imunologia , Membranas Intracelulares/ultraestrutura , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Toxoplasma/ultraestrutura , Vacúolos/imunologia , Vacúolos/ultraestruturaRESUMO
Tumor necrosis factor alpha (TNF-alpha) plays a crucial role in the pathogenesis of the central nervous system infections. In an in vitro reconstructed blood-brain barrier model, a significant dysregulation of receptor mediated endocytosis of low density lipoproteins (LDL) and transferrin (Tf) is demonstrated at delayed phase of direct TNF-alpha activation. Concomitant with the increase in LDL uptake, we demonstrate a decrease of Tf-receptor mediated endocytosis. The potential role of TNF action in the differential or opposite routing of macromolecules is also characterized by a stimulation of their transcytosis. These findings may provide a new insight into the inflammatory effect of TNF-alpha on brain capillary endothelial cells.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Receptores da Transferrina/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Transporte Biológico , Capilares/citologia , Capilares/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Bovinos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Endotélio Vascular/citologia , Inulina/farmacocinética , Lipoproteínas LDL/farmacocinética , Substâncias Macromoleculares , Ratos , Sacarose/farmacocinéticaRESUMO
Inhibition of acyl-coenzyme A: cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) reduces intracellular cholesteryl esters that are substrates for steroidogenesis in adrenal cells. The adrenal side effects of ACAT inhibitors remain a key point for their development as antiatherosclerotic agents. The aim of this study was to characterize the effects of a novel and powerful ACAT inhibitor, F 12511 (S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthio-phenylacetanilide, on the NCI-H295R cell line, which has functional properties comparable to those of normal human adrenal cells. F 12511 incubated with cultured cells for 4-72 hr strongly inhibited cholesteryl oleate formation. The concentrations required to produce 50% inhibition (IC50) values) ranged from 20 to 50 nM; in the presence of low-density lipoproteins (LDL), this effect was paralleled by a decrease in cholesteryl ester mass and an increase in intracellular free cholesterol. At concentrations 100-fold larger than the IC(50) value for up to 48 hr, F 12511 reduced neither the basal release of cortisol and aldosterone nor the production of cortisol stimulated by forskolin. F 12511 did not modify the mRNA levels of the steroidogenic enzyme genes cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), or cytochrome P450 21-hydroxylase (P450c21) or those of the LDL receptor and high-density lipoprotein scavenger receptor class B, type I (SR-BI) genes, either in the presence or absence of adenosine 3',5'-cyclic monophosphate stimulation for 24 hr. Exposure to F 12511 at up to 3 microM for 24 or 48 hr did not result in significant change in morphological and ultrastructural characteristics; the cytoplasm contained large numbers of mitochondria with intact crystae, and the same typical features of secretory activity were observed in NCI-H295R control cells. Exposure to 3 microM of F 12511 for 96 hr also did not affect cell viability. These data demonstrate that reduction of the substrate for steroidogenesis by the ACAT inhibitor F 12511 impairs neither steroid production nor transcription of genes involved in steroidogenesis and lipoprotein uptake in the pluripotent human adrenal cell line NCI-H295R.
Assuntos
Glândulas Suprarrenais/efeitos dos fármacos , Anilidas/farmacologia , Inibidores Enzimáticos/farmacologia , Esterol O-Aciltransferase/antagonistas & inibidores , Neoplasias do Córtex Suprarrenal , Glândulas Suprarrenais/enzimologia , Sítios de Ligação , Transporte Biológico , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Receptores de Lipoproteínas/metabolismo , Esteroides/metabolismo , Esterol O-Aciltransferase/metabolismo , Células Tumorais CultivadasRESUMO
Tumor necrosis factor-alpha (TNF-alpha) plays a crucial role in the pathogenesis of the central nervous system infections. The aim of the present study was to analyze quantitatively the changes in the blood-brain barrier (BBB) permeability after the intracarotid injection of TNF-alpha. Recombinant human TNF-alpha was injected into the left internal carotid artery of anesthetized newborn pigs (n = 48) in the doses of 0, 1000, 10 000 and 100 000 IU, respectively. Before, as well as 1, 2, 4, 8, and 16 h after the challenge, the extravasation of a small (sodium fluorescein (SF), mw 376), and a large (Evan's blue-albumin (EBA), mw 67 000) tracer was determined concomitantly by spectrophotometry in the cerebral cortex of the animals. There was a time- and dose-dependent increase in BBB permeability both for SF and EBA; however, significant (P < 0.05) BBB opening for albumin only developed 2 h after the challenge. In the morphological study the same excitable tracers, identical experimental protocol and groups were used. Cryostat sections of brain tissue were viewed for optical sectioning with a confocal laser scanning microscope equipped with an argon/krypton ion laser. A diffuse BBB opening for SF and a moderate perivascular extravasation for EBA were found in the cortices of TNF-alpha-treated animals. We conclude that significant increases in intravascular TNF-alpha-concentration during neonatal infections may result in vasogenic brain edema formation.