RESUMO
We describe enhanced expression and enzymatic activity of ecto-ATPase and ecto-5'nucleotidase on CMV infected endothelial cells as compared to uninfected cells. These ectoenzymes play a major role in modulation of platelet activation and aggregation. Furthermore, adenosine has a modulatory effect upon inflammation. Addition of ATP, ADP or AMP to cultures of CMV infected or uninfected endothelial cells revealed increased turnover of AMP in CMV infected endothelial cells. In addition, the superoxide production by stimulated polymorphonuclear cells was inhibited in the presence of CMV infected endothelial cells as compared to uninfected cells, probably due to the enhanced activity of ecto-5'nucleotidase and associated to production of adenosine.
Assuntos
5'-Nucleotidase/genética , Adenosina Trifosfatases/genética , Infecções por Citomegalovirus/patologia , Endotélio Vascular/metabolismo , 5'-Nucleotidase/metabolismo , Nucleotídeos de Adenina/farmacologia , Adenosina Trifosfatases/metabolismo , Células Cultivadas , Infecções por Citomegalovirus/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/virologia , Granulócitos/metabolismo , Humanos , Imuno-Histoquímica , Superóxidos/metabolismo , Regulação para CimaRESUMO
Injection of rabbit anti-rat thymocyte serum (ATS) i.v into rats induces a transient glomerulopathy characterized by immune aggregates localized in the mesangium and along the glomerular capillary wall, as detected by immunofluorescence (IF) techniques. Neither light microscopical alterations in the kidney, nor proteinuria could be detected in these animals and no autologous IgG could be observed in the glomeruli during the observation period (45 days). Results from ex vivo perfusion studies showed identical localization of immune aggregates--which, in electron microscopy, appear to be localized subepithelially. The ATS used, was monospecific in that no other specificities could be detected after thorough absorption with rat tissue extracts including tubular brushborder antigens, so it is concluded from these data that ATS is able to participate in the formation of immune complexes in situ by recognizing epitopes both in the mesangium and at the epithelial side of the glomerular basement membrane.
Assuntos
Soro Antilinfocitário , Glomerulonefrite/imunologia , Doenças do Complexo Imune/imunologia , Linfócitos T/imunologia , Animais , Feminino , Imunofluorescência , Glomerulonefrite/patologia , Doenças do Complexo Imune/patologia , Rim/patologia , Microscopia Eletrônica , Ratos , Ratos EndogâmicosRESUMO
Polyclonal rabbit antirat thymocyte serum (ATS) has been shown to form in situ glomerular immune aggregates following perfusion into normal rat kidney ex vivo. This may be due to the presence of T-cell-like epitopes in the rat kidney, or it may be a result of contaminating anti-connective-tissue antibodies in ATS. To exclude the latter possibility we investigated binding to the rat kidney of three different (mouse) monoclonal antirat thymocyte antibodies (anti-T-cell MoAbs), directed to Thy 1.1 antigen, as well as (control) anti-B-cell MoAbs. The MoAbs were incubated in vitro with kidney sections or perfused into the blood-free rat kidney ex vivo. It was shown using immunofluorescence (IF) and immunoelectron microscopy (IEM) (peroxidase technique) that the anti-T-cell MoAbs used, in contrast to anti-B-cell MoAbs, are able to bind with glomerular capillary walls, and with mesangial structures after incubation in vitro or perfusion ex vivo. Although staining patterns are not completely identical, the reaction product is clearly demonstrated throughout the glomerular basement membrane (GBM) and along the plasma membranes of endothelial and epithelial cells, after contact with either of the three anti-T-cell MoAbs used. It is concluded that the presence of T-cell-like epitopes in the rat kidney may lead to immune complex formation following contact with anti-T-cell MoAbs. The nephritogenicity of rabbit ATS, as well as of some batches of clinically used ATS, may also be explained by this mechanism rather than by the usually assumed presence of contaminating antibodies in these polyclonal antisera.
Assuntos
Anticorpos Monoclonais/análise , Especificidade de Anticorpos , Soro Antilinfocitário/análise , Sítios de Ligação de Anticorpos , Rim/imunologia , Animais , Feminino , Imunofluorescência , Mesângio Glomerular/imunologia , Mesângio Glomerular/metabolismo , Mesângio Glomerular/ultraestrutura , Rim/metabolismo , Rim/ultraestrutura , Nefrite/imunologia , Ratos , Ratos EndogâmicosRESUMO
We studied glomerular ATPase activity, as detectable at the light microscopic (LM) level in cryostat sections of the rat kidney, after unilateral local X-irradiation. The biochemically detectable reduction in glomerular ATPase activity after X-irradiation could be demonstrated at the LM level by application of a modified cerium-based technique. Results show a clear reduction of reaction product in glomeruli in X-irradiated kidneys as compared with the contralateral control kidney. Technical parameters (i.e., tissue fixation, second thickness, cerium concentration of the incubation mixture, and percentage H2O2 added for the amplification step) were established for optimal reproducibility of the staining results. We show that this modified staining protocol allows detection of differences of ATPase activity in contrast to conventional histochemical methods. Inhibition studies with various phosphatase inhibitors and competitive substrate inhibition experiments revealed that the enzyme is specific for nucleoside di- and triphosphatases. Since reduced glomerular adenine nucleotidase activity has recently been recognized as an early event in (experimental) glomerulonephritis, we feel that the new staining protocol presented here may be highly relevant for routine tissue section screening in nephropathological research.
Assuntos
Adenosina Trifosfatases/metabolismo , Glomérulos Renais/enzimologia , Coloração e Rotulagem/métodos , Animais , Cério , Histocitoquímica , Glomérulos Renais/efeitos da radiação , Masculino , RatosRESUMO
The effect of 17-beta-oestradiol (OE2) upon the activity of the glomerular anti-thrombotic ecto-enzyme ADPase was studied in cyclic and ovariectomized (OVX) Wistar rats. On day 0 (i.e. at the time of ovariectomy or 11 days after ovariectomy) rats received OE2-releasing Silastic implants or empty implants and were sacrificed on day 3, 10 or 21. Cryostat kidney sections were histochemically stained for ecto-ADPase activity using enzyme-histochemistry and glomerular reaction product was quantitatively evaluated by computerized image analysis. Both the histological distribution of reaction product in each glomerulus, as reflected by the relative glomerular area covered with reaction product, as well as enzyme activity, as reflected by staining intensity of the reaction product, were scored. The results show significantly decreased histological distribution after OVX; OVX, however, did not change enzyme activity. It further appeared that OE2 (partly) prevented the decrease of histological distribution in OVX rats, while the enzyme activity was significantly increased by exogenous OE2. In cyclic rats, OE2 did not change histological distribution, although OE2 significantly increased enzyme activity in these rats. It is concluded that glomerular ecto-ADPase expression in the rat kidney is influenced by one or more ovarian factor(s), a very likely candidate being oestradiol. These results may thus point to a dual action of OE2 upon haemostasis: In addition to the known enhancement of procoagulatory plasma factors by OE2, also anti-aggregatory effects may be stimulated by OE2 as reflected by upregulation of vessel wall associated ecto-ADPase activity.
Assuntos
Apirase/biossíntese , Estradiol/farmacologia , Glomérulos Renais/efeitos dos fármacos , Ovário/fisiologia , Análise de Variância , Animais , Feminino , Histocitoquímica , Glomérulos Renais/metabolismo , Ovariectomia , Ratos , Ratos WistarRESUMO
Female Wistar rats were injected with (mouse) monoclonal antibodies (Moabs) of different IgG subclasses directed to rat thymocytes or rat tumor cells. Following intravenous injection of antithymocyte Moabs, glomerular binding of mouse IgG was observed during the first 4 days along the GBM and in the mesangium. No staining for mouse IgG was detected in anti-tumor Moab injected rats. Animals injected with IgG 2a anti-thymocyte Moab developed glomerulonephritis and a massive proteinuria in contrast to rats injected with IgG 1 Moab which is non-complement fixing. The glomerulonephritis lesion consisted of microaneurysms and focal and segmental proliferation. Deposits of complement and fibrin could be detected exclusively in rats injected with IgG 2a anti-thymocyte Moab during the whole observation period of 14 days. This is the first demonstration of overt glomerulonephritis lesions on the injection of monoclonal antibodies.
Assuntos
Glomerulonefrite/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Feminino , Imunofluorescência , Glomerulonefrite/patologia , Imunoglobulina G/imunologia , Injeções Intravenosas , Proteinúria/imunologia , RatosRESUMO
The mechanism underlying increased sensitivity for endotoxin in pregnancy, as reflected by intravascular thrombus formation in various organs i.e. the placenta, is unknown. We studied the influence of endotoxin infusion at day 14 upon the vascular antithrombotic ADPase present in the labyrinth of the rat placenta just before term (day 21). Pregnant Wistar rats were infused with either endotoxin (1 microgram/kg body weight) or saline through permanent vena jugularis catheters and their placenta and kidneys were examined at day 21 using light electron microscopy and enzyme cytochemistry at the ultrastructural level. Also, placenta perfusion ex vivo was done using platelets and ADP to test the thrombotic tendency of placental vessels in endotoxin treated and control rats. The results show in both maternal as well as the fetal vessels of the placental labyrinth vascular occlusions and decreased membrane ADPase activity exclusively in endotoxin treated and not in saline infused pregnant animals. Alternate placenta and kidney perfusion ex vivo resulted in intraplacental and intraglomerular platelet aggregation again exclusively in endotoxin-treated rats. It is concluded that vascular ADPase may be affected by endotoxin due to the pregnant condition, resulting in a functional defect in antithrombotic potention which may promote intravascular formation of microthrombi in situ.
Assuntos
Apirase/antagonistas & inibidores , Endotoxinas/toxicidade , Placenta/efeitos dos fármacos , Trombose/induzido quimicamente , Animais , Feminino , Técnicas In Vitro , Troca Materno-Fetal , Microcirculação/enzimologia , Perfusão , Placenta/irrigação sanguínea , Placenta/enzimologia , Gravidez , Ratos , Ratos EndogâmicosRESUMO
In the present study the possible role of decidual cells in the pregnancy-associated increased sensitivity of glomerular ecto-ADP-ase to endotoxin was investigated. Early (day 5) pregnant (E-Pr; n = 10), pseudopregnant (E-PSP; n = 10), (day 5), pseudopregnant rats with a decidualized uterus (E-DEC; n - 10), as well as late (day 14) pregnant (L-Pr; n = 10), pseudopregnant (L-PSP: n = 10) (day 14), and pseudopregnant rats with a decidualized uterus (E-DEC; n = 10) were infused with either endotoxin (1.0 mg/kg bw) or saline. Three days later rats were killed and specimens of the left kidney were snap-frozen. Cryostat kidney sections (4 microns) were stained for ecto-ADP-ase activity and quantitatively evaluated. The results show that only glomerular ecto-ADP-ase activity of both groups of pregnant rats (E-Pr and L-Pr) was significantly decreased after endotoxin infusion as compared to saline infusion. In the other groups of rats, no significant differences in ecto-ADP-ase activity were observed between saline and endotoxin infusion. It is concluded that decidual cells do not play a role in the increased sensitivity of ecto-ADP-ase to endotoxin during pregnancy.
Assuntos
Apirase/efeitos dos fármacos , Decídua/citologia , Endotoxinas/farmacologia , Glomérulos Renais/enzimologia , Análise de Variância , Animais , Apirase/metabolismo , Decídua/fisiologia , Feminino , Processamento de Imagem Assistida por Computador , Gravidez , Pseudogravidez , Ratos , Ratos WistarRESUMO
Heterologous antibodies directed to brushborder antigens of rat kidney tubules are nephritogenic in that an immediate immune complex glomerulopathy occurs after injection ot these antibodies into normal rats. Since previous observations suggested the presence of anti-T-cell specificity within these antibodies, we now compared the specificities of these antibodies with those of heterologous anti-rat thymocyte antibodies, using immunofluorescence (IF), cytotoxicity and migration inhibition (MIF) assays. The results suggest that anti-brushborder anti-serum contains anti-thymocyte specificities, while antithymocyte antiserum contains anti-brushborder specificity. These specificities could be removed selectively from both of the antisera by absorption with insoluble tissue extracts containing the appropriate antigens, i.e. thymocyte antigens or brushborder antigens respectively, indicating that two specificities are involved rather than only cross-reacting antibodies. Since it is unlikely for several other reasons that this feature is simply the result of an immunologic cross-reaction due to antibodies raised by immunization with purified kidney tissue antigens, this dual specificity of anti-brushborder antibody might play a role in the pathogenesis of experimental glomerulopathy.
Assuntos
Anticorpos , Especificidade de Anticorpos , Nefrite/imunologia , Linfócitos T/imunologia , Animais , Soro Antilinfocitário/imunologia , Chinchila , Citotoxicidade Imunológica , Feminino , Imunofluorescência , Cobaias , Rim/imunologia , Fatores Inibidores da Migração de Macrófagos/farmacologia , Microvilosidades/imunologia , Coelhos , RatosRESUMO
Autologous immune complex glomerulopathy (AICG) is induced by immunizing rats with a crude brushborder fraction of rat kidney tubules (Fx1A) or with the purified GP 330 antigen. In these animals, anti-brushborder antibodies develop, leading to subepithelial immune complexes along the glomerular capillary wall. In rats with AICG, thymocytes sensitized against Fx1A as well as thymocytes recognizing anti-Fx1A are present. These latter cells might play a role in the specific tolerance against the pathogenetic antigen GP 330. To substantiate this notion, immunofluorescence studies were performed in which the number of anti-GP 330 binding cells was quantified in thymus cell suspensions of rats with AICG, in control rats and in naive rats with different genetic background. It is shown that increased numbers of anti-GP 330-binding thymocytes in rats with AICG are associated with a decline in the serum anti-brushborder titer. Furthermore, it appears that the number of anti-GP 330-binding thymocytes in naive rats of the non-responder Brown Norway strain is significantly higher as compared to the PVG/c and Lewis strains, which are susceptible for AICG. The correlation between the numbers of anti-GP 330-binding thymocytes and the susceptibility for AICG suggests a role for these cells in maintaining the tolerance against the Fx1A (GP 330) antigen.
Assuntos
Glomerulonefrite Membranosa/imunologia , Glicoproteínas de Membrana/imunologia , Linfócitos T/imunologia , Animais , Anticorpos , Autoantígenos/administração & dosagem , Feminino , Imunofluorescência , Glomerulonefrite Membranosa/etiologia , Complexo Antigênico da Nefrite de Heymann , Imunização , Microvilosidades , Proteinúria/etiologia , Ratos , Ratos EndogâmicosRESUMO
A novel coating solution for the improvement of biocompatibility of polyurethane-based vascular prostheses was tested in rabbits and rats in vivo. Segments of coated and uncoated vascular prostheses were implanted into the peritoneal cavity of rats, followed by induction of experimental haemorrhage; otherwise whole vascular prostheses were implanted in the carotid artery of rabbits using microsurgical procedures. While in both rats and rabbits, the uncoated material showed abundant formation of fibrinoid thrombi, clear reduction of thrombus formation was seen in all ADPase coated materials following implantation in vivo.
Assuntos
Apirase , Materiais Biocompatíveis , Prótese Vascular , Teste de Materiais , Poliuretanos , Trombose/prevenção & controle , Animais , Masculino , Neutrófilos/patologia , Coelhos , Ratos , Ratos Endogâmicos Lew , Trombose/patologiaRESUMO
Peripheral blood mononuclear cells (PBC) from non-nephrotic and nephrotic patients with different glomerulopathies were tested for their potential to produce vascular permeability increasing factor (VPF) after stimulation with Concanavalin-A (Con-A) in vitro. Supernatants from cultures of PBC from patients with IgA nephropathy were injected intradermally into the skins of normal Wistar rats which were given Evans blue dye solution intravenously. The mean extravasation of dye after 60 minutes was taken as a standard for the induction of local vascular permeability. Using a routine vascular permeability assay based on this principle similar studies were done with supernatants from cultures of PBC from nephrotic subjects with minimal change disease (MCD), or membranous nephropathy (MGN) and from healthy donors. The results show that cultures of PBC from non-nephrotic subjects with IgA nephropathy as well as from nephrotic MCD patients produced VPF in their supernatants whereas lymphocytes from nephrotic MGN subjects or normal donors did not. It is concluded that the production of VPF in stimulated PBC cultures from patients with IgA nephropathy or MCD might reflect altered T-cell function in these diseases, and that there is no direct relationship between VPF production and increased glomerular permeability.
Assuntos
Permeabilidade Capilar , Imunoglobulina A/análise , Nefropatias/sangue , Linfocinas/sangue , Nefrose Lipoide/sangue , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Humanos , Nefropatias/imunologia , Pessoa de Meia-Idade , Nefrose Lipoide/imunologia , Ratos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Ecto ATP-diphosphohydrolase (apyrase) activity of human endothelial cells following aspirin treatment has been studied in-vitro. It was shown by HPLC analysis of supernatant samples that pre-incubation of the cultures with aspirin resulted in a significantly increased turnover of supplemented ATP into its degradation products (ADP and AMP). Enhanced expression of ectoenzyme after aspirin treatment could be observed as demonstrated by immunofluorescence-staining with monoclonal anti-apyrase antibodies. This suggests enhancement of endothelial ATP-diphosphohydrolase activity induced by aspirin. The present data obtained in human vascular cells in-vitro are in line with results from previous animal studies in-vivo, suggesting a novel cyclooxygenase-independent antithrombotic activity of aspirin.
Assuntos
Apirase/metabolismo , Aspirina/farmacologia , Endotélio Vascular/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Fluoresceína-5-Isotiocianato/química , Humanos , Espectrometria de Fluorescência , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismoRESUMO
In this pilot study ADP-ase coated polyurethane (PU) vascular prostheses and noncoated (control) PU vascular prostheses (all vascular prostheses: ID 1.5 mm, length 1.5 cm) were implanted into the carotid artery of the rabbit to test whether ADP-ase might function as an adequate anti-thrombogenic coating. The prostheses were evaluated after 1 hour (n = 4) and 3 weeks (n = 8). After 1 hour, there was extensive accumulation of thrombus on the inner surface of the control PU vascular prostheses, in contrast to the ADP-ase coated prostheses. At 3 weeks, all control PU vascular prostheses (n = 8) were occluded, whereas only 1 of the 8 ADP-ase coated PU vascular prostheses. The patient ADP-ase coated PU prostheses showed already extensive endothelial healing. These results indicate the potential of ADP-ase to function as an effective antithrombogenic coating of small-caliber and microvascular PU prostheses.
Assuntos
Apirase , Prótese Vascular , Trombose das Artérias Carótidas/prevenção & controle , Oclusão de Enxerto Vascular/prevenção & controle , Poliuretanos , Animais , Masculino , Microscopia Eletrônica de Varredura , Projetos Piloto , Coelhos , Fatores de Tempo , Grau de Desobstrução Vascular/fisiologiaRESUMO
INTRODUCTION: Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant rats induced preeclampsia-like symptoms such as albuminuria and placental ischemia. We hypothesized that ATP inhibits trophoblast invasion and spiral artery remodeling and affects macrophages and natural killer (NK) cells present in the rat mesometrial triangle. METHODS: Pregnant rats were infused with ATP or saline (control) on day 14 of pregnancy. Rats were sacrificed on day 15, 17 or 20 of pregnancy and placentas with mesometrial triangle were collected. Sections were stained for trophoblast cells, α-smooth muscle actin (spiral artery remodeling), NK cells and various macrophage populations. Expression of various cytokines in the mesometrial triangle was analyzed using real-time RT-PCR. RESULTS: ATP infusion decreased interstitial trophoblast invasion on day 17 and spiral artery remodeling on day 17 and 20, increased activated tartrate resistant acid phosphatase (TRAP)-positive macrophages on day 15, decreased NK cells on day 17 and 20, and decreased inducible nitric oxide synthase (iNOS)-positive and CD206-positive macrophages and TNF-α and IL-33 expression at the end of pregnancy (day 20). DISCUSSION: Interstitial trophoblast invasion and spiral artery remodeling in the rat mesometrial triangle were decreased by infusion of ATP. These ATP-induced modifications were preceded by an increase in activated TRAP-positive macrophages and coincided with NK cell numbers, suggesting that they are involved. CONCLUSION: Trophoblast invasion and spiral artery remodeling may be inhibited by ATP-induced activated macrophages and decreased NK cells in the mesometrial triangle in rat pregnancy.
Assuntos
Trifosfato de Adenosina/fisiologia , Placentação , Prenhez/imunologia , Trofoblastos/fisiologia , Útero/imunologia , Trifosfato de Adenosina/administração & dosagem , Animais , Feminino , Interleucina-33 , Interleucinas/metabolismo , Células Matadoras Naturais/fisiologia , Macrófagos/fisiologia , Masculino , Gravidez , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismo , Útero/irrigação sanguínea , Útero/metabolismoRESUMO
INTRODUCTION: Poor placentation (disturbed and decreased trophoblast invasion) is a hallmark of preeclampsia (PE), which is a major complication of pregnancy. Unfortunately, the cause and mechanism of disturbed trophoblast invasion is still unknown. OBJECTIVES: The pro-inflammatory agent ATP has been shown to induce PE-like signs, after a single infusion in pregnant rats. These PE-like characteristics include proteinuria and decreased fetal weight. Since purinergic ATP receptors are expressed on trophoblast cells, we aimed to study the effect of ATP infusion on trophoblast invasion in pregnant rats in this pilot study. METHODS: Pregnant rats received a single ATP (n=4) or saline (control,ni=5) infusion via a permanent jugular vein cannula on day 14 of pregnancy. At the time of maximal trophoblast invasion (day 17 of pregnancy) rats were sacrificed and placentas with mesometrial triangle were collected, fixed in zinc-buffer and embedded in paraffin. 4 µm sections were stained with monoclonal α-cytokeratin antibodies. In the mesometrial triangle, the maternal part of the rat placenta, the percentage of surface area of trophoblast invasion was evaluated using computerized image analysis. Also, the depth and width of invasion were analyzed by subdividing the mesometrial triangle in three concentric depth levels of equal width. In addition, trophoblast invaded versus non-invaded spiral arteries in the mesometrial triangle were quantified. RESULTS: In the mesometrial triangle, no changes in percentage of surface area of trophoblast invasion and percentage of invaded spiral arteries were observed after ATP infusion. However, the pattern of trophoblast invasion appeared to be disturbed in ATP infused rats, with a decreased depth of invasion and an increased width of invasion, resulting in a trend towards a decreased depth/width ratio of trophoblast invasion in ATP infused rats. CONCLUSION: In this (pilot) study we showed an altered trophoblast invasion pattern in the mesometrial triangle of the placenta, although no significant differences in the total surface area of trophoblast invasion were seen in experimental versus control pregnant animals. e mechanism by which ATP induces this altered trophoblast invasion pattern and its potential contribution to the pathophysiology of this experimental PE in the pregnant rat awaits further investigation.