RESUMEN
BACKGROUND: Angiotensin-converting enzyme (ACE) downregulates the activity of bradykinin, a potent proinflammatory and immunostimulatory peptide liberated from an internal portion of kininogens. Here, we asked whether periodontitis is worsened in patients under antihypertensive treatment with ACE inhibitors. METHODS: Periodontal parameters were recorded from 30 individuals taking ACE inhibitors (case) and 35 taking a non-ACE inhibitor medication (control). Data were analyzed by nonparametric and parametric statistical tests. RESULTS: Most sociodemographic figures were similar in both groups. However, family income was statistically higher in the control group, and the percentage of sites with visible plaque (PL) was statistically higher in the case group (P = 0.043 and P = 0.005, respectively). The prevalence of individuals with chronic periodontitis varied from 31.5% in the control group to 63.4% in the case group (P = 0.001). Patients in the case group presented a 3.2-fold higher risk of having sites with pocket depth ≥5 mm and a 2.9-fold higher risk of having sites with clinical attachment loss ≥5 mm in comparison with those in the control group (P = 0.009 and P = 0.001, respectively; adjusted for family income and visible PL). CONCLUSION: Angiotensin-converting enzyme inhibitors may increase the prevalence and extent of chronic periodontitis in Brazilian patients.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/efectos adversos , Antihipertensivos/efectos adversos , Periodontitis Crónica/inducido químicamente , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Antihipertensivos/uso terapéutico , Estudios de Casos y Controles , Periodontitis Crónica/patología , Femenino , Bolsa Gingival/inducido químicamente , Bolsa Gingival/patología , Humanos , Hipertensión/tratamiento farmacológico , Masculino , Persona de Mediana EdadRESUMEN
C4-binding protein (C4-bp), a new component of the complement system, was isolated from human plasma by precipitation with polyethyleneglycol, followed by chromatography on ion exchangers. C4-bp was identified on sodium dodecyl- sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by two independent criteria: its ability to bind to C4b, and immunoprecipitation with a monospecificantiserum. Purified C4-bp is a 10.7 s glycoprotein. It consists of several disulfide bonded subunits of mol wt 70,000 daltons. Under nonreducing conditions, its mol wt has been estimated on SDS-PAGE as 540- 590,000 daltons. C4-bp moves as a slow B-globulin at pH 8.6 in the absence of free divalent cations, but when the buffers contain Ca(++)-lactate, C4-bp is a gamma globulin. Purified C4-bp binds to purified C4b. The reaction proceeds in the presence or absence of divalent cations and is not inhibited by diisopropylfluorophosphate. The C4b/C4-bp complexes have sedimentation coefficients between 15 and 17 s on sucrose gradient ultracentrifugation, and can be readily identified by crossed immunoelectrophoresis (CIE). The complexes move faster toward the anode than either protein. C4-bp is multivalent. Saturation is reached at molecular ratios of C4b/C4- bp of between 4 and 5. The interaction between C4b and C4-bp may complicate the electrophoretic patterns of these proteins in normal human serum, if the complement system is activated before or during the run. However, in EDTA-plasma, native C4 and C4-bp do not form stable complexes and can be identified in separate peaks after CIE.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Complemento C4/metabolismo , Glicoproteínas/aislamiento & purificación , Electroforesis de las Proteínas Sanguíneas , Proteínas Sanguíneas/aislamiento & purificación , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Humanos , Sueros Inmunes , Inmunoelectroforesis , UltracentrifugaciónRESUMEN
The parasitic protozoan Trypanosoma cruzi employs multiple molecular strategies to invade a broad range of nonphagocytic cells. Here we demonstrate that the invasion of human primary umbilical vein endothelial cells (HUVECs) or Chinese hamster ovary (CHO) cells overexpressing the B(2) type of bradykinin receptor (CHO-B(2)R) by tissue culture trypomastigotes is subtly modulated by the combined activities of kininogens, kininogenases, and kinin-degrading peptidases. The presence of captopril, an inhibitor of bradykinin degradation by kininase II, drastically potentiated parasitic invasion of HUVECs and CHO-B(2)R, but not of mock-transfected CHO cells, whereas the B(2)R antagonist HOE 140 or monoclonal antibody MBK3 to bradykinin blocked these effects. Invasion competence correlated with the parasites' ability to liberate the short-lived kinins from cell-bound kininogen and to elicit vigorous intracellular free calcium ([Ca(2+)](i)) transients through B(2)R. Invasion was impaired by membrane-permeable cysteine proteinase inhibitors such as Z-(SBz)Cys-Phe-CHN(2) but not by the hydrophilic inhibitor 1-trans-epoxysuccinyl-l-leucyl-amido-(4-guanidino) butane or cystatin C, suggesting that kinin release is confined to secluded spaces formed by juxtaposition of host cell and parasite plasma membranes. Analysis of trypomastigote transfectants expressing various cysteine proteinase isoforms showed that invasion competence is linked to the kinin releasing activity of cruzipain, herein proposed as a factor of virulence in Chagas' disease.
Asunto(s)
Endotelio Vascular/metabolismo , Endotelio Vascular/parasitología , Receptores de Bradiquinina/metabolismo , Trypanosoma cruzi/fisiología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Bradiquinina/análogos & derivados , Bradiquinina/antagonistas & inhibidores , Bradiquinina/farmacología , Antagonistas de los Receptores de Bradiquinina , Células CHO , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Captopril/farmacología , Células Cultivadas , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/patología , Enfermedad de Chagas/fisiopatología , Cricetinae , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Quininógenos/metabolismo , Cininas/metabolismo , Cininas/farmacología , Peptidil-Dipeptidasa A/metabolismo , Proteínas Protozoarias , Receptor de Bradiquinina B2 , Receptores de Bradiquinina/genética , Transfección , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/patogenicidad , Venas UmbilicalesRESUMEN
A prominent feature of the life cycle of intracellular parasites is the profound morphological changes they undergo during development in the vertebrate and invertebrate hosts. In eukaryotic cells, most cytoplasmic proteins are degraded in proteasomes. Here, we show that the transformation in axenic medium of trypomastigotes of Trypanosoma cruzi into amastigote-like organisms, and the intracellular development of the parasite from amastigotes into trypomastigotes, are prevented by lactacystin, or by a peptide aldehyde that inhibits proteasome function. Clasto-lactacystin, an inactive analogue of lactacystin, and cell-permeant peptide aldehyde inhibitors of T. cruzi cysteine proteinases have no effect. We have also identified the 20S proteasomes from T. cruzi as a target of lactacystin in vivo. Our results document the essential role of proteasomes in the stage-specific transformation of a protozoan.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Trypanosoma cruzi/crecimiento & desarrollo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/ultraestructura , Inhibidores de Cisteína Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Complejos Multienzimáticos/química , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/ultraestructura , Complejo de la Endopetidasa Proteasomal , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimologíaRESUMEN
The anti-hypertensive drug captopril is used commonly to reduce blood pressure of patients with severe forms of Chagas disease, a cardiomyopathy caused by chronic infection with the intracellular protozoan Trypanosoma cruzi. Captopril acts by inhibiting angiotensin-converting enzyme (ACE), the vasopressor metallopeptidase that generates angiotensin II and promotes the degradation of bradykinin (BK). Recent studies in mice models of Chagas disease indicated that captopril can potentiate the T helper type 1 (Th1)-directing natural adjuvant property of BK. Equipped with kinin-releasing cysteine proteases, T. cruzi trypomastigotes were shown previously to invade non-professional phagocytic cells, such as human endothelial cells and murine cardiomyocytes, through the signalling of G protein-coupled bradykinin receptors (B(2) KR). Monocytes are also parasitized by T. cruzi and these cells are known to be important for the host immune response during infection. Here we showed that captopril increases the intensity of T. cruzi infection of human monocytes in vitro. The increased parasitism was accompanied by up-regulated expression of ACE in human monocytes. While T. cruzi infection increased the expression of interleukin (IL)-10 by monocytes significantly, compared to uninfected cells, T. cruzi infection in association with captopril down-modulated IL-10 expression by the monocytes. Surprisingly, studies with peripheral blood mononuclear cells revealed that addition of the ACE inhibitor in association with T. cruzi increased expression of IL-17 by CD4(+) T cells in a B(2) KR-dependent manner. Collectively, our results suggest that captopril might interfere with host-parasite equilibrium by enhancing infection of monocytes, decreasing the expression of the modulatory cytokine IL-10, while guiding development of the proinflammatory Th17 subset.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Captopril/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Monocitos/efectos de los fármacos , Trypanosoma cruzi/inmunología , Adulto , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/parasitología , Linfocitos T CD4-Positivos/patología , Células Cultivadas , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/fisiopatología , Regulación de la Expresión Génica , Interacciones Huésped-Parásitos/efectos de los fármacos , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-10/biosíntesis , Interleucina-10/genética , Interleucina-17/genética , Interleucina-17/metabolismo , Masculino , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/parasitología , Monocitos/patología , Sistema Renina-Angiotensina/efectos de los fármacos , Balance Th1 - Th2 , Trypanosoma cruzi/patogenicidad , Virulencia/efectos de los fármacosRESUMEN
C4-binding protein (bp), a glycoprotein with specific binding affinity for the activated form of C4 (C4b), has recently been isolated from human serum and partially characterized. This report demonstrates that C4-bp is incorporated into soluble immune complexes after complement activation in vitro. The reaction requires Ca++ ions and the presence of C4 in serum. Immunopathological studies of various forms of glomerulonephritis revealed intense C4-bp deposition in glomeruli from patients with immune-complex type of pathogenesis. C4-bp deposition was in close correlation with that of C4. These observations, together with the in vitro association of C4-bp to immune complexes, support the notion that the deposits in glomeruli represent the local accumulation of immune complexes.
Asunto(s)
Complejo Antígeno-Anticuerpo , Proteínas Sanguíneas/inmunología , Proteínas Portadoras/inmunología , Complemento C4/metabolismo , Glicoproteínas/inmunología , Proteínas Portadoras/sangre , Técnica del Anticuerpo Fluorescente , Glomerulonefritis/inmunología , Glicoproteínas/sangre , Humanos , Enfermedades del Complejo Inmune/inmunología , Técnicas In VitroRESUMEN
Plasma albumin reacts with nitric oxide (NO) to form the bioactive adduct, S-nitroso-albumin (S-NO-albumin). The limited intracellular access of S-NO-albumin suggests the need for a vascular transfer mechanism of NO from a large plasma S-NO-albumin pool to effect biologic function. To study the role of low molecular weight (LMW) thiols in NO transfer in vivo, we administered intravenous S-NO-albumin (1-300 nmol/kg) to rabbits before and after an intravenous infusion of L-cysteine or N-acetyl-L-cysteine. S-NO-albumin produced dose-dependent hypotension that was significantly augmented by prior infusion of either LMW thiol. LMW thiol infusion significantly accelerated the rate of onset and reduced the duration of action of the hypotension induced by S-NO-albumin. The hemodynamic effects of S-NO-albumin after pretreatment with LMW thiols were mimicked by administration of the corresponding LMW S-nitrosothiol. The transfer of NO from albumin to L-cysteine was directly measured in rabbit plasma using a novel technique that couples high performance liquid chromatography to electrochemical detection. These data demonstrate that NO exchange between plasma protein thiol-bound NO and available LMW thiol pools (transnitrosation) occurs in vivo.
Asunto(s)
Mercaptoetanol , Óxido Nítrico/metabolismo , Compuestos Nitrosos/sangre , Compuestos Nitrosos/metabolismo , S-Nitrosotioles , Albúmina Sérica Bovina/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Acetilcisteína/administración & dosificación , Acetilcisteína/análogos & derivados , Acetilcisteína/metabolismo , Acetilcisteína/farmacología , Animales , Transporte Biológico , Presión Sanguínea/efectos de los fármacos , Cromatografía Líquida de Alta Presión/métodos , Cisteína/administración & dosificación , Cisteína/análogos & derivados , Cisteína/sangre , Cisteína/metabolismo , Cisteína/farmacología , Nitrosación , Compuestos Nitrosos/administración & dosificación , Compuestos Nitrosos/farmacología , Unión Proteica , Conejos , Compuestos de Sulfhidrilo/sangre , Resistencia Vascular/efectos de los fármacosRESUMEN
Recent evidence suggests that sulfhydryl species can react with oxides of nitrogen under physiologic conditions and thereby stabilize endothelium-derived relaxing factor (EDRF) activity, but the presence of a specific in vivo thiol carrier for nitric oxide (NO) remains controversial. The single free sulfhydryl of serum albumin is the most abundant thiol species in plasma (approximately 0.5 mM) and is particularly reactive towards NO. To examine the potential role of serum albumin in endogenous nitric oxide metabolism, we synthesized S-nitroso-BSA (S-NO-BSA), a model S-nitroso-protein, and examined its effects on platelet function and coronary and systemic vascular tone in 16 mongrel dogs. Intravenous bolus S-NO-BSA markedly reduced mean arterial pressure in a dose-dependent manner and proved seven and a half-fold less potent than intravenous nitroglycerin and 10-fold less potent than intravenous S-nitroso-cysteine (half-maximal response of 75 nmol/kg compared to 10 and 7.5 nmol/kg, respectively; P < 0.05); when given by intravenous infusion (half-maximal response = 10 nmol/kg per min), however, S-NO-BSA and nitroglycerin were equipotent. Intravenous bolus S-NO-BSA had a greater duration of action than either nitroglycerin or S-nitroso-cysteine and produced marked prolongation of the template bleeding time associated with dose-dependent inhibition of ex vivo platelet aggregation (half-maximal response approximately 70 nmol/kg). Intracoronary S-NO-BSA increased coronary blood flow (mean +/- SEM) less effectively than nitroprusside, acetylcholine, or S-nitroso-cysteine (165% +/- 24% vs. 315% +/- 82%, 483% +/- 55%, or 475% +/- 66%, respectively; P < 0.05) although with much longer duration of action. On a molar basis, S-nitroso-cysteine proved more effective than S-nitroso-BSA, nitroprusside, or acetylcholine as an epicardial coronary vasodilator. Thus, serum albumin reacts with oxides of nitrogen to form a stable S-nitroso-thiol with properties reminiscent of authentic EDRF supporting the view that protein associated thiol may participate in the action and metabolism of EDRF.
Asunto(s)
Óxido Nítrico/metabolismo , Óxido Nítrico/fisiología , S-Nitrosotioles , Albúmina Sérica Bovina/metabolismo , Acetilcolina/farmacología , Animales , Tiempo de Sangría , Plaquetas/fisiología , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/fisiología , Cisteína/análogos & derivados , Cisteína/farmacología , Perros , Femenino , Semivida , Masculino , Relajación Muscular/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Óxido Nítrico/farmacología , Nitroglicerina/farmacología , Nitroprusiato/farmacología , Agregación Plaquetaria/efectos de los fármacos , Albúmina Sérica Bovina/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
Emerging evidence suggests a role for purinergic signaling in the activation of multiprotein intracellular complexes called inflammasomes, which control the release of potent inflammatory cytokines, such as interleukin (IL) -1ß and -18. Porphyromonas gingivalis is intimately associated with periodontitis and is currently considered one of the pathogens that can subvert the immune system by limiting the activation of the NLRP3 inflammasome. We recently showed that P. gingivalis can dampen eATP-induced IL-1ß secretion by means of its fimbriae in a purinergic P2X7 receptor-dependent manner. Here, we further explore the role of this purinergic receptor during eATP-induced IL-1ß processing and secretion by P. gingivalis-infected macrophages. We found that NLRP3 was necessary for eATP-induced IL-1ß secretion as well as for caspase 1 activation irrespective of P. gingivalis fimbriae. Additionally, although the secretion of IL-1ß from P. gingivalis-infected macrophages was dependent on NLRP3, its adaptor protein ASC, or caspase 1, the cleavage of intracellular pro-IL-1ß to the mature form was found to occur independently of NLRP3, its adaptor protein ASC, or caspase 1. Our in vitro findings revealed that P2X7 receptor has a dual role, being critical not only for eATP-induced IL-1ß secretion but also for intracellular pro-IL-1ß processing. These results were relevant in vivo since P2X7 receptor expression was upregulated in a P. gingivalis oral infection model, and reduced IFN-γ and IL-17 were detected in draining lymph node cells from P2rx7(-/-) mice. Furthermore, we demonstrated that P2X7 receptor and NLRP3 transcription were modulated in human chronic periodontitis. Overall, we conclude that the P2X7 receptor has a role in periodontal immunopathogenesis and suggest that targeting of the P2X7/NLRP3 pathway should be considered in future therapeutic interventions in periodontitis.
Asunto(s)
Infecciones por Bacteroidaceae/metabolismo , Porphyromonas gingivalis/patogenicidad , Receptores Purinérgicos P2X7/fisiología , Animales , Infecciones por Bacteroidaceae/microbiología , Proteínas Portadoras/fisiología , Caspasa 1/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cysteine peptidase inhibitor genes (ICP) of the chagasin family have been identified in protozoan (Leishmania mexicana and Trypanosoma brucei) and bacterial (Pseudomonas aeruginosa) pathogens. The encoded proteins have low sequence identities with each other and no significant identity with cystatins or other known cysteine peptidase inhibitors. Recombinant forms of each ICP inhibit protozoan and mammalian clan CA, family C1 cysteine peptidases but do not inhibit the clan CD cysteine peptidase caspase 3, the serine peptidase trypsin or the aspartic peptidases pepsin and thrombin. The functional homology between ICPs implies a common evolutionary origin for these bacterial and protozoal proteins.
Asunto(s)
Proteínas Bacterianas/farmacología , Inhibidores de Cisteína Proteinasa/genética , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas Protozoarias/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Eucariontes/enzimología , Evolución Molecular , Leishmania major/genética , Leishmania mexicana/genética , Mamíferos , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
A novel immunoassay (PINC-ELISA) was designed using proteinase inhibitors of the cystatin superfamily (PINC) in the solid phase, to promote the selective capture of cysteine proteinases. The method was applied in the identification of papain-like antigens from parasitic protozoa. PINC of human origin, namely recombinant cystatin C (r-cystatin C) or low molecular weight kininogen were used in the assays to adsorb proteases contained in cell lysates from various trypanosomatids. The PINC-ELISA was at first optimized with the major cysteine proteinase from Trypanosoma cruzi (known as GP57/51 or cruzipain), an antigen whose serodiagnostic properties were previously established. Cruzipain is selectively adsorbed from crude extracts of T. cruzi onto PINC-coated wells; the finding that antibodies bind to epitopes located away from the sites of interaction with r-cystatin or low molecular weight kininogen has allowed for the screening of antibodies in chagasic sera, the methodology being advantageous in that it dispensed prior purification of the proteinase antigen. The PINC-ELISA was then carried out with lysates originating from Leishmania m. amazonensis (amastigotes) or Leishmania donovani (promastigotes). Complexes between solid-phase r-cystatin C and antigenic ligands in the lysates were again detected. The Leishmania molecules which bound to r-cystatin C, were respectively recognized by serum antibodies from mice chronically infected with L. amazonensis or from patients with visceral leishmaniasis. Direct evidence for the presence of cysteine proteinases in lysates from L. donovani was then obtained, using synthetic fluorogenic substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Cistatinas/metabolismo , Cisteína Endopeptidasas/análisis , Animales , Anticuerpos Monoclonales/inmunología , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Leishmania/enzimología , Ratones , Pruebas de Precipitina , Trypanosoma cruzi/enzimologíaRESUMEN
Purified GP57/51, a Trypanosoma cruzi glycoprotein earlier identified as a major antigen in infected humans, was subjected to N-terminal sequence analysis. Alignment of the first 30 amino acids revealed that its N-terminal region is virtually identical to that reported for a cysteine-proteinase isolated from the Tulahuen strain, including the presence of active site cysteine at position 25. The finding of serine at position 24 of GP57/51 (Y strain) has further increased the homology between this protozoan antigen with other members of the eukaryotic family of cysteine proteases, including human cathepsin L. Functional analysis of GP57/51 indicated that the antigen is indeed an active thiol proteinase, which is active across a wide pH range (5-7.5). This was shown using either human IgG or gelatin substrates co-polymerized into polyacrylamide gels prepared for electrophoresis, and also by enzyme assays peformed with the synthetic substrate Z-phe-arg-NMec. The enzyme was activated by thiol containing reagents, and was strongly inhibited by low concentrations of E-64 (IC50 0.1 microM), cystatin (IC50 1 microM), leupeptin (IC50 0.1 microM) and antipain (IC50 0.1 microM). Monoclonal antibodies directed against distinct epitopes of GP57/51 absorbed the hydrolytic activity from purified preparations, demonstrating that the antigenic and enzymatic activities were indeed expressed by the same molecular entities. The subcellular localization of immunoreactive molecules was investigated by electron microscopy; immunogold staining was conspicuously found in vesicles belonging to the endosomal-lysosomal system, in tissue culture trypomastigotes as well as in epimastigotes. The possibility that this highly antigenic protease is actively secreted and/or leaked out of damaged parasites is under investigation; its release to tissues and to the circulation may contribute to pathology, considering that it (i) can degrade proteins across a wide pH range and (ii) stimulates immune T cells from chronic chagasic patients.
Asunto(s)
Antígenos de Protozoos/química , Cisteína Endopeptidasas/química , Trypanosoma cruzi/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Antígenos de Protozoos/análisis , Antígenos de Protozoos/aislamiento & purificación , Antígenos de Protozoos/metabolismo , Cisteína Endopeptidasas/metabolismo , Electroforesis en Gel de Poliacrilamida , Epítopos , Concentración de Iones de Hidrógeno , Lisosomas/enzimología , Lisosomas/inmunología , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/ultraestructuraRESUMEN
The therapeutic potential of synthetic inhibitors to the major cysteine-proteinase from Trypanosoma cruzi (cruzain or cruzipain) was recently demonstrated in animal models of Chagas' disease. A possible limitation of this strategy would be the emergence of parasite populations developing resistance to cysteine-proteinase inhibitors. Here, we describe the properties of a phenotypically stable T. cruzi cell line (R-Dm28) that displays increased resistance to Z-(SBz)Cys-Phe-CHN2, an irreversible cysteine-proteinase inhibitor which preferentially inactivates cathepsin L-like enzymes. Isolated from axenic cultures of the parental cells (IC50 1.5 microM), R-Dm28 epimastigotes exhibited 13-fold (IC50) 20 microM) higher resistance to this inhibitor and did not display cross-resistance to unrelated trypanocidal drugs, such as benznidazol and nifurtimox. Western blotting (with mAb), affinity labeling (with biotin-LVG-CHN2) and FACS analysis of R-Dm28 log-phase epimastigotes revealed that the cruzipain target was expressed at lower levels, as compared with Dm28c. Interestingly, this deficit was paralleled by increased expression of an unrelated Mr 30 000 cysteine-proteinase whose activity was somewhat refractory to inhibition by Z-(SBz)Cys-Phe-CHN,. N-terminal sequencing of the affinity-purified biotin-LVG-proteinase complex allowed its identification as a cathepsin B-like enzyme. Increased antigenic deposits of this proteinase were found in the grossly enlarged and electron dense reservosomes from R-Dm28 epimastigotes. Our data suggest that R-Dm28 resistance to toxic effects induced by the synthetic inhibitor may result from decreased availability of the most sensitive cysteine-proteinase target, cruzipain. The deficit in metabolic functions otherwise mediated by this cathepsin L-like proteinase is likely compensated by increased expression/accumulation of a cathepsin B-like target.
Asunto(s)
Catepsina B/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Antígenos de Protozoos/farmacología , Catepsina B/análisis , Línea Celular , Cisteína Endopeptidasas/análisis , Farmacorresistencia Microbiana , Citometría de Flujo , Glicoproteínas/farmacología , Immunoblotting , Inmunohistoquímica , Nifurtimox/farmacología , Nitroimidazoles/farmacología , Proteínas Protozoarias , Tripanocidas/farmacología , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/metabolismoRESUMEN
Cysteine-proteinases from parasitic protozoa have been recently characterized as factors of virulence and pathogenicity in several human and veterinary diseases. In Chagas' disease, the chronic infection caused by Trypanosoma cruzi, structure-functional studies on cysteine proteases were thus far limited to the parasite's major isoform, a cathepsin L-like lysosomal protease designated as cruzipain, cruzain or GP57/51. Encoded by a large gene family, cruzipain is efficiently targeted by synthetic inhibitors, which prevent parasite intracellular growth and differentiation. We have previously demonstrated that the multicopy cruzipain gene family includes polymorphic sequences, which could encode functionally different isoforms. We report here a comparative kinetic study between cruzain, the archetype of the cruzipain family, and an isoform, termed cruzipain 2, which is expressed preferentially by the mammalian stages of T. cruzi. Heterologous expression of the catalytic domain of cruzipain 2 in Saccharomyces cerevisae yielded an enzyme that differs markedly from cruzain with respect to pH stability, substrate specificity and sensitivity to inhibition by natural and synthetic inhibitors of cysteine proteases. We suggest that the structural-functional diversification imparted by genetic polymorphism of cruzipain genes may have contributed to T. cruzi adaptation to vertebrate hosts.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Lisosomas/enzimología , Datos de Secuencia Molecular , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Trypanosoma cruzi/genéticaRESUMEN
Peptidyl diazomethane (PDAM) derivatives, a class of irreversible inhibitors for cysteine proteinase, were screened for the ability to impair Trypanosoma cruzi invasion and intracellular development in primary cultures of heart muscle cells (HMC). T. cruzi GP57/51, a purified cysteinyl proteinase, and the substrate Z-Phe-Arg-NHMec were used to determine inhibition rate constants (k'+2) by continuous kinetic assays. The k'+2 values ranged from 25,400 to 2,800. The best inhibitors of GP57/51 had bulky hydrophobic residues in the P1 position (in addition to P2), the S1 sub-site specificity of the enzyme being thus similar to mammalian cathepsin L. The effects of these PDAM on parasite infectivity were then investigated. The ability to invade HMC was markedly impaired when trypomastigotes were briefly exposed to 10 microM of Z-(S-Bzl)Cys-Phe-CHN2. Striking effects were observed when PDAM were added to HMC cultures that had been previously infected with trypomastigotes: Z-(S-Bzl)Cys-Phe-CHN2 with an IC50 of 0.4 microM, and less markedly Z-Phe-Phe-CHN2 and Z-Tyr-Phe-CHN2 (or Z-Phe-Tyr-CHN2) blocked amastigote replication as well as their transformation into trypomastigotes, thereby arresting intracellular development. Bz-Phe-Gly-CHN2, in contrast, failed to display antiparasite activity. Direct characterization of the target cysteinyl proteinase was sought, by incubating viable amastigotes or infected HMC with Z-[125I]Tyr-Phe-CHN2. Affinity labeling implicated GP57/51 as the major cysteinyl proteinase target for this probe. We propose that T. cruzi intracellular development is critically dependent on GP57/51 (cruzipain). Selective inhibitors for this cysteinyl proteinase may have therapeutic potential.
Asunto(s)
Antígenos de Protozoos/efectos de los fármacos , Cisteína Endopeptidasas/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Glicoproteínas/efectos de los fármacos , Trypanosoma cruzi/enzimología , Animales , Antígenos de Protozoos/metabolismo , Células Cultivadas , Cisteína Endopeptidasas/metabolismo , Glicoproteínas/metabolismo , Immunoblotting , Cinética , Ratones , Proteínas Protozoarias , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/patogenicidadRESUMEN
Thrombin activity is increased in the setting of acute myocardial infarction (AMI) and has been shown to increase further after the administration of thrombolytic therapy for acute infarction. This increase in thrombin activity may play an important role in the 15% to 25% rate of failure to achieve initial reperfusion and in the 5% to 15% rate of early reocclusion after initially successful thrombolysis. To investigate potential mechanisms of thrombin formation in vivo, to understand better the balance of coagulation and fibrinolysis during treatment with recombinant tissue-type plasminogen activator (rt-PA), and to investigate the role of hemostatic markers as predictors of clinical events, we measured 3 markers of procoagulant activity: fibrinopeptide A (FPA), thrombin-antithrombin III complexes (TAT), and prothrombin fragment 1.2 (F1.2), and a marker of fibrinogenolytic activity (B beta 1-42) in patients enrolled in the Thrombolysis in Myocardial Infarction (TIMI)-5 study. This trial was a randomized, dose-ranging, pilot trial of hirudin versus heparin as adjunctive antithrombotic therapy with rt-PA administered to patients with AMI. Correlation of markers at 1 hour with clinical outcomes revealed that increased FPA and TAT levels were associated with increased mortality and TIMI grades 0, 1, or 2 flow at 90 minutes; increased F1.2 levels were associated with TIMI grade 0 or 1 flow at 90 minutes; and increased levels of all 3 procoagulant markers were associated with hemorrhagic events. Late (12 to 24 hours) increases in F1.2, TAT, and B beta 1-42 may be predictive of recurrent ischemia. These results suggest that selected markers of procoagulant and fibrinogenolytic activity may be useful in predicting clinical outcomes in patients treated with thrombolytic therapy for AMI.
Asunto(s)
Antitrombina III/análisis , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Fibrinopéptido A/análisis , Heparina/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Fragmentos de Péptidos/análisis , Péptido Hidrolasas/análisis , Protrombina/análisis , Terapia Trombolítica , Biomarcadores , Fibrinolíticos/uso terapéutico , Hemostasis , Terapia con Hirudina , Humanos , Proyectos Piloto , Proteínas Recombinantes , Resultado del TratamientoRESUMEN
Development of a highly specific test system for the diagnosis of Chagas' disease (CD) was sought using Gp25, a surface glycoprotein recently isolated from Trypanosoma cruzi culture forms. Radioimmunoprecipitation assays were performed to screen 567 sera for IgG antibodies to Gp25. Correct diagnosis was attained in 97.8% of the 321 sera collected from chagasic patients in several endemic areas of South America. Sera from patients with various clinical forms of chronic disease displayed similar levels of antibodies (Abs) to Gp25. Moreover, there was little cross-reactivity when assayed against 246 sera from non-chagasic individuals, including 105 samples from individuals infected with unrelated trypanosomatids. Cross-reactivity was found in two such sera; these were used to identify a minor protein contaminant as the nonspecific antigen. Residual cross-reactive molecules were eliminated from Gp25 by affinity purification on Concanavalin A (Con A) columns. We recommend this antigen-antibody system for use in routine screening of blood donors.
Asunto(s)
Antígenos de Protozoos/inmunología , Antígenos de Superficie/inmunología , Enfermedad de Chagas/diagnóstico , Inmunoglobulina G/análisis , Trypanosoma cruzi/inmunología , Adulto , Antígenos de Protozoos/aislamiento & purificación , Antígenos de Superficie/aislamiento & purificación , Cardiomiopatía Chagásica/diagnóstico , Enfermedad de Chagas/inmunología , Reacciones Cruzadas , Humanos , Leishmaniasis/inmunología , Persona de Mediana Edad , Radioinmunoensayo , Tripanosomiasis Africana/inmunologíaRESUMEN
With the exception of assays for the detection of antibodies promoting complement-mediated lysis of Trypanosoma cruzi, serologic tests have generally failed to assess the effectiveness of chemotherapy for Chagas' disease. Conventional serology, although useful for the diagnosis of infection, is not capable of determining which patients have been cured. Here we demonstrate that a high proportion of antibodies detected by conventional serology (using fixed epimastigotes or trypomastigotes or crude extracts obtained therefrom) are directed against the carbohydrate residue galactosyl alpha 1- > 3 galactose (Gal alpha 1- > 3 Gal), a determinant also recognized by antibodies from noninfected healthy volunteers. In a study of 14 cured patients with long-term followup, we found that the persistently positive reactions detected using conventional serology were largely eliminated following immunoadsorption with melibiose. Because of their wide distribution among microorganisms of intestinal and pulmonary microflora, these carbohydrate determinants may keep stimulating lymphocytes previously primed by T. cruzi Gal alpha 1- > 3 Gal epitopes, thereby accounting for false-positive results in cured patients. Consistent with this proposition, enzyme-linked immunosorbent assays performed with two distinct T. cruzi antigen preparations that lack the Gal alpha 1- > 3 Gal epitope, namely purified GP57/51 and trypomastigote-shed antigens, were indeed capable of determining a cure after chemotherapy, albeit to a different degree. Collectively, the data indicate that conventional immunoassays prepared with highly specific T. cruzi antigens can be useful in the assessment of a cure after chemotherapy.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Enfermedad de Chagas/diagnóstico , Glicoproteínas/inmunología , Trypanosoma cruzi/inmunología , Enfermedad Aguda , Adsorción , Adulto , Animales , Especificidad de Anticuerpos , Enfermedad de Chagas/tratamiento farmacológico , Niño , Enfermedad Crónica , Reacciones Cruzadas , Cisteína Endopeptidasas , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Nifurtimox/uso terapéutico , Nitroimidazoles/uso terapéutico , Resultado del Tratamiento , Tripanocidas/uso terapéutico , Trypanosoma cruzi/químicaRESUMEN
OBJECTIVE: To project the cost-effectiveness of fluconazole for prophylaxis against AIDS-related primary systemic fungal infections. DESIGN: A Markov model with data from the literature. PATIENTS: Hypothetical cohort of 100,000 AIDS patients. INTERVENTION: No prophylaxis, and fluconazole prophylaxis beginning when a patient's CD4 count declined to below 200/mm3, below 100/mm3, or below 50/mm3. RESULTS: The no-prophylaxis policy was associated with a discounted life expectancy of 28.20 months and direct medical costs of $36,100 per person. The < 200/mm3 strategy increased costs to $40,500 and life expectancy to 28.42 months, producing a ratio of $240,000 per year of life saved (YLS). Compared with the no-prophylaxis and < 200/mm3 policies, the intermediate alternatives were less economically efficient. A reduction in fluconazole's cost from $206 to $80 decreased the ratio to $50,000 for the < 200/mm3 strategy. Doubling fungal infection incidence lowered this ratio to $96,000/YLS. CONCLUSIONS: Fluconazole prophylaxis is unlikely to be cost-effective unless its cost is lowered, or it is focused on patients in regions endemic for fungal infections.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Antifúngicos/uso terapéutico , Costos de los Medicamentos , Fluconazol/uso terapéutico , Micosis/prevención & control , Infecciones Oportunistas Relacionadas con el SIDA/economía , Infecciones Oportunistas Relacionadas con el SIDA/mortalidad , Antifúngicos/economía , Recuento de Linfocito CD4 , Análisis Costo-Beneficio , Fluconazol/economía , Humanos , Cadenas de Markov , Modelos Biológicos , Modelos Teóricos , Micosis/economía , Micosis/mortalidad , Análisis de Supervivencia , Estados Unidos/epidemiologíaRESUMEN
BACKGROUND: Disagreement exists among decision makers regarding the allocation of limited HIV patient care resources and, specifically, the comparative value of preventing opportunistic infections in late-stage disease. METHODS: A Monte Carlo simulation framework was used to evaluate a state-transition model of the natural history of HIV illness in patients with CD4 counts below 300/mm3 and to project the costs and consequences of alternative strategies for preventing AIDS-related complications. The authors describe the model and demonstrate how it may be employed to assess the cost-effectiveness of oral ganciclovir for prevention of cytomegalovirus (CMV) infection. RESULTS: Ganciclovir prophylaxis confers an estimated additional 0.7 quality-adjusted month of life at a net cost of $10,700, implying an incremental cost-effectiveness ratio of roughly $173,000 per quality-adjusted life year gained. Sensitivity analysis reveals that this baseline result is stable over a wide range of input data estimates, including quality of life and drug efficacy, but it is sensitive to CMV incidence and drug price assumptions. CONCLUSIONS: The Monte Carlo simulation framework offers decision makers a powerful and flexible tool for evaluating choices in the realm of chronic disease patient care. The authors have used it to assess HIV-related treatment options and continue to refine it to reflect advances in defining the pathogenesis and treatment of AIDS. Compared with alternative interventions, CMV prophylaxis does not appear to be a cost-effective use of scarce HIV clinical care funds. However, targeted prevention in patients identified to be at higher risk for CMV-related disease may warrant consideration.