RESUMEN
An antigenic conserved B domain was previously identified within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, the r-potDomain B, a 6× His-tag polypeptide belonging to the conserved B domain from the potato apyrase, and synthetic peptides LbB1LJ and LbB2LJ derived from the B domain from Leishmania NTPDase 1 were used as molecular tools for studies of the Leishmania amazonensis NTPDase 1. Widespread subcellular location of the specific NTPDase 1 was detected by Western blots of promastigote fractions and ultrastructural immunocytochemical microscopy using immune sera raised against these biomolecules. In addition, the L. amazonensis-infected BALB/c mice were evaluated at 12 to 120 days after infection, which progresses showing typical nodular lesion. High antibody reactivity with either r-potDomain B, LbB1LJ, or LbB2LJ was found in L. amazonensis-infected BALB/c mice indicating the antigenicity of the B domain from NTPDase 1 isoform. The IgG1 antibody reactivity significantly increased at 90-120 days postinfection, 18- to 24-fold when compared to the 12th day, and remained elevated even at 120th after infection, coinciding with the most active stage of the disease. In contrast, significantly higher IgG2a antibody reactivity with each biomolecule was observed at 40th day, about two- to fourfold higher than those found at 12th or 20th day, and decreased along 120-day period. Apparently, the conserved B domain is capable to induce IgG2a production in early disease stages. All together, these results suggest that r-potDomain B or synthetic peptides could be molecular starting points in experimental protocols of immunotherapy and/or vaccination for leishmaniasis.
Asunto(s)
Antígenos CD/metabolismo , Apirasa/metabolismo , Leishmania/enzimología , Leishmaniasis/parasitología , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Antígenos de Protozoos , Apirasa/genética , Regulación Enzimológica de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Anotación de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
An ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83% ATPase and 87% of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1:50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80% seropositivity) and its potato apyrase counterpart (50% seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response.
Asunto(s)
Apirasa/metabolismo , Leishmania braziliensis/enzimología , Leishmaniasis Cutánea/parasitología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/sangre , Apirasa/genética , Apirasa/inmunología , Western Blotting , Humanos , Inmunoprecipitación , Isoenzimas , Leishmania braziliensis/inmunología , Leishmania braziliensis/ultraestructura , Leishmaniasis Cutánea/inmunología , Microscopía Electrónica , Datos de Secuencia MolecularRESUMEN
In order to unravel the physiopathology of leishmaniasis in humans, it is necessary to better understand how Leishmania are able to survive for years within immunologically active granulomas. In the present study, we used a macaque (Macaca mulatta) model of infection with Leishmania braziliensis as a means of assessing the usefulness of this primate system. This model more closely mirrors human protective immunity to Leishmania than the murine model; therefore, we used it to study the host inflammatory granulomatous response involved in the control of cutaneous leishmaniasis. Infected primates developed localized long-term skin ulcerations, but complete spontaneous clinical healing occurred in all infected animals. The infection induced the recruitment and activation of inflammatory mast cells, granulocytes, mononuclear phagocytes, and lymphocytes at the site of infection. During the acute reaction, polymorphonuclear leukocytes were more prominent than other cell types and apparently destroyed many parasites; macrophages then rapidly engulfed dying neutrophils together with their parasitic cargo. In the chronic phase, persisting parasites induced a typical T helper (Th) cytokine, type 1-mediated, immunity-induced granulomatous reaction. By this time, more or less differentiated macrophage accumulations were found, and these evolved to become mature tissue granulomas consisting of all the specific cell types found within human granulomas. In the healing stage, fibroblasts proliferated at the periphery and finally invaded the granulomas with fibrotic substitution. These findings point to the feasibility of using this model to elucidate the potentially disabling Th1-cell mechanisms that may eventually render the host granulomatous response inadequate for fighting L. braziliensis infections.
Asunto(s)
Leishmania braziliensis/fisiología , Leishmaniasis Cutánea/inmunología , Macaca mulatta/inmunología , Enfermedades de los Monos/parasitología , Piel/inmunología , Piel/parasitología , Animales , Antígenos de Protozoos/análisis , Quimiotaxis de Leucocito , Citometría de Flujo , Granuloma/inmunología , Granuloma/parasitología , Inmunofenotipificación , Leishmania braziliensis/ultraestructura , Recuento de Leucocitos , Macaca mulatta/parasitología , Masculino , Microscopía Electrónica , Modelos Animales , Enfermedades de los Monos/inmunología , Linfocitos T/inmunologíaRESUMEN
The nerve biopsies of 11 patients with pure neuritic leprosy were submitted to routine diagnostic procedures and immunoperoxidase staining with antibodies against axonal (neurofilament, nerve growth factor receptor (NGFr), and protein gene product (PGP) 9.5) and Schwann cell (myelin basic protein, S-100 protein, and NGFr) markers. Two pairs of non-adjacent histological cross-sections of the peripheral nerve were removed for quantification. All the fascicles of the nerve were examined with a 10X-ocular and 40X-objective lens. The immunohistochemistry results were compared to the results of semithin section analysis and clinical and electroneuromyographic data. Neurofilament staining was reduced in 100% of the neuritic biopsies. NGFr positivity was also reduced in 81.8%, PGP staining in 100% of the affected nerves, S100 positivity in 90.9%, and myelin basic protein immunoreactivity in 90.9%. Hypoesthesia was associated with decreased NGFr (81.8%) and PGP staining (90.9%). Reduced potential amplitudes (electroneuromyographic data) were found to be associated with reduced PGP 9.5 (63.6%) and nerve fiber neurofilament staining (45.4%) by immunohistochemistry and with loss of myelinated fibers (100%) by semithin section analysis. On the other hand, the small fibers (immunoreactive dots) seen amid inflammatory cells continued to be present even after 40% of the larger myelinated fibers had disappeared. The present study shows an in-depth view of the destructive effects of leprosy upon the expression of neural markers and the integrity of nerve fiber. The association of these structural changes with the clinical and electroneuromyographic manifestations of leprosy peripheral neuropathy was also discussed.
Asunto(s)
Antígenos Bacterianos/análisis , Glucolípidos/análisis , Lepra/diagnóstico , Mycobacterium leprae/inmunología , Fibras Nerviosas Mielínicas/patología , Proteínas del Tejido Nervioso/análisis , Neuritis/diagnóstico , Adulto , Antígenos Bacterianos/inmunología , Biomarcadores/análisis , Biopsia , ADN Bacteriano/análisis , Electromiografía , Femenino , Glucolípidos/inmunología , Humanos , Técnicas para Inmunoenzimas , Inmunohistoquímica , Lepra/patología , Masculino , Mycobacterium leprae/genética , Proteína Básica de Mielina/análisis , Neuritis/patología , Proteínas de Neurofilamentos/análisis , Reacción en Cadena de la Polimerasa , Receptores de Factor de Crecimiento Nervioso/análisis , Proteínas S100/análisisRESUMEN
The nerve biopsies of 11 patients with pure neuritic leprosy were submitted to routine diagnostic procedures and immunoperoxidase staining with antibodies against axonal (neurofilament, nerve growth factor receptor (NGFr), and protein gene product (PGP) 9.5) and Schwann cell (myelin basic protein, S-100 protein, and NGFr) markers. Two pairs of non-adjacent histological cross-sections of the peripheral nerve were removed for quantification. All the fascicles of the nerve were examined with a 10X-ocular and 40X-objective lens. The immunohistochemistry results were compared to the results of semithin section analysis and clinical and electroneuromyographic data. Neurofilament staining was reduced in 100 percent of the neuritic biopsies. NGFr positivity was also reduced in 81.8 percent, PGP staining in 100 percent of the affected nerves, S100 positivity in 90.9 percent, and myelin basic protein immunoreactivity in 90.9 percent. Hypoesthesia was associated with decreased NGFr (81.8 percent) and PGP staining (90.9 percent). Reduced potential amplitudes (electroneuromyographic data) were found to be associated with reduced PGP 9.5 (63.6 percent) and nerve fiber neurofilament staining (45.4 percent) by immunohistochemistry and with loss of myelinated fibers (100 percent) by semithin section analysis. On the other hand, the small fibers (immunoreactive dots) seen amid inflammatory cells continued to be present even after 40 percent of the larger myelinated fibers had disappeared. The present study shows an in-depth view of the destructive effects of leprosy upon the expression of neural markers and the integrity of nerve fiber. The association of these structural changes with the clinical and electroneuromyographic manifestations of leprosy peripheral neuropathy was also discussed.
Asunto(s)
Adulto , Femenino , Humanos , Masculino , Antígenos Bacterianos/análisis , Glucolípidos/análisis , Lepra/diagnóstico , Mycobacterium leprae/inmunología , Fibras Nerviosas Mielínicas/patología , Proteínas del Tejido Nervioso/análisis , Neuritis/diagnóstico , Antígenos Bacterianos/inmunología , Biopsia , Biomarcadores/análisis , ADN Bacteriano/análisis , Electromiografía , Glucolípidos/inmunología , Técnicas para Inmunoenzimas , Inmunohistoquímica , Lepra/patología , Proteína Básica de Mielina , Mycobacterium leprae/genética , Neuritis/patología , Proteínas de Neurofilamentos/análisis , Reacción en Cadena de la Polimerasa , Receptores de Factor de Crecimiento Nervioso/análisis , /análisisRESUMEN
This paper aims to test the influence of route of infection (intravitreal and instillation) on the course of ocular toxoplasmosis in mice, using the Toxoplasma gondii Me-49 strain. All mice inoculated intravitreally or by instillation presented the same pattern of infection. Using either route, parasites were observed in the retinal vessel with the formation of a glial reaction in the inner plexiforme layer and discontinuity of the pigmented epithelium of the retina 7 days after infection. However, when the intravitreal route was used a more intense inflammatory infiltrate was observed in the retina. The results suggest that inoculation route remarkably influences the inflammatory pattern in ocular toxoplasmosis and that the instillation route should be preferentially used in experimental infections in the murine ocular model of infection by T. gondii, specially with small animals where there is extensive needle damage, which is not observed in the instillation route.
Asunto(s)
Toxoplasmosis Ocular/fisiopatología , Toxoplasmosis Ocular/parasitología , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Retina/parasitología , Retina/patologíaRESUMEN
Leishmania proteinase activity is known as parasite differentiation marker, and has been considered relevant for leishmanial survival and virulence. These properties suggest that Leishmania proteinases can be promising targets for development of anti-leishmania drugs. Here, we analyze the activities of four proteinases during the early phase of the Leishmania amazonensis promastigotes differentiation into amastigotes induced by heat shock. We have examined activities of cysteine-, metallo-, serine-, and aspartic-proteinase by hydrolysis of specific chromogenic substrates at pH 5.0 and at the optimal pH for each enzyme. Our results show that metallo-, serine-, and aspartic-proteinases activities were down-regulated during the shock-induced transformation of promastigotes into amastigotes. In contrast, cysteine-proteinase activity increased concomitantly with the promastigote differentiation. Immunocytochemical localization using two anti-cysteine-proteinase monospecific rabbit antibodies detected the enzyme in several cell compartments of both parasite stages. Our results show different proteinase activity modulation and expression during the early phases of the shock-induced parasite transformation.
Asunto(s)
Leishmania mexicana/enzimología , Péptido Hidrolasas/metabolismo , Animales , Compuestos Cromogénicos/metabolismo , Vesículas Citoplasmáticas/enzimología , Flagelos/enzimología , Calor , Concentración de Iones de Hidrógeno , Hidrólisis , Sueros Inmunes/inmunología , Immunoblotting , Inmunohistoquímica , Leishmania mexicana/crecimiento & desarrollo , Leishmania mexicana/ultraestructura , Proteínas Protozoarias/metabolismo , ConejosRESUMEN
Extracellular proteolytic activity was detected in a Leishmania ( L.) amazonensis culture supernatant and a 56-kDa protein was purified using (NH4)2SO4 precipitation followed by affinity chromatography on aprotinin-agarose. A rabbit serum obtained against the 56-kDa extracellular serine protease was used in order to analyze its location in L. ( L.) amazonensis parasites. Immunocytochemistry studies revealed that the enzyme is mainly found in the flagellar pocket and cytoplasmic vesicles of promastigote forms, whereas in amastigotes, it is located in electron-dense structures resembling megasomes. These results indicate that the 56-kDa serine protease is released into the extracellular environment through the flagellar pocket; and its intracellular location suggests either a correlated enzymatic activity or intracellular trafficking.
Asunto(s)
Leishmania braziliensis/enzimología , Serina Endopeptidasas/metabolismo , Animales , Interacciones Huésped-Parásitos , Immunoblotting , Leishmania braziliensis/crecimiento & desarrollo , Leishmania braziliensis/ultraestructura , Lisosomas/enzimología , Lisosomas/parasitología , Serina Endopeptidasas/aislamiento & purificaciónRESUMEN
The Vero cell line, a non-phagocytic cell, has supported the intracellular mechanism of Leishmania (L.) chagasi. This strain (MHOM/BR/501/MS00) was isolated from a human case of visceral leishmaniasis in Mato Grosso do Sul, Brazil and cultivated in Schneider's Drosophila medium with 20% of heat inactivated fetal calf serum. It was allowed to infect the Vero cells at a ratio of 10 to 20 promastigotes per cell. Within six hours of incubation, promastigote forms were found attached to Vero cells without any particular orientation. The number of amastigotes per cell increased during the incubation period. Results showed that promastigotes of L. (L.) chagasi could interact, transform to amastigote forms and multiply in non-phagocytic cells, demonstrating a new model to study the intracellular cycle of this protozoan.
Asunto(s)
Leishmania infantum/fisiología , Células Vero/parasitología , Animales , Chlorocebus aethiops , Medios de Cultivo , Leishmania infantum/crecimiento & desarrollo , Microscopía Electrónica/veterinaria , Células Vero/ultraestructuraRESUMEN
Our aim was to study the migration of retinal pigmented epithelium (RPE) into the retinal layer during infection of C57BL/6 mice with Toxoplasma gondii. Eyes from infected and non-infected animals were analyzed on the 60th day of infection by light and transmission electron microscopy. Non-infected eyes showed a normal morphology. In contrast, we observed free parasites in the retinal vasculature, the presence of mononuclear inflammatory infiltrate (MNII) and parasites in the vasculature of choroids in infected eyes. No inflammatory infiltrate was observed; RPE cells were identified near the MNII in nuclear and plexiforme layers. RPE cells were also found on the ganglion cell layer and in the outer segments of the photoreceptor. The morphology showed that RPE cells caused a discontinuity in the nuclear and plexiforme layers. Clusters of parasites were found surrounded by RPE cells and MNII in the inner plexiforme layers. Ultrastructural analysis showed that RPE cells migrated through the epithelium into the inner retinal layers. We did not observe Toxoplasma cysts in many eyes in which pathological changes were detected. Only 8.3% of the animals had Toxoplasma cysts in the inner nuclear layer in the absence of inflammatory cells. The migration of RPE cells can be triggered by a disruption of the RPE monolayer or injury to the neural retina, as in the case of toxoplasmosis.
Asunto(s)
Epitelio Pigmentado Ocular/patología , Epitelio Pigmentado Ocular/parasitología , Toxoplasma/patogenicidad , Toxoplasmosis Ocular/patología , Toxoplasmosis Ocular/parasitología , Animales , Movimiento Celular , Coroides/parasitología , Coroides/patología , Ojo/parasitología , Ojo/patología , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos C57BL , Microscopía , Microscopía Electrónica , Retina/parasitología , Retina/patología , Vasos Retinianos/parasitología , Vasos Retinianos/patologíaRESUMEN
An ATP diphosphohydrolase was identified in the plasma membranes isolated from promastigote forms of Leishmania amazonensis. Both ATP and ADP were hydrolysed at similar rates by the enzyme. Other nucleotides such as UTP, GTP and CTP were also degraded, revealing a broad substrate specificity. Adding ATP and ADP simultaneously, the amount of hydrolysis achieved was compatible with the presence of a single enzyme. ATPase activity was not affected by addition of vanadate, ouabain, thapsigargin, dicyclohexylcarbodiimide, oligomycin and bafilomycin A, thus excluding involvement of P-, F- and V-type ATPases. The effects of pH in the range 6.5-8.5 were examined using ATP or p-NPP as substrate. At pH 7.4, the phosphatase activity decreased, and did not show a significant contribution to ATP hydrolysis. In addition, the enzyme was not inhibited by levamisole and ammonium molybdate, excluding alkaline phosphatase and nucleotidase activities, respectively. Sodium azide (5-10 mM) caused inhibition of the ATP and ADP hydrolysis in a dose-dependent manner. Calcium was the best activating metal ion for both ATPase and ADPase activities. Ultrastructural cytochemical microscopy showed ATP diphosphohydrolase on the surface and flagellar pocket of the parasite. We have proposed that L. amazonensis ATP diphosphohydrolase may participate in the salvage pathway of nucleosides.
Asunto(s)
Apirasa/metabolismo , Leishmania/enzimología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apirasa/antagonistas & inhibidores , Apirasa/aislamiento & purificación , Calcio/química , Membrana Celular/enzimología , Membrana Celular/ultraestructura , Inhibidores Enzimáticos/farmacología , Femenino , Concentración de Iones de Hidrógeno , Leishmania/ultraestructura , Levamisol/farmacología , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Molibdeno/química , Azida Sódica/química , Especificidad por SustratoRESUMEN
Rabbit serum against the cysteine-proteinases papain has been employed for the cellular localization of cysteine-proteinases of in Leishmania amazonensis promastigotes. By immunocytochemistry, immune complexes were found in the plasma membrane and in the flagella pocket of the parasite. The antiserum immunoprecipitated major iodinated proteins with molecular masses of 66, 45, 28 and 24 kDa and a wide partitioning of the Triton X-114 detergent phase. The presence of cysteine-proteinase at the cell surface membrane was also suggested by the detection of proteolytic activity in living cells (19.0 microg azocasein min(-1) 10(-7) promastigotes (1.0 S.D. )).
Asunto(s)
Cisteína Endopeptidasas/análisis , Leishmania mexicana/enzimología , Animales , Membrana Celular/enzimología , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Flagelos/inmunología , Sueros Inmunes , Leishmania mexicana/ultraestructura , Microscopía Inmunoelectrónica , Peso Molecular , ConejosRESUMEN
The effects of glycoinositolphospholipid (GIPL), from the pathogenic protozoan Trypanosoma cruzi, and its isolated glycan and lipid (dihydroceramide) components, were investigated in J774 cells and primary macrophages. Isolated GIPL ceramide, but not intact GIPL or its glycan, induced intense fluid phase endocytosis when added exogenously. In the presence of the cytokine IFN-gamma, GIPL ceramide induced marked apoptosis in J774 cells and macrophages, independent of nitric oxide secretion. When cells were preincubated with the GIPL-derived glycan chain, addition of intact GIPL induced macrophage apoptosis in the presence of IFN-gamma. Synthetic C2-dihydroceramide also induced apoptosis in the presence of IFN-gamma. Induction of apoptosis in T. cruzi-infected macrophages by GIPL ceramide plus IFN-gamma led to increased parasite release compared with IFN-gamma treatment alone. Viable parasites released comprised both infective trypomastigote and spheromastigote forms. These results identify a novel pathway by which T. cruzi glycosylphosphatidylinositol family molecules affect host macrophages, with implications for the infectious process.
Asunto(s)
Apoptosis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glucolípidos/farmacología , Interferón gamma/fisiología , Macrófagos Peritoneales/parasitología , Fosfolípidos/farmacología , Trypanosoma cruzi/química , Animales , Ceramidas/farmacología , Sinergismo Farmacológico , Endocitosis/efectos de los fármacos , Femenino , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/patología , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Polisacáridos/farmacología , Trypanosoma cruzi/inmunología , Trypanosoma cruzi/patogenicidad , Células Tumorales Cultivadas , Virulencia , omega-N-Metilarginina/farmacologíaRESUMEN
With ultrastructural cytochemistry we localized the activity of the plasma membrane enzyme markers Mg2+ ATPase and Ca2+ ATPase during the interaction between Leishmania amazonensis and in vitro primary culture fibroblasts. The expression of the enzymes was followed during the parasite adhesion and its interiorization. After the interiorization step, a striking difference was seen between the two enzymes studied when the parasite was found within the parasitophorous vacuole in the fibroblast cytoplasm. The activity of the Ca2+ ATPase found at the Leishmania amazonensis plasma membrane during the attachment step of the infection remained also present inside the phagosome, whereas the Mg2+ ATPase activity disappeared. So far, all the reports in the literature referred the presence of Ca2+ ATPase in Leishmania parasite only in the crude ghost plasma membrane. The Ca2+ ATPase present at the parasite plasma membrane may be involved in the regulation of calcium levels inside the phagosome. Further characterization of this Ca2+ ATPase at the plasma membrane of the parasite, when still inside the phagosome, should permit a better understanding of its functional role in maintaining the parasite surface membrane structure necessary for its existence.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Leishmania mexicana/enzimología , Animales , Adhesión Celular , Membrana Celular/enzimología , Células Cultivadas , Fibroblastos/parasitología , Fibroblastos/ultraestructura , Histocitoquímica , Interacciones Huésped-Parásitos , Humanos , Técnicas In Vitro , Leishmania mexicana/patogenicidad , Leishmania mexicana/ultraestructura , Leishmaniasis Cutánea/etiología , Leishmaniasis Cutánea/parasitología , Microscopía Electrónica , Fagosomas/parasitología , Fagosomas/ultraestructuraRESUMEN
We have applied both enzyme cytochemistry and immunological labeling techniques to characterize the enzyme 5'-nucleotidase (5'-Nase), at the ultrastructural level, in promastigote forms of four Leishmania species: Leishmania amazonensis, Leishmania mexicana, Leishmania donovani and Leishmania chagasi. The cerium phosphate staining was localized at the surface of the cell body, the flagellum and the flagellar pocket membranes of all the parasites studied. The immunogold labelling technique confirmed these results. In this report we localized 5'-Nase in L. chagasi and L. amazonensis which have been implicated respectively in visceral and cutaneous forms of leishmaniasis. In addition, we confirmed the localization of this phosphomonoesterase in the other two species studied. The superior quality of the images, obtained with both methodologies, confirms that these parasites possess mechanisms capable of hydrolyzing nucleotide monophosphates, and that the expression of 5'-Nase is associated with the outer surface of the plasma membrane.
Asunto(s)
5'-Nucleotidasa/análisis , Leishmania/enzimología , Animales , Histocitoquímica , Inmunohistoquímica , Leishmania/ultraestructuraRESUMEN
The degree of maturation of cells of the Mononuclear Phagocyte System (MPS), during in vivo and in vitro infection by Leishmania mexicana amazonensis, was evaluated in this study. The macrophages' differentiation was assayed by cytochemical characterization at the ultrastructural level, using two well-established markers: 5'-nucleotidase enzyme activity, for revealing the mature cells; and the peroxidase activity present in the cell granules to demonstrate immature mononuclear phagocytes. Only a few macrophages, demonstrating 5'-nucleotidase positive reaction in both the plasma membrane and within their cytoplasmic vesicles, were found scattered in the chronic inflammation at the L. m. amazonensis lesions in albino mice. However, by the peroxidase activity analysis, we were also able to demonstrate the presence of immature MPS cells, which predominate, together with parasitized vacuolated macrophages, in chronic lesions induced in this system by L. m. amazonensis. The implications of these results on the pathogenesis of murine cutaneous leishmaniasis are discussed.
Asunto(s)
Leishmaniasis/enzimología , Macrófagos/enzimología , Nucleotidasas/metabolismo , Peroxidasas/metabolismo , Fagocitos/enzimología , 5'-Nucleotidasa , Animales , Diferenciación Celular , Femenino , Leishmaniasis/patología , Macrófagos/ultraestructura , RatonesRESUMEN
Celulas peritoneais de camundongos cultivadas in vitro foram infectadas com inoculos identicos de amastigotas (AM) e de promastigotas, respectivamente, virulentas (VP), avirulentas (AVP) e fixadas (FP) de uma mesma cepa de Leishmania m. mexicana.As monocamadas coradas com May-Grunwald-Giemsa foram examinadas em diferentes intervalos de tempo. Ao nivel de 3a. hora pos-infeccao o numero de macrofagos contendo parasitas variou entre 60,5% (VP) e 84% (AM) nas monocamadas expostas aos parasitas viaveis, comparados com 6,5% para aquelas expostas aos parasitas fixados.Entretanto, nesse tempo de interacao, houve alteracoes degenerativas parasitarias e o numero de macrofagos contendo parasitas intatos variou significantemente entre as celulas infectadas com AM (84%) e aquelas infectadas com VP (42%) ou AVP (40%). O numero medio de parasitas intatos/macrofago tambem diferiu significantemente entre as celulas infectadas com AM e aquelas infectadas com promastigotas viaveis ou fixadas. A analise quantitativa de parasitas intatos e degenerados indicou uma multiplicacao e destruicao parasitaria nas monocamadas infectadas com VP e sobrevivencia e multiplicacao parasitarias naquelas infectadas com AM. Em contraste, nao houve evidencia de multiplicacao de parasitas nas celulas infectadas com AVP. Exses resultados sugerem que os eventos cruciais que determinam a evolucao da infeccao ocorrem durante as primeiras 24 horas da interacao parasito-hospedeiro. Esses eventos sao aparentemente influenciados nao somente pela cepa do parasita ou do hospedeiro, mas tambem por variacoes ambientais induzidas em uma dada cepa parasitaria