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F J Ramalho-Pinto

Publications and source records attributed to F J Ramalho-Pinto.

At least 19 recordsLinked to original sources
PubMed1998

Differential sensitivity of New World Leishmania spp. promastigotes to complement-mediated lysis: correlation with the expression of three parasite polypeptides.

American tegumentary leishmaniasis is caused by Leishmania of the subgenera Leishmania and Viannia. In this paper, we demonstrate that promastigotes of these two subgenera display distinct characteristic patterns of complement sensitivity during growth in vitro. Using fresh normal human serum in lytic assays, we show that while promastigotes of two species of the subgenus Leishmania differentiate into forms that are more resistant to the lytic action of complement, promastigotes of three species of the subgenus Viannia remain sensitive to complement mediated lysis during all stages of their growth in vitro. Complement resistance of the subgenus Leishmania is temporary, reaching its peak at the beginning of the stationary phase of growth, and decreasing thereafter. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we detected in L. amazonensis (subgenus Leishmania), but not in L. guyanensis (subgenus Viannia), three polypeptides whose expression parallels the resistance of promastigotes to complement-mediated lysis.

Animals↗
PubMed1997

Leishmania amazonensis promastigotes evade complement killing by interfering with the late steps of the cascade.

During their growth in vitro, promastigotes of Leishmania amazonensis undergo differentiation from complement-susceptible to complement-resistant forms. Here, we demonstrate that both forms bind comparable amounts of C3 on their surfaces, with the predominant molecule species being the haemolytically active C3b. Likewise, equivalent amounts of C9 are deposited on both forms of promastigotes. However, while C9-bearing complexes are exposed on the cell surface of resistant promastigotes, they are cryptic in the susceptible stage of the parasites. The membrane fraction of complement-resistant promastigote lysates has the ability to inhibit complement-mediated haemolysis, blocking C9, but not C3 deposition to complement-activating complexes. Moreover, the membrane fraction of complement-resistant promastigote lysates can inhibit the late steps of guinea-pig erythrocyte lysis much more efficiently than complement-susceptible ones. Our results indicate that L. amazonensis promastigotes evade complement killing by inhibiting the cytolytic pathway of the complement cascade.

Animals↗
PubMed1996

Proteasome activity is required for the stage-specific transformation of a protozoan parasite.

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.

Acetylcysteine↗
PubMed1996

Cytolytic activity in the genus Leishmania: involvement of a putative pore-forming protein.

We describe here that parasites of the genus Leishmania contain a cytolytic activity which acts optimally at pH 5.0 to 5.5 and at 37 degrees C in vitro. or the four species examined, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) major presented considerable hemolytic activity, whereas Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis showed little and no hemolytic activity, respectively. The cytolytic factor of L. amazonensis promastigotes was characterized as a protein with no protease-, phospholipase-, or detergent-like activity, probably localized inside membranous vesicles. The use of osmotic protectants revealed the colloid-osmotic nature of hemolysis, which is indicative of pore formation in the membranes of target cells. This putative pore-forming protein also damaged nucleated cells, including macrophages, causing an increase in their membrane permeability with leakage of cytoplasmic proteins. Both promastigotes and amastigotes express this lytic activity, suggesting that the cytolysin may have a function in both stages of this parasite. The pH and temperature required for optimal activity indicate that it might be more effective within the mammalian host, particularly inside the macrophage parasitophorous vacuole. In promastigotes of L. amazonensis, the expression of lytic activity seems to be regulated during their growth in vitro, being maximal at the early stationary phase.

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PubMed1996

Complement resistance of Leishmania amazonensis promastigotes is independent of parasite proteases and lysis of sensitive forms is not due to natural antibodies in normal human serum.

Leishmania amazonensis promastigotes cultivated in vitro differentiate from complement-sensitive to complement-resistant forms. In order to determine the possible involvement of parasite proteases in this process, L. amazonensis promastigotes were collected daily and their proteolytic enzyme patterns analyzed using polacrylamide gels copolymerized with gelatin. Although promastigotes at different growth stages showed differences in protease patterns, these changes did not correlate with their susceptibility to complement. The major protease of promastigotes, gp63, was expressed at the same level throughout culture, regardless of the complement resistance of the promastigotes. Furthermore, inhibitors specific for the classes of proteases found in L. amazonensis promastigotes did not interfere with the complement-mediated killing of promastigotes. We also investigated the binding of natural antibodies to promastigotes. at different stages of growth using ELISA. Although complement-sensitive promastigotes bound significantly more antibodies from fresh normal human serum than complement-resistant promastigotes, equivalent amounts of C3 were detected on their surfaces following complement activation. Moreover, serum depleted of anti-Leishmania antibodies was an efficient in killing promastigotes and the intact serum. These data suggest that the resistance of L. amazonensis to complement killing involves strategies other than that of the regulated expression of endogenous proteases capable of inactivating complement components, or the differential ability to bind natural antibodies that might interfere with complement deposition on the parasite surface.

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PubMed1994

Trypanosoma cruzi infection in rats induced early lesion of the heart noradrenergic nerve terminals by a complement-independent mechanism.

The acute phase of the experimental Chagas' disease in rats induces extensive lesion of the heart sympathetic nerve terminals. Because of evidence indicating the involvement of immune reactions in neuron destruction provoked by Chagas' disease, we tested the effects of depleting the complement system by cobra venom factor upon the sympathetic denervation. The serum hemolytic activity against sensitized sheep erythrocytes ensured the efficacy of the anticomplementary treatment. Glyoxylic acid-induced histofluorescence and electron-microscopic methods allowed the study of the heart noradrenergic nerves. T. cruzi infection caused marked rarefaction of fluorescent nerve terminals at day 10 of infection and the ultrastructural study guaranteed that this rarefaction involved lesion of noradrenergic terminals. The complement depletion failed to prevent this early heart noradrenergic denervation, excluding the participation of complement-mediated lysis as a main mechanism.

Animals↗
PubMed1994

Binding of GPI-PLD-treated DAF to the surface of Schistosoma mansoni schistosomula.

Decay accelerating factor (DAF,CD55) is a 70-kDa glycosylphosphatidylinositol (GPI)-anchored protein that protects human erythrocytes (HuE) from complement-mediated damage by regulation of the C3-convertase. Purified human DAF can be incorporated into sheep red blood cell (SRBC) membrane and confer complement resistance on these DAF-deficient cells. Here, we demonstrate that normal HuE or their stroma (HuES) incubated at 37 degrees C for 24 h release soluble DAF in a biologically active form into the culture medium. This soluble DAF neither inserts into SRBC plasma membranes nor presents the cross-reacting determinant (CRD) characteristic of the hydrolysis by phosphatidylinositol-specific phospholipases C (PI-PLC) but binds to schistosomula of S. mansoni protecting them from antibody-mediated complement-dependent damage. To study the binding of DAF to schistosomula in vitro, we have used purified human DAF labeled with 125I(125I-DAF), intact or treated with either PI-PLC or GPI-PLD (glycosylphosphatidylinositol-specific phospholipase D). We have found that GPI-PLD-treated DAF binds to the surface of parasites more readily than intact or PI-PLC-treated DAF. Immunoprecipitation of the samples with a monoclonal anti-human DAF antibody (IA10) revealed that schistosomula incubated with GPI-PLD-treated 125I-DAF emit a stronger signal than their counterparts. This result indicates that the surface of schistosomula is capable of acquiring GPI-PLD-treated DAF more effectively than intact or PI-PLC-treated molecules.

Animals↗
PubMed1994

Identification of a putative pore-forming hemolysin active at acid pH in Leishmania amazonensis.

Several organisms, including the protozoa Entamoeba histolytica and Trypanosoma cruzi, have been shown to contain pore-forming proteins (PFP) thought to play a role in the pathogenesis of the diseases they generate. In the present report, we show that promastigotes of Leishmania amazonensis express a hemolysin that appears to cause colloid-osmotic lysis, typical of pore formation. This hemolysin affects red blood cells of different species at 37 degrees C, but not at 0 degrees C, with maximum activity at pH 5.0. The hemolytic activity is heat-labile, but lysis is not affected by protease inhibitors. These results suggest the involvement of a protein with no proteolytic or detergent activity. Hemolysis is inhibited by polyethyleneglycol, suggesting its colloid-osmotic nature. Hemolytic extracts of the parasite contain a polypeptide that reacts with antibodies to perforin from mouse cytotoxic T lymphocytes or to C9 from human complement. In addition, genomic DNA of L. amazonensis contains a fragment that hybridizes to a perforin cDNA probe. The circumstantial evidence suggests that the L. amazonensis hemolytic activity may be mediated by a PFP homologous to perforin and C9.

Animals↗
PubMed1992

Mechanisms of evasion of Schistosoma mansoni schistosomula to the lethal activity of complement.

Schistosomula of Schistosoma mansoni became resistant to antibody-dependent complement damage in vitro after pre-incubation with normal human erythrocytes (NHuE) whatever the ABO or Rh blood group. Resistant parasites were shown to acquire host decay accelerating factor (DAF), a 70 kDa glycoprotein attached to the membrane of NHuE by a GPI anchor. IgG2a mAb anti-human DAF (IA10) immunoprecipitated a 70 kDa molecule from 125I-labeled schistosomula pre-incubated with NHuE and inhibited their resistance to complement-dependent killing in vitro. Incubation of schistosomula with erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNHE) or SRBC, which are DAF-deficient, did not protect the parasites from complement lesion. Supernatant of 100,000 x g collected from NHuE incubated for 24 h in defined medium was shown to contain a soluble form of DAF and to protect schistosomula from complement killing. Schistosomula treated with trypsin before incubation with NHuE ghosts did not become resistant to complement damage. On the other hand, pre-treatment with chymotrypsin did not interfere with the acquisition of resistance by the schistosomula. These results indicate that, in vitro, NHuE DAF can be transferred to schistosomula in a soluble form and that the binding of this molecule to the parasite surface is dependent upon trypsin-sensitive chymotrypsin-insensitive polypeptide(s) present on the surface of the worm.

Animals↗
PubMed1991

Role of human decay-accelerating factor in the evasion of Schistosoma mansoni from the complement-mediated killing in vitro.

Decay-accelerating factor (DAF) is a 70-kD membrane glycoprotein that prevents complement (C)-mediated hemolysis by blocking the assembly or accelerating the decay of C3 convertase. Purified DAF is known to incorporate into the membrane of DAF-deficient cells, inhibiting lysis. Since Schistosoma mansoni is a blood-dwelling parasite, we investigated whether DAF can be transferred from human erythrocytes to the worm and protect it against C-mediated killing in vitro. We have found that schistosomula (schla) incubated with normal human erythrocytes (N-HuE), but not with DAF-deficient erythrocytes, become resistant to C damage in vitro. Protected parasites acquire a 70-kD surface protein which can be immunoprecipitated by anti-DAF antibodies. The acquired resistance is abrogated by treatment of N-HuE-incubated parasites with anti-DAF antibody. These results indicate that, in vitro, N-HuE DAF can be transferred to schla, and suggest its participation in preventing their C-mediated killing. This could represent an important strategy of parasites to evade the host's immune response in vivo.

Animals↗
PubMed1991

Protective immunity to Schistosoma mansoni in mice is dependent on antibody and complement but not on radiosensitive leukocytes.

The role of complement in the control of the Schistosoma mansoni infection in mice was investigated in vivo. The number of schistosomula recovered from the lung 5 days post-infection was used as a parasitological criterion of immunity. A significant difference in worm burden was observed between normal and immune mice. In contrast, when cobra venom factor (CVF) was injected into normal or immune mice 3 h before challenge, a significant increase in worm burden was noticed compared to untreated mice. We also investigated the protective mechanisms in mice that had been exposed to 650 rads of 60Co gamma radiation before challenge infection. Our results show that gamma-irradiated immune mice, depleted of more than 90% of their circulating or tissue leukocytes, are still able to destroy most of the parasites of a challenge infection with cercariae, suggesting that the radiosensitive leukocytes are not essential in the effector mechanisms of this protective immunity to S. mansoni. These results provide evidence of a role for the complement system, in association with radioresistant effector cells, in protective immunity occurring in the first hours after infection with S. mansoni.

Animals↗
PubMed1989

Lymphokine production by blood or spleen mononuclear cells from patients with schistosomiasis mansoni.

Peripheral blood mononuclear cells (PBMN) from individuals with active or former intestinal schistosomiasis mansoni or splenocytes from patients with the hepatosplenic form of the disease were evaluated for their ability to generate chemotactic factors for neutrophils in response to schistosomal antigenic preparations derived from adult worms (SWAP), eggs (SEA), or phytohemagglutinin (PHA). When supernatants from cultures of stimulated PBMN from normal donors were assayed, only those obtained from cells which had been cultured in presence of PHA displayed chemotactic activity for neutrophils. In contrast, supernatants from cultures of SWAP or SEA stimulated PBMN from patients with intestinal or hepatosplenic schistosomiasis were shown to contain chemotactic activity for neutrophils from normal individuals. PBMN from persons who previously had been infected with Schistosoma mansoni but had received chemotherapy years before presented a pattern of response to SWAP or SEA similar to those from patients with active infections. The response of splenocytes from patients with hepatosplenic schistosomiasis was considerably different from PBMN from individuals with active or with treated schistosomiasis. Splenocytes from most of those patients with hepatosplenic disease failed to produce chemotactic factors for neutrophils in response to stimulation with at least 1 of the schistosome antigens tested. These results indicate that the lymphocytes from schistosomiasis mansoni patients are able to recognize and are stimulated by adult worm and egg antigens to produce chemotactic substances for neutrophils, and that this ability persists for many years after chemotherapy with schistosomicidal drugs. At the hepatosplenic stage, immunoregulatory mechanisms, which may prevent the production of chemotactic factors by splenocytes and/or their activity upon neutrophils in vitro, seem to occur.

Adult↗
PubMed1987

Decay accelerating factor (DAF) as the host antigen with protective activity to complement killing of schistosomula.

The acquisition of host antigens by Schistosoma mansoni was studied by evaluating the resistance of schistosomula to the complement attack mediated by lethal antibody. Schistosomula cultured for 24 hours with intact human erythrocytes (N-HuE) or ghosts of any type of ABO or Rh blood group, showed a marked resistance to complement damage. Sheep red blood cells, pronase-treated N-HuE or erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, which are complement-sensitive cells, were unable to protect schistosomula. Schistosomula protected by N-HuE became again susceptible to complement killing after incubation with a monoclonal antibody anti-DAF. These results indicate that, in vitro, host DAF from N-HuE can be acquired by schistosomula surface in a biological active form that protects the parasite from the complement lesion.

Animals↗
PubMed1987

Levels of lethal antibody during the course of infection with Schistosoma mansoni in rats and mice.

Schistosoma mansoni infected hosts produce an IgG that mediates the complement-dependent killing of schistosomula in vitro. In this study, we followed the levels of serum lethal antibody during infection of rats and mice. Rats presented detectable lethal activity early in the course of infection with a peak in the 6-8th week of infection. This activity declined to non-detectable levels within 2 weeks, remaining low up to the 20-26th week. In mice, lethal antibody was not detected before 7-12 weeks of infection, but raised to higher levels, as compared to non-infected animals, up to 20-24 weeks after infection. We correlate lethal antibody and protective immunity suggesting that the antibody-mediated complement-dependent cytotoxicity to schistosomula play a role in the immunity to reinfection.

Animals↗
PubMed1984

Subclasses of rat IgG active in the killing of schistosomula of Schistosoma mansoni in vitro and in vivo.

Schistosomula of Schistosoma mansoni are known to be killed in vitro by complement and IgG (lethal antibody). To investigate whether this mechanism reflects the in vivo situation, we isolated IgG subclasses from sera of infected rats and assayed their ability to promote the complement-mediated killing of schistosomula in vitro as well as to protect normal recipients from a challenge infection. We found that a serum fraction containing only IgG2a + IgG2b has lethal activity to schistosomula in vitro, whereas a fraction containing only IgG1 + IgG2c fails to kill schistosomula in the presence of complement. The assay of protective activity has shown that the same fraction containing the lethal activity (IgG2a + IgG2b) was able to reduce the number of schistosomula recovered from lungs. These results provide evidence of the participation of IgG2a and/or IgG2b, but not IgG1 or IgG2c, in protective immunity to S. mansoni in rats, possibly through a complement-mediated mechanism.

Animals↗
PubMed1981

Suppressor T cells in the specific control of the carrier response to schistosomula in mice infected with Schistosoma mansoni.

The carrier effect, using TNP-labelled schistosomula was used to measure the helper T-cell activity against the schistosomula surface in CBA mice exposed to 30 cercariae of Schistosoma mansoni. After infection the helper T-cell activity reached a peak in 8--10 days, but by 6 weeks it had declined to background levels. Five x 10(7) spleen cells from chronically (12-week) infected mice when injected into 9-day infected mice caused a specific suppression of the helper T-cell response to schistosomula. Subsequent fractionation of the spleen cell population using a nylon wool column and specific depletion of T cells from the spleen cell population with anti-Thy 1.2 antisera and complement, showed that the suppressive activity was due to T cells. We conclude that during infection of mice with S. mansoni a population of suppressor T cells is generated which partially regulates antibody production against schistosome surface antigens.

Animals↗
PubMed1979

Murine Schistosomiasis mansoni: anti-schistosomula antibodies and the IgG subclasses involved in the complement- and eosinophil-mediated killing of schistosomula in vitro.

Protein A-sepharose affinity chromatography was used to isolate IgG subclasses from the serum of CBA mice chronically infected with Schistosoma mansoni. The subclasses were tested for the presence of two antibodies which are responsible for the death of young schistosomula in vitro; 'lethal antibody' (LA), which kills schistosomula in co-operation with complement and 'eosinophil adherence antibody' (EAA) which causes the death of schistosomula by promoting the adherence of eosinophils to the parasite. LA and EAA were detected only in the IgG fraction of the serum. LA was concentrated in the IgG2a fraction and EAA in the IgG1 fraction. The development of IgG subclasses specific for schistosomula was followed in mice exposed to twenty cercariae by the fluorescent antibody technique. IgG1, IgG2a and IgG2b antibodies were detected 2 weeks after infection and their titres rose steadily to reach high levels by weeks 12 or 14. IgM antibody was not detected until week 6 and IgA until week 10; both were present at lower concentrations than the IgG1 antibodies.

Animals↗