Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll.
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Biomedical subjects
A Sher
Publications and source records attributed to A Sher.
The genome of Schistosoma mansoni: isolation of DNA, its size, bases and repetitive sequences.
DNA has been prepared from adults and cercariae of Schistosoma mansoni utilizing a technique that involves centrifugation through cesium chloride. The DNA isolated from S. mansoni adults and that isolated from cercariae were found to be indistinguishable in all analyses. No modified bases were detected by chromatography or comparative endonuclease restriction. Cot analysis demonstrated that the haploid genome of S. mansoni is 0.26 pg (2.7 X 10(8) base pairs) and that the genome contains both moderately and highly repeated components. Some of the repetitive fraction of DNA consists of tandemly repeated ribosomal genes of which there are 500-1000 copies per genome (1.8-3.6% of the total DNA). Four other non-ribosomal repetitive sequences (comprising at least a further 2.0% of the total DNA) have been isolated from a DNA clone bank and their arrangement within the S. mansoni genome investigated by restriction and Southern blot analysis. These cloned segments of DNA appear in many different locations within the genome and thus are reminiscent of the interspersed DNA sequences described in higher eukaryotic organisms.
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Cytotoxicity of cytosine arabinoside on mouse progenitor stem cells.
In vitro colonogenic assay of mouse marrow granulocytic progenitor cells (spleen and agar colony forming cells) was carried out to determine the cytotoxic effect of 1-B-D-arabinofuranosyl cytosine on granulopoiesis. Mainly, the drug cytotoxicity on progenitor cells depended upon exposure time. Endotoxin stimulated colony forming cells showed enhanced cytotoxicity with shorter (25 min) exposure time as compared to a longer exposure of 60 min. A 60 min exposure was found to give maximum cytotoxic effect on spleen colony forming cells. With prolonged incubation, the cytotoxic effect of the drug on spleen colony forming and agar colony forming cells was more or less the same. Incubation of granulocytic precursor cells with the cytotoxic drug, in vitro, produced effects similar to those previously reported with in vivo drug administration. Therefore, some of these techniques can be employed as improved screening models for evaluating the cytotoxic agents in clinical trials.
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Decreasing immunogenicity of developing schistosome larvae.
Cercariae, skin stage schistosomula, and lung stage schistosomula of Schistosoma mansoni were attenuated by gamma irradiation and tested for their ability to induce protective immunity against cercarial challenge in C57Bl/6J mice. The highest levels of resistance were induced by cercariae administered either percutaneously or intramuscularly. Skin stage schistosomula inoculated intramuscularly gave less protection while lung stage schistosomula from syngeneic donors were the least immunogenic. A similar ranking in immunogenicity was observed when the anti-skin stage schistosomular antibody responses induced by the different parasite stages were compared. In contrast, none of the immunization protocols were found to stimulate antibodies capable of recognizing lung stage schistosomula. These results suggest that, as schistosome larvae mature from the cercarial to the lung stage, they undergo a substantial loss in immunogenicity. This change may help explain the failure of older larvae to be immunologically destroyed in infected hosts.
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Enhanced resistance to acute infection with Trypanosoma cruzi in mice treated with an interferon inducer.
For an exploration of the effects of interferon-inducible resistance mechanisms in acute American trypanosomiasis, the synthetic interferon inducer tilerone hydrochloride was administered to mice of the C57BL/6J strain, which is highly resistant to Trypanosoma cruzi, 18 to 24 h before infection with a potentially lethal dose of bloodstream trypomastigotes. Although all of the control mice died within 30 days of the acute infection, approximately 50% of the tilerone-treated animals were able to survive indefinitely (P less than 0.05). The tilerone-treated mice demonstrated significant levels of serum interferon and splenic natural killer cells at the time of infection. Macrophages isolated from the peritoneal cavities of tilerone-treated C57BL/6J mice appeared to kill significant numbers of trypanosomes during 2 to 3 days of in vitro culture, indicating that activated macrophages may contribute to the enhanced resistance to T. cruzi infection in these mice. Beige mice treated with tilerone did not survive T. cruzi infection as well as tilerone-treated heterozygotes did, suggesting a role for natural killer cells in interferon-induced resistance. These results suggest that interferon or effector mechanisms enhanced by interferon induction can play a significant role in influencing resistance to T. cruzi infection.
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Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae. II. Analysis of immunity in hosts deficient in T lymphocytes, B lymphocytes, or complement.
Laboratory mice with genetic or induced immunodeficiencies were vaccinated with irradiated cercariae of Schistosoma mansoni and assayed 4 wk later for their resistance to challenge infection. Athymic nude mice failed to develop immunity to challenge or detectable antibody responses to schistosomula, whereas heterozygote (nu/+) controls and nude mice with thymus grafts displayed highly significant levels of resistance and anti-schistosomulum antibody. Similarly, no resistance or antibody production as observed in vaccinated mice that had been made deficient in B lymphocytes by the injection of anti-mu-chain antisera from birth. In contrast, normal levels of vaccine-induced resistance were observed in mice genetically deficient in the fifth component of complement (C5) and in mice decomplemented before challenge by treatment with cobra venom factor. These results indicate that the resistance to challenge infection induced by irradiated cercariae is both thymus and B lymphocyte dependent and therefore is likely to result from specific immune responses directed against schistosome worms. The data also argue against a role for complement in the effector mechanism of vaccine-induced immunity.
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Macrophages as effector cells of protective immunity in murine schistosomiasis. II. Killing of newly transformed schistosomula in vitro by macrophages activated as a consequence of Schistosoma mansoni infection.
Plastic adherent peritoneal cells from Schistosoma mansoni-infected mice have previously been shown to exhibit nonspecific tumoricidal activity in vitro. In this report we show that these same cell populations kill significant numbers of skin-stage schistosomula in vitro in the absence of added antibody. Larval killing by these activated cells could be enhanced by the use of suspension rather than monolayer cultures and by addition of heat-inactivated immune mouse serum to the cultures. Adherence of cells to schistosomula was also enhanced under the same conditions, suggesting that cell binding to the larvae might be critical in the development or expression of microbicidal activity. In support of this hypothesis, the same level of enhancement of cell binding and larval damage was observed upon substitution of concanavalin A for immune mouse serum. Killing of schistosomula appeared to be mediated solely by activated macrophages in the peritoneal cell suspensions from S. mansoni-infected mice, because partially purified preparations of eosinophils were virtually inactive in these assays. Likewise, inflammatory macrophages from uninfected mice were unable to kill schistosomula under the same conditions, emphasizing the importance of activation in the development of killing capability. The finding that macrophages activated as a consequence of S. mansoni infection are able to kill larval schistosomes in vitro suggests that these cells may play a role in concomitant immunity to schistosomiasis in vivo.
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Macrophages as effector cells of protective immunity in murine schistosomiasis. III. Loss of susceptibility to macrophage-mediated killing during maturation of S. mansoni schistosomula from the skin to the lung stage.
Newly transformed skin-stage and lung-stage schistosomula were compared in terms of their susceptibility to killing mediated by activated mouse macrophages in vitro. Although skin-stage schistosomula were readily killed by macrophages activated as a consequence of either BCG or Schistosoma mansoni infection and used either as cell monolayers or in suspension, lung-stage larvae appeared to be totally resistant to this effector mechanism and survived normally when reinjected into mice. Resistance of schistosomula to in vitro damage by macrophages was evident as early as 18 hr after host infection and was complete in worms recovered at 42 hr. The insusceptibility of lung-stage larvae is apparently not due to a defect in effector cell-target contact, because the induction of extensive macrophage adherence to the worms by the addition of anti-mouse red blood cell antisera to the cultures had no effect on parasite viability. These findings provide additional support for the concept that schistosomula during their development to the lung stage undergo a generalized change affecting their susceptibility to a variety of different immunologic effector mechanisms.
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Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae. I. Analysis of antibody and T-lymphocyte responses in mouse strains developing differing levels of immunity.
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Transformation of hemopoietic stem cells by phytohemagglutinin (PHA) II. Mechanism of action.
It has long been known that phytohemagglutinin (PHA) stimulates transformation and growth of immune competent lymphocytes. Lymphoid cell colonies have previously been shown in the spleen with PHA treated lymph node cells from donor mice were injected into irradiated mice. This communication reports the results of in vivo effects of PHA stimulation on agar colony forming units and spleen colony forming units (CFU). C3H/Hej mice injected with 1 ml of PHA-M were found to secrete colony stimulating factors (PHA-CSF) which lead to an increase in the number of agar colony forming units (granulopoiesis). Serum obtained on day 6 after PHA injection showed enhanced granulopoiesis which was four times higher than in the controls. It was found that 1.0 ml and 0.15 ml of PHA-CSF were more effective in promoting the growth of agar colony forming units in agar plates. Intravenous injection of PHA increased the number of spleen-as well as agar colony forming units (CFU). Day 3 appeared to be optimal for in vivo effects of PHA on granulopoietic cells. An increase in the colony forming units was obtainable when previously treated bone marrow and spleen cells taken on day 3 were injected into irradiated mice. There was a fourfold increase of the colony forming units (CFUs) in the spleen and a twofold one in bone marrow cells. As compared to the controls, the increase in agar colony forming units (CFUc) of bone marrow was fourfold and that of the spleen tenfold. On day 5 some effect was still noticeable but it was lower than on day 3. On weight and cell count basis it was found that on day 6 PHA had a significant in vivo effect on the spleen. On the basis of our findings it can be concluded that PHA supports the survival of transplanted stem cells by stimulating their differentiation into unipotent erythroid progenitor cells. It may also be concluded that PHA activates the immune competent stem cells (mostly T lymphocytes) and displays a supporting function for a better stem cell survival and differentiation into the erythroid progenitor cells. The activated lymphocytes secrete a colony-stimulating like factor which stimulates granulopoiesis and also helps in the differentiation of the stem cells.
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Enzymatic treatment transforms trypomastigotes of Trypanosoma cruzi into activators of alternative complement pathway and potentiates their uptake by macrophages.
In the absence of bound antibody, trypomastigote bloodstream forms of Trypanosoma cruzi fail to activate the alternative complement pathway. We now demonstrate that treatment with trypsin and, to a lesser extent, with sialidase converts these protozoa into activators of the pathway, as judged by their lysis in normal sera or sera genetically deficient in fourth or second component of complement (C4 or C2) and their Mg2+-dependent consumption of C3 as measured by crossed immunoelectrophoresis. In addition, after pretreatment with enzyme and incubation in C5-deficient serum, trypomastigotes were shown to possess both C3 and properdin factor B (B) on their surface as judged by immunofluorescence. Requirement for the late components C5-C9 was suggested by the failure of C5-deficient sera to lyse trypsin-treated parasites. The inability to activate the alternative complement pathway was regained by these organisms after incubation in vitro. This restoration of insusceptibility was inhibited when puromycin was included in the culture medium. Treatment of the trypomastigotes with trypsin also potentiated their uptake by mouse peritoneal macrophages without apparent interference with their capacity to differentiate and multiply inside the cell. These findings suggest that untreated trypomastigotes normally escape recognition by the alternative pathway in vivo because of the presence on their surface of trypsin- and sialidase-sensitive regulatory molecules, the expression of which is dependent on protein synthesis.
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The response of mice immune to Schistosoma mansoni to a challenge infection which bypasses the skin: evidence for two mechanisms of immunity.
Mice which had developed immunity to reinfection with Schistosoma mansoni following exposure to 20 cercariae and mice which had been immunized against S. mansoni by exposure to 400 highly irradiated (20 krad) cercariae, were tested for their ability to resist a percutaneous cercarial challenge and an intravenous challenge with 5-day-old lung-stage schistosomula derived from the same cercariae. Although both types of immune mice showed a marked resistance to a cercarial challenge, only the infected mice showed a comparable immunity to an intravenous challenge with lung schistosomula. These results confirm earlier studies which suggest that the major attrition of a cercarial challenge in infected mice occurs at the post-lung stage, whilst the attrition of a challenge infection in mice immunized with highly irradiated cercariae takes place in the skin. They provide further evidence for two separate mechanisms of immunity against S. mansoni in mice.
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Activation of the alternative complement pathway by Trichomonas vaginalis.
The mechanism underlying the lysis of Trichomonas vaginalis by normal human or guinea pig serum was investigated. The involvement of the complement system was demonstrated by the failure of human serum deficient in C3 or C8 to mediate parasite killing and by the ablation of lytic activity observed when fresh sera were heated at 56 degrees C or treated with ethylenediaminetetraacetate. Fixation of human C3 on the parasite surface was demonstrated by indirect immunofluorescence. The involvement of the alternative complement pathway was demonstrated (i) by the inability of properdin-depleted human serum to lyse T. vaginalis and (ii) by the normal killing observed with guinea pig serum lacking C4 and with normal human or guinea pig serum treated with ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid and Mg2+ to selectively inhibit the classical pathway.
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Immunogenicity of haptenated schistosomula in vitro.
Skin-stage schistosomula of Schistosoma mansoni modified with 2,4,6-trinitrophenyl (TNP) induce primary anti-TNP plaque-forming cell (PFC) responses in normal mouse spleen cells in vitro. The PFC are TNP-specific, and the response is dependent on adherent cells and T lymphocytes. In contrast, comparably haptenated lung stage larvae are weakly or nonimmunogenic in this system, and their inability to stimulate anti-TNP PFC cannot be attributed to a toxic or suppressive effect. These observations suggest that maturation of schistosomula in vivo is accompanied by a decline in their immunogenicity, which, along with other adaptive mechanisms, may promote the survival of parasites in the host environment.
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Cell-mediated cytotoxicity to Trypanosoma cruzi. II. Antibody-dependent killing of bloodstream forms by mouse eosinophils and neutrophils.
Mouse mononuclear cells, neutrophils, and eosinophils were tested for their capacity to mediate antibody-dependent cytotoxicity against bloodstream trypomastigotes of Trypanosoma cruzi. Granulocyte populations were found to be far more effective than nonadherent mononuclear cells in an in vitro assay in which the number of motile parasites was measured. Eosinophils and neutrophils were observed to be equally efficient on a cell-per-cell basis in killing the trypomastigotes. These results were verified in parallel experiments in which trypomastigotes, after incubation with antibody and effector cells, were reinjected into susceptible mice and the survival of the animals was determined.
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Light and electron microscopic appearance of rat peritoneal mast cells adhering to schistosomula of Schistosoma mansoni by means of complement or antibody.
Metrizamide-gradient purified rat peritoneal mast cells were allowed to adhere to schistosomula of S. mansoni that had been preincubated in either minimum essential medium with 5% fetal calf serum (MEM/FCS), heat-inactivated serum from rats 8 weeks after cercarial infection (IRS) fresh normal rat serum as a source of complement (NRS), or IRS and NRS. Adherence was evaluated in sections 0.3 micrometers thick. The preincubation conditions favoring cell adherence were IRS + NRS greater than NRS greater than IRS greater than MEM/FCS. Greater adherence was seen at 60 min than at 10 min. Discharge of mast cell granulates was seen infrequently, was not greater in adherent than nonadherent cells, was not increased by any preincubation condition, and did not occur against the parasite's surface, as would be expected if antigen, antibody, and surface receptors were aggregated there. Electron microscopic examination showed that attachment to the parasite occurred through electron-dense material which was usually fibrillar in appearance. Membrane fusion, such as is seen between human neutrophils and schistosomula, did not occur.
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Studies of the antibody-dependent killing of schistosomula of Schistosoma mansoni employing haptenic target antigens. II. In vitro killing of TNP-schistosomula by human eosinophils and neutrophils.
Purified human eosinophils and neutrophils were compared for their capacity to mediate anti-TNP antibody-dependent killing of schistosomula of Schistosoma mansoni surface labeled with TNBS. In contrast with findings from studies employing human anti-schistosome sera and unmodified larvae, neutrophils in addition to eosinophils were found to be potent killers of schistosomula. Indeed, neutrophils proved to be consistently more effective than eosinophils in killing haptenated larvae throughout the dose response of each of the components (i.e., the TNBS surface label, rabbit anti-TNP sera and cells) of the in vitro reaction. The capacity of neutrophils to kill haptenated parasites is not likely to be due to the use of rabbit antibodies in the assays, since these cells were ineffective in killing unlabeled schistosomula when either rabbit or human anti-schistosome sera were employed. Furthermore, the ability of neutrophils to destroy TNP-labeled schistosomula does not appear to result from sublethal damage induced by the labeling procedure, since haptenated parasites were found to be no more susceptible than unmodified worms to killing mediated by anti-schistosome sera plus eosinophils or complement and, like unlabeled parasites, could not be killed by anti-schistosome sera plus neutrophils. The lethal interaction of neutrophils and haptenated schistosomula was preceded by a rapid and marked adherence of the cells to the worms, an effect that was not observed in the nonlethal interaction of neutrophils with schistosomula induced by anti-schistosome sera. The results argue, therefore, that the capacity of different granulocyte populations to kill schistosomula may be determined by the kinetics and intensity with which the effector cells adhere to the parasites. The findings further suggest that a key factor influencing the differential adherence of the cells to the parasites and their subsequent death may be the nature of the specific antigen-antibody interaction occurring at the surface of the larvae.
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Schistosomiasis: immunologic properties of developing schistosomula.
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