Schistosoma mansoni: In vitro schistosomicidal activity of piplartine

Schistosomiasis is one of the world’s greatly neglected tropical diseases, and its control is largely dependent on a single drug, praziquantel. Here, we report the in vitro effect of piplartine, an amide isolated from Piper tuberculatum (Piperaceae), on Schistosoma mansoni adult worms. A piplartine concentration of 15.8 μM reduced the motor activity of worms and caused their death within 24 h in a RPMI 1640 medium. Similarly, the highest sub-lethal concentration of piplartine (6.3 μM) caused a 75% reduction in egg production in spite of coupling. Additionally, piplartine induced morphological changes on the tegument, and a quantitative analysis carried out by confocal microscopy revealed an extensive tegumental destruction and damage in the tubercles. This damage was dose-dependent in the range of 15.8–630.2 μM. At doses higher than 157.6 μM, piplartine induced morphological changes in the oral and ventral sucker regions of the worms. It is the first time that the schistosomicidal activity has been reported for piplartine.     

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stage-specific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. mansoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.     

Schistosoma mansoni: surface membrane stability in vitro and in vivo

The human complement component C3b is known to bind in vitro to the surfaces of all developmental stages of schistosomes as a consequence of complement activation by the alternative pathway. C3b bound to Schistosoma mansoni parasites has now been used in combination with fluorescent labeled antibodies against C3b to label the surfaces of living schistosomes. Binding of complement components and labeled antibodies to adult schistosomes rendered their surface membrane homogeneously fluorescent. At the ultrastructural level, the label was seen as a dense deposit lying on the tegumental membrane. Surface damage was not observed in labeled adults by electron microscopy. Fluorescent schistosomes were cultured in vitro for periods of up to 2 weeks, during which time the parasites remained fully viable and their surface membrane was still fluorescent. The electron dense deposit persisted, and tegumental damage at the electron microscope level was minimal or absent. Consequently, adult schistosomes would seem able to survive in vitro in the absence of rapid and general turnover of their surface membrane. Loss of fluorescence was observed consistently only at the anterior end of the parasite, including the suckers, a finding which indicates that membrane turnover may occur at different rates on different parts of the body. Fluorescent 3-week-old juveniles and 6-day-old lung stage parasites were cultured under the same conditions with similar results: they remained viable and fluorescent for at least 2 weeks. Results with skin schistosomula were different in the sense that many worms died during culture, and those which survived lost large parts of their fluorescent surface. A few of the surviving and fluorescent schistosomula developed the elongate shape typical of lung stage parasites. Fluorescent viable skin schistosomula were injected intravenously into mice and subsequently recovered from the lungs after varying periods. Fluorescence was lost in a patchy way within a few minutes from some individuals and within several hours from most of the worms. These data permit the following conclusions: C3b is a suitable tracer for membrane renewal in all developmental stages of schistosomes. Very slow membrane renewal in vitro and very rapid renewal in vivo are both compatible with parasite survival.     

Haptenation of adult Schistosoma mansoni and assessment of humorally mediated damage in vitro

Adult Schistosoma mansoni were haptenated with DNP under mild conditions using DNP-liposomes, as devised by Levi-Schaffer et al. (1983, Am. J. Trop. Med. Hyg. 32: 343) for haptenating schistosomula. The effects of complement, alone or with anti-DNP antibodies, on tegumental morphology of DNP-haptenated adult worms were assessed by both scanning and transmission electron microscopy, as a simple model system for possible in vitro effects of complement and anti-schistosomal antibodies on normal adult worms. Complement-mediated cytotoxicity, as measured by tegumental changes in both normal and haptenated worms, was observed and indicates a possible role for complement in humorally mediated damage of adult schistosomes. The damage to haptenated adults in the presence of both complement and anti-DNP was greater than that to nonhaptenated worms similarly treated. However, anti-DNP plus complement caused less damage to normal worms than did complement alone, perhaps the result of blocking by antibodies bound to the tegument via Fc receptors. Evidence herein presented implicates both classical and alternative complement activation by the adult worm's tegument. Tegumental tubercle smoothing was one manifestation of damage, as assessed by SEM. Smoothing appears to progress from spine blunting to disappearance, arguing for resorption rather than shedding as the mechanism for spine loss. Because the spines are mostly actin bundles, we suggest that complement-mediated intrategumental calcium ion flux could lead to spine resorption resulting from actin filament depolymerization and/or calcium-induced severing of actin filaments and dissociation of actin filament cross-linking proteins. This process could have adaptive value to the adult worm, in that the spines may be a repository of readily mobilizable actin molecules for tegumental maintenance and repair.     

Tegumental alterations in juvenile Schistosoma haematobium harboured in hamsters following artemether treatment

We report the findings of a detailed temporal study on tegumental alterations in juvenile Schistosoma haematobium, induced by artemether, using scanning electron microscopy. Hamsters infected with S. haematobium cercariae for 28 days were treated intragastrically with a single dose of 300 mg/kg artemether. Groups of two hamsters were killed 24 h, 72 h and 7 days after treatment, and schistosomula were recovered from livers by perfusion and subsequent systematic examination of the tissue, before routinely processing for scanning electron microscopic examination. Most schistosomula collected 24 h after artemether administration showed severe tegumental damage, usually including swelling, fusion, vesiculation, peeling and collapse of enlarged sensory structures. After 72 h, tegumental damage had increased and schistosomula generally showed contraction with extensive swelling, erosion and peeling of the tegument. Seven days post-treatment, severe tegumental damage was only seen in a single male specimen with swelling of the worm body and destruction of the oral sucker. The other schistosomula showed only light to moderate damage, suggesting that schistosomula surviving the treatment began to recover. Our findings of tegumental damage following artemether treatment correlate with the efficacy of this novel antischistosomal drug in killing the juvenile stages of S. haematobium and complement recent findings with S. japonicum and S. mansoni.     

Tegumental changes in adult Schistosoma mansoni harbored in mice treated with artemether

A detailed temporal examination was made of alterations induced by artemether in the tegument of adult Schistosoma mansoni worms using scanning electron microscopy (SEM). Mice infected with S. mansoni cercariae 42 days previously were treated intragastrically with artemether at a single dose of 400 mg/kg. Groups of 3 mice were killed at 24 hr, 72 hr, and 7 days after treatment; the worms were collected by perfusion and examined by SEM. Twenty-four hours after artemether treatment, focal damage to the tubercles on the tegumental surface of male worms was seen. In both male and female worms, there was focal swelling and fusion of tegumental ridges, and sometimes peeling. After 72 hr, the damage to the tegument had increased, especially in female worms, with extensive swelling, fusion, and peeling of the tegumental ridges. In the most severely damaged worms, host leukocytes were seen to be adhered to the damaged tegument. Damage to the oral sucker was also occasionally seen in both male and female worms. Seven days after treatment, the appearance of the tegument had returned to normal in some male and female worms, whereas others still showed apparent damage. The results demonstrate that artemether damages the tegument of adult S. mansoni, and the intensity of damage is more severe in female worms than in males.     

β-Lapachone: A naphthoquinone with promising antischistosomal properties in mice

The activity of β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione, β-lap) against different stages of Schistosoma mansoni was investigated in mice. Mice infected with 50 cercariae (BH strain) were intraperitoneally treated at a dose of 50 mg/kg for 5 consecutive days, starting on the 1st, 14th, 28th and 45th days after infection, to evaluate the effect of β-lap on skin schistosomula, lung schistosomula, young worms (before oviposition) and adult worms (after oviposition), respectively. All animals were euthanized 60 days after infection. β-Lap significantly reduced (p < 0.001) the number of worms in 29.78%, 37.2%, 24.2% and 40.22% when administered during the phases of skin schistosomula, lung schistosomula, young worms and adult worms, respectively. Significant reduction was also achieved in terms of female burden. In all groups, there was significant reduction in the number of eggs and granulomas in the hepatic tissue. When the intervention was performed during the phase of adult worms, β-lap reduced the size of hepatic granulomas and changed the oogram pattern, lowering the percentage of immature eggs and increasing the percentage of mature and dead eggs. Our data indicate that β-lap has moderate antischistosomal properties. Its molecule may also be used as a prototype for synthesis of new naphthoquinone derivatives with potential schistosomicidal properties. Further studies with different formulations containing β-lap are needed to clearly establish the best dose and route of administration and its mechanism of action against schistosomes.     

Ultrastructural alterations in adult Schistosoma mansoni caused by artemether

Progress has been made over the last decade with the development and clinical use of artemether as an agent against major human schistosome parasites. The tegument has been identified as a key target of artemether, implying detailed studies on ultrastructural damage induced by this compound. We performed a temporal examination, employing a transmission electron microscope to assess the pattern and extent of ultrastructural alterations in adult Schistosoma mansoni harboured in mice treated with a single dose of 400 mg/kg artemether. Eight hours post-treatment, damage to the tegument and subtegumental structures was seen. Tegumental alterations reached a peak 3 days after treatment and were characterized by swelling, fusion of distal cytoplasma, focal lysis of the tegumental matrix and vacuolisation. Tubercles and sensory organelles frequently degenerated or collapsed. Typical features of subtegumental alterations, including muscle fibres, syncytium and parenchyma tissues, were focal or extensive lysis, vacuolisation and degeneration of mitochondria. Severe alterations were also observed in gut epithelial cells and vitelline cells of female worms. Our findings of artemether-induced ultrastructural alterations in adult S. mansoni confirm previous results obtained with juvenile S. mansoni and S. japonicum of different ages.     

Functional properties of a rat monoclonal IgE antibody specific for Schistosoma mansoni

A rat monoclonal antibody of IgE isotype (B48-14) raised against Schistosoma mansoni has been generated by the fusion of mesenteric lymph node cells from LOU/M rats immunized with a preparation of adult schistosome worms and IR973F nonsecreting rat myeloma cells. Investigation of the in vitro effector functions of this IgE antibody showed a high level of cytotoxicity against S. mansoni schistosomula in the presence of eosinophils, macrophages, and platelets. A significant level of protection (40 to 60%) against a challenge infection with S. mansoni cercariae was achieved by passive transfer experiment of B48-14 IgE to naive recipient rats. By immunoprecipitation, B48-14 IgE antibodies were shown to react with an antigen of 26 kDa present in excretion-secretion products of schistosomula, previously described as a potential immunogen eliciting a protective IgE response against schistosomiasis.     

Characterization of proteins and immunogens released by adult Schistosoma mansoni

The proteins released in vitro by metabolically radiolabeled adult Schistosoma mansoni were identified by 2-dimensional gel electrophoresis. To determine the origin of these proteins, adult worms were fractionated into surface membrane, tegument, and remaining body components, and the electrophoretic patterns of the proteins in the 3 fractions were compared to those of the released proteins. The immunogens present in these fractions then were identified by immunoprecipitation with sera from humans infected with S. mansoni. This analysis indicated that essentially all of the proteins released from the worm were immunogenic, whereas most of the major membrane and tegumental proteins were not reactive with the immune sera. Thus, it appears that the adult worm is defended against immune attack by detection of the host's antibody response against released proteins rather than against proteins-exposed on the worm's surface.     

Fasciola hepatica and Schistosoma mansoni: immunofluorescent antigen localization and cross-reactivity

In an attempt to identify the tissue sources of biochemically purified antigenic fractions of Fasciola hepatica and Schistosoma mansoni, antisera were tested against plastic-embedded sections of worms of various ages by an indirect fluorescent-antibody-labeling technique. Antibodies prepared against antigens purified by chromatography of F. hepatica whole worm extract through concanavalin A-Sepharose 4B labeled the parenchyma and tegument of adult F. hepatica strongly while antibodies developed against antigens purified by antibody-affinity chromatography against antibodies of S. mansoni labeled only the parenchyma. Antigens common to these two groups clearly originated from F. hepatica parenchyma. Certain of these common antigens are known to provide significant protection in mice to challenge with S. mansoni cercariae, and in the present study antisera against F. hepatica extracts cross-labeled S. mansoni adult male parenchyma. Reciprocal cross-reactions between antisera against S. mansoni and the parenchyma of adult F. hepatica were also noted. FhFIIb, an extract of F. hepatica which Tailliez described as not cross-reacting with S. mansoni, was found to contain no F. hepatica parenchymal antigens. Antigenic fractions of F. hepatica and S. mansoni collected from the surface of worms after incubation in nonionic detergent were unexpectedly found to contain much parenchymal antigen, suggesting leakage of internal components into the supernatant during preparation. Antisera to F. hepatica developed during a natural infection in rabbits labeled tegumental components and gut strongly but did not react with parenchymal tissue. Antisera against extracts of adult schistosomes labeled the parenchyma of male worms and the glycocalyx of the cercarial tegument, indicating the presence of common antigens in the adult and the cercarial stage. Reciprocal reactions between anticercarial sera and adult sections provided further evidence of shared antigenicity. Antisera against S. mansoni egg antigens strongly labeled sections of eggs in liver tissue and cross-reacted with cercarial glycocalyx, indicating the existence of common antigens between these two stages. The antisera also cross-reacted with what appeared to be non-membrane-bound protein in the tegument of F. hepatica. The soluble egg antigen extract shared antigenicity with the parenchyma of both S. mansoni and F. hepatica but circumoval precipitin had no cross-reactivity with this tissue. Thus S. mansoni eggs contain nondiffusable components sharing antigenic specificity with adult parenchymal tissue.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in tegumental surface during development of Schistosoma mansoni

The tegumental surface of immature Schistosoma mansoni was studied with the scanning electron microscope. The surfaces of immature males and females bear no resemblance to that of adult worms and are characterized by having many tegumental folds. The tegumental surfaces of immature males and females are similar, and the dorsal and ventral surfaces of the male are similar before formation of the gynecophoral canal. Transition of the tegumental surface from the juvenile to the adult form begins after worms are in copula and have grown to several millimeters in length.     

Human neutrophils endocytose multivalent ligands from the surface of schistosomula of Schistosoma mansoni before membrane fusion

Human buffy coat cells adhering to schistosomula of Schistosoma mansoni that were preincubated in fluorochrome-conjugated concanavalin A (Con A), wheat germ agglutinin, lentil lectin, or purified IgG from a hyperimmunized rabbit, were examined by fluorescence and transmission electron microscopy and by freeze-fracture. All four fluorochrome- conjugated multivalent ligands were homogeneously distributed on the parasite surface after preincubation. Within 1-3 h after the addition of cells, large areas of nonfluorescence, 10-20 micrometer in diameter, were seen on the parasite surface. In addition, the fluorochromes were observed in granules within the cells. Electron microscope autoradiography of worms preincubated with 125I-Con A showed silver grains evenly distributed over the tegumental membrane. After the addition of cells, grains were seen over phagolysosomes in the cytoplasm of neutrophils adhering to the parasites. In addition, no grains were present over large areas of the tegumental membrane, which still retained its normal architecture, or over fusions between the neutrophil plasma membrane and the outer tegumental membrane. Rabbit IgG formed an electron-dense layer on the tegumental membrane which was endocytosed by neutrophils. Both neutrophils and eosinophils fused with the parasite in areas containing no electron-dense material on the surface. It is concluded that human neutrophils will endocytose a variety of multivalent ligands from the surface of schistosomula, which probably accounts for the failure of neutrophils to kill the parasite and acts to clear the parasite surface of both antigen and antibody. Presumably, the components of the parasite surface which have originally bound the ligands are also endocytosed since surface components labeled by galactose oxidase and NaB3H4 are taken into cells when examined by light microscope autoradiography. Finally, membrane fusion occurs in areas devoid of multivalent glands, which suggests that these ligands serve to bring the cells and parasites close together, but the actual fusigens probably reside in the lipids in the outer tegumental membrane.     

Schistosoma mansoni: dose-related tegumental surface changes after in vivo treatment with praziquantel

The in vivo effects of a range of concentrations of praziquantel (10, 25, 50, 100, 200 and 500 mg/kg body weight of mouse) on the tegumental surface of adult Schistosoma mansoni were studied using scanning electron microscopy. Worms were recovered from mice at 1 and 4 h post-treatment. In general, irrespective of the dose level, male worms exhibited more pronounced and extensive surface alterations which included surface bleeding, swellings, wrinkling and constrictions and surface lesions, particularly on the spined tubercles. In male worms, in particular, the number of worms exhibiting damage and the amount of tegumental surface damage depended, mainly, on the concentration of praziquantel, although, at any given dose level, the extent of the damage increased with time post-treatment.     

Amino acid transport in schistosomes: Characterization of the permeaseheavy chain SPRM1hc

Schistosomes are human parasitic flatworms that constitute an important public health problem globally. Adult parasites live in the bloodstream where they import nutrients such as amino acids across their body surface (the tegument). One amino acid transporter, Schistosome Permease 1 light chain, SPRM1lc, a member of the glycoprotein-associated family of transporters (gpaAT), has been characterized in schistosomes. Only a single member of the SLC3 family of glycoproteins that associate with gpaATs is found following extensive searching of the genomes of Schistosoma mansoni and S. japonicum. In this report, we characterize this schistosome permease heavy chain (SPRM1hc) gene and protein. The 72-kDa gene product is predicted to possess a single transmembrane domain, a (betaalpha)(8) (TIM barrel) conformation and a catalytic triad. Xenopus oocytes functionally expressing SPRM1hc with SPRM1lc import phenylalanine, arginine, lysine, alanine, glutamine, histidine, tryptophan, and leucine. Biochemical characterization demonstrates that in Xenopus extracts and in schistosome extracts SPRM1hc is associated into a high molecular weight complex with SPRM1lc that is disrupted by reducing agents. Quantitative real-time PCR and Western analysis demonstrate that SPRM1hc is expressed in each schistosome life stage examined (eggs, cercariae, schistosomula, adult males and females). SPRM1hc is widely distributed throughout adult male and female worms as determined by immunolocalization. Consistent with the hypothesis that SPRM1hc functions to facilitate nutrient uptake from host blood, immunogold electron microscopy confirms that the protein is distributed on the host-interactive tegumental membranes. We propose that surface-exposed, host-interactive, nutrient-transporting proteins like the SPRM1 heterodimer are promising vaccine candidates.     

Lung-stage expression of a major schistosome surface antigen

The topographical expression of a glycoprotein of 180,000 molecular weight on the surface of lung-stage Schistosoma mansoni schistosomula was determined by immunofluorescence microscopy using a monoclonal antibody. Postfixation treatment with graded ethanols enhanced specific immunofluorescent staining of adult worms, and was required for detection of the antigen on the surfaces of lung-stage schistosomula. The epitope recognized by the monoclonal antibody was also present on the surfaces of adult Schistosoma haematobium, but not on those of Schistosoma japonicum.     

Update on paramyosin in parasitic worms

Paramyosin was first identified as a structural component of invertebrate muscle. Analysis of crude, native, adult schistosome worm preparations identified a highly immunogenic protein which was later identified as paramyosin. Early vaccination/challenge studies with native paramyosin produced encouraging levels of protective efficacy against schistosomes, which led to the question as to how a sub-tegumental (muscular) protein could provide a target for vaccine-mediated immunological attack. Immunolocalisation studies of schistosomes confirmed the presence of paramyosin within the post-acetabular glands of cercariae and on the tegumental surface of lung schistosomula. Here we present an update on the more recent research on paramyosin in parasitic worms that has focused primarily in two directions: (i) further testing of the vaccine potency of paramyosin against schistosomes and other parasitic worms; and (ii) characterisation of the protein at the molecular and biochemical levels.     

A monoclonal antibody raised against adult Schistosoma mansoni which recognizes a surface antigen on schistosomula

A monoclonal antibody has been raised by immunizing a mouse with an isolated tegumental preparation of adult Schistosoma mansoni. The hybridoma designated NIMP/M.47, secreted an IgG2a antibody which was positive by indirect immunofluorescence with live schistosomula of S. mansoni, but not with live schistosomula of S. bovis, or with other living life cycle stages of S. mansoni. In complement-dependent, or cell-mediated in vitro cytotoxicity assays, the monoclonal antibody mediated levels of schistosomular killing as high as those obtained with sera from infected mice. No significant protection, however, was obtained in passive transfer experiments. NIMP/M47 was specific for a 20,000 dalton polypeptide in the schistosomular surface, which was also recognized by serum from infected mice.     

Extraction and partial characterization of non-histone nuclear proteins of Schistosoma mansoni.

A pool of nuclear proteins from adult worms of Schistosoma mansoni was analyzed for amino acid composition and found to be compatible with high mobility group (HMG) proteins. One of the schistosome HMG proteins was identified as HMG 2 by one-dimensional and two-dimensional PAGE. Stage-specific differences in the HMG-like protein composition were encountered when adult worms were compared to schistosomula, the larval form. Immobilization of the adult male and female nuclear proteins onto nitrocellulose, followed by hybridization against 32P-F-10, a schistosome sex specific gene encoding a major egg shell protein, revealed distinct banding patterns. On the other hand, a synthetic oligonucleotide, derived from the 3' untranslated end of the F-10 gene and possibly containing one regulatory element of the gene, bound mainly to male low MW proteins.     

Identification of Schistosoma mansoni glycoproteins recognized by protective antibodies from mice immunized with irradiated cercariae

The humoral immune responses of mice patently infected with Schistosoma mansoni and of mice vaccinated with radiation-attenuated cercariae were compared by radioimmunoassays and one- and two-dimensional polyacrylamide gel analyses of radioimmunoprecipitates. The binding observed with antibodies of mice vaccinated twice with radiation-attenuated cercariae over a period of 7 to 11 wk was less than 50% of the binding observed with antibodies of mice patently infected for 20 wk, but three to four times greater than that obtained with antibodies of mice infected for 6 wk, irrespective of whether the test antigen extracts were derived from schistosomula or adult worms. Sera of vaccinated mice precipitated a restricted number of predominantly high m.w. glycoproteins of both schistosomula and adult worms metabolically labeled with [35S] methionine. Each of the glycoproteins of 36 hr in vitro-cultured schistosomula that was precipitated by the sera of vaccinated mice was also precipitated by sera of infected mice. In contrast, sera of vaccinated mice uniquely precipitated a 38,000 m.w. glycoprotein of schistosomula cultured for 5 days and a 94,000 m.w. glycoprotein of adult male worms. Although radiation-attenuated larvae do not reach the adult stage, mice vaccinated with these still elicit a strong immune response against egg glycoproteins. In particular, an egg glycoprotein of 85,000 to 70,000 and isoelectric point of 4.8 showed an enhanced reactivity with sera of vaccinated mice in comparison with infected mice. These results show that the antibody response in mice vaccinated with radiation-attenuated larvae differs qualitatively and quantitatively from that of infected mice.