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Although several reports have demonstrated physiological and behavioral changes in adult rats due to neonatal immune challenges, little is known about their effects in adolescence. Since neonatal exposure to lipopolysaccharide (LPS) alters the neural substrates involved in cognitive disorders, we tested the hypothesis that it may also alter the response to novel environments in adolescent rats. At 3 and 5 days of age, male Wistar rats received intraperitoneal injections of either vehicle solution or E. coli LPS (0.05mg/kg) or were left undisturbed. In the mid-adolescent period, between 40 and 46 days of age, the rats were exposed to the following behavioral tests: elevated plus-maze, open-field, novel-object exploration task, hole-board and the modified Porsolt forced swim test. The results showed that, in comparison with control animals, LPS-treated rats exhibited (1) less anxiety-related behaviors and enhanced patterns of locomotion and rearing in the plus-maze and the open-field tests, (2) high levels of exploration of both objects in the novel-object task and of corner and central holes in hole-board test, and (3) more time spent diving, an active behavior in the forced swim test. The present findings suggest that neonatal LPS exposure has long-lasting effects on the behavior profile adolescent rats exhibit in response to novelty. This behavioral pattern, characterized by heightened exploratory activity in novel environments, also suggests that early immune stimulation may contribute to the development of impulsive behavior in adolescent rats.

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.

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.

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.

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.

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.

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.

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.

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.

Defined conditions are described for the in vitro production of large numbers of tail-free viable schistosomules. These consist of (1) the centrifugation of cold cercarial suspension and the incubation of the packed cercariae in a minimal volume of medium at 30 C for 40 min to effect tail loss and glandular secretion; (2) the isolation of the bodies by resuspension and sedimentation and (3) the induction of surface changes by incubating the bodies in inactivated serum or a defined tissue culture medium for a further 40 min interval at 37 C with mild agitation. The resultant schistosomules are characterized by the depletion of their penetration gland contents, loss of tail, fluoride and water sensitivities, complement insensitivity, negative “Cercarien-hullen Reaktion,” and loss of the surface coat as demonstrated by periodic acid-Schiff (PAS) staining and electron microscope observations.

Summary 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.

The effect of different incubation media and of temperature on the induction of water sensitivity has been investigated in intact and tailless Schistosoma mansoni cercariae. Removal of the cercarial tail by vortex stirring and elevation of the temperature of the medium from 27 to 37°C resulted in the rapid onset of permeability changes in the larvae. The rate of change was greater in water than in TC-199 or Hanks' BSS media. Lowering the pH of the medium or increasing the concentration of Ca2+ ions decreased the rate of permeability change: raising the pH of the medium or the addition of 10−5 M EDTA enhanced the rate.Raising the temperature of the medium also increased the rate of permeability change in intact cercariae although the rates obtained varied with the different media tested, being greatest in TC-199.It is concluded that both temperature elevation and loss of the cercarial tail influence the onset and rate of permeability changes in cercarial bodies during the transformation to 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.

Eosinophils from the peritoneal cavity of normal rats, in the presence of fresh normal rat serum (NRS), adhered to schistosomula of Schistosoma mansoni in vitro and killed the majority of parasites within 18 h. The reaction differed from the previously described antibody-mediated eosinophil adherence to schistosomula which occurs in heat-inactivated immune rat serum (IRS) and where adherence is mediated through Fc receptors. Adherence of eosinophils in fresh NRS was shown to be due to the activation of complement at the schistosomular surface by the alternative pathway, and it was effected through C3 receptors. The ability of eosinophils to kill in Fc-mediated adherence. This enhancement of killer activity may be due to the generation by complement activation of eosinophil chemotactic factors which increase the concentration of cells at the target surface. It is suggested that eosinophil adherence mediated through complement activation could be the principla mechanism of destroying schistosomula in the host.

The capacity of rat peritoneal eosinophils to kill schistosomula of Schistosoma mansoni coated with antibody plus complement (C) (fresh immune rat serum) has been compared with that recorded when the parasites are coated with C alone (fresh normal rat serum) or antibody alone (heat-inactivated immune rat serum). Time course studies of adherence and killing, together with serum and cell dilution experiments, have revealed the superior efficiency of the system involving eosinophils, antibody, and C. Cell migration assays have shown that the combined factors generated when both the classical and alternative C pathways are activated at the parasite surface are more potent eosinophil chemoattractants than those generated from the alternative pathway alone. Moreover, faster and higher levels of eosinophil-mediated killing accompany the generation of such factors; 70 to 80% mortality being recorded within 3 hr as compared to 25% or less when the factors are absent. Experiments with fIRS chelated with EGTA indicate that more C3 molecules are probably bound to the parasite surface when C is activated via the classical rather than the alternative pathway, and that factors stimulating eosinophil migration generated from the classical pathway promote faster adherence and, as a consequence, higher levels of eosinophil-mediated killing. IgE does not seem to play an important role in eosinophil-mediated cytotoxicity in vitro, in the presence of fIRS, but the possible participation of the anaphylatoxins and ECF-A is discussed. It is suggested that the rapid and enhanced parasite killing effected by the combination of eosinophils, antibody, and C may be of paramount importance in the destruction of the schistosomula of a secondary infection in the immune host.

SUMMARY 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 × 107 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.

Over the last ten years there has been a steadily increasing effort in the field of schistosome immunology, and the greater part of this activity has had the long term aim of producing an effective anti-schistosome vaccine. It seems likely that the development of such a vaccine will depend on an improved understanding of the biology of the schistosome surface. Available evidence suggests that surface antigens of the young parasite act as targets for the host's immune attack. These target antigens apparently become hidden from the host as the parasite develops; in this way host immunity is evaded. In this paper we review the current information on the nature of the schistosomular surface and the changes which take place there as a result of host-parasite interaction. Unlike the nematode, the schistosome is not bounded by a chitinous-like cuticle but by a syncytial layer of cytoplasm bounded by a living plasma membrane (Fig. 1) which has important physiological and absorptive functions.

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.

— Two enzymes that selectively hydrolyse kinins at pH 7.5 were obtained in partially purified form from the supernatant fraction of homogenates of previously frozen rabbit brain by gel filtration on Sephadex G-100. The enzymes were detected and their activity estimated by bioassay with the isolated guinea pig ileum The products of the enzymic reactions were identified by high voltage electrophoresis at pH 3.5 and by the determination with the amino acid analyser of the amino acids released from the kinins. One enzyme, kinin-converting enzyme, catalyses the hydrolysis of kinin-10 (Lysbradykinin) and kinin-11 (Met-Lys-bradykinin) into kinin-9 (bradykinin). It also hydrolyses the aminoacyl-8-naphthylamides of methionine, lysine, arginine and leucine. The conversion of kinin-10 to kinin-9 was inhibited by puromycin (Ki 3.5 × 10−5 M) These properties are similar to those of brain arylamidases described in the literature. Kininase, the second enzyme, inactives kinins 9, 10 and 11 by peptide-bond hydrolysis. Similar rates of release of arginine and phenylalanine were observed for the three kinins, suggesting that kininase acts at the carboxy-terminus of these peptides. Our results suggest that brain contains proteases which apparently selectively metabolize polypeptide hormones that exert definite pharmacological effects on the central and peripheral nervous systems.

The mechanism of tail loss from cercariae of Schistosoma mansoni was investigated under experimental conditions. Tail loss from 90% or more of cercariae occurred in less than 10 min when packed organisms were incubated at 30 C in a minimal volume of water. Proteolytic secretions from the acetabular glands did not play a significant part in this process since the addition of known protease inhibitors nor the addition of secretions collected from other cercariae influenced the rate of the process. Tail loss was inhibited when the packed cercariae were incubated in saline at concentrations of 0.05 M or above though glandular secretion occurred at an equal rate in both water and saline. Tail loss was also inhibited by the chelating agents ethylenediaminotetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA). A marked feature of cercariae packed in water was their clumping and adhesion to the walls of the containing vessel by secretions from the postacetabular glands. Such clumping did not occur in saline or chelant solutions. It is suggested that the most probable mechanism for tail loss is a simple mechanical trauma effected by the movement of the tail acting against the resistance of the secretion-fixed body. During incubation under conditions which remove the “coat” from the body of the cercaria, the “coat” of the tail remains intact.

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.

Rat eosinophils form an intimate association with the surfaces of parasitic helminths, in vitro, in the presence of immune serum. The parasite presents a non-phagocytosable surface to the cell. The initial response of the eosinophil is degranulation which leads to the formation of large cytoplasmic vacuoles. Peroxidase, an enzyme localized in the matrix of the crystalloid secretion granules, is discharged into these vacuoles as a consequence of degranulation. The vacuoles eventually become connected to the adherent basal plasma membrane of the eosinophil, and peroxidase is secreted directly onto the surface of the parasite. There is no morphological evidence to suggest that this particular secretion affects the integrity of the parasite surface.

SummaryRat peritoneal eosinophils adhere to live Schistosoma mansoni schistosomula in vitro in the presence of fresh normal rat serum, or in heat-inactivated serum from rats immune to the parasite. When the eosinophils are present in sufficient numbers the worms show ultrastructural evidence of surface damage and are ultimately killed. It is believed that the appearance of focal lesions in the tegument of the schistosomulum follows the secretion of enzymes by the eosinophils onto the parasite surface. The cells have been observed within these lesions and later between the basal plasma membrane of the tegument and the underlying interstitial material. It is suggested that the cells are responsible for prising the tegument away from the body of the worm. The detached tegument shows evidence of further degradation. Adherent eosinophils which have released their secretions appear to degenerate and are eventually replaced by macrophages. Remnants of both the expended eosinophils and the disrupted tegument have been identified within the macrophages. Adherence of eosinophils through C3–C3 receptor interaction results in earlier and more severe damage to the schistosomula than when adherence occurs through Fe receptors. Rat eosinophils also adhere to C3-coated, glutaraldehyde-flxed schistosomula and C3-coated Sepharose beads. However, evidence of enzyme secretion is only obtained when the target is a schistosomulum.

Eosinophils from the peritoneal washings of normal rats adhered to live or formalin-fixed schistosomula of Schistosoma mansoni in vitro, in the presence of heat-inactivated serum from infected rats. Eosinophil adherence caused permeability changes in the schistosomula as revealed by 51Cr release and methylene blue uptake. The serum factor which mediated adherence resided in the 7S fraction after Sephadex G-200 gel filtration. Protein A from Staphylococcus aureus which binds specifically to the Fc piece of IgG inhibited adherence, thereby demonstrating that IgG was the antibody responsible for this reaction and that the Fc portion was the site of interaction between eosinophil and antibody; rat eosinophils were shown to possess Fc receptors. The antibody mediating adherence reached high titres in the sera of rats 5-8 weeks after exposure to 500 cercariae, but thereafter there was a gradual decline in titre. Surface membrane from adult S. mansoni inhibited adherence, indicating the presence of cross-reacting antigens in adult worms and schistosomula.

It is believed that a T-cell helper response against the schistosome surface is a necessary prerequisite for the development of protective immunity in schistosomiasis. Accordingly, the carrier effect has been used to assay eleven antigenic preparations of Schistosoma mansoni for their helper T-cell priming against surface components of the schistosomula. Three weeks after i.v. injection of the preparation, CBA mice were injected with schistosomula coated with trinitrophenol (TNP) and 4 days later, their spleens were assayed for plaque-forming cells (PFC) against TNP. Formalin-fixed schistosomula and crude adult worm tegumental membrane induced the highest response; only thirty schistosomula or 10mug of crude membrane protein were needed to generate a T-cell helper response equivalent to that induced by a living infection. All other antigenic preparations, including formalin-fixed cercariae, live miracidia, eggs and adult worm culture fluid, generated some response indicating the presence of shared carrier components.

1. 1. The activity of cercarial extract on the fibrous proteins elastin and keratin, as well as synthetic substrates has been studied. 2. 2. When cercarial extract was subjected to column chromatography three proteolytic components were obtained. 3. 3. The major proteolytic fraction was purified by salt fractionation and column chromatography. 4. 4. The purified fraction shows a chymotryptic-like specificity on synthetic substrates. Inhibitors of α-chymotrypsin also inhibit the cercarial enzyme.

Extracts from Schistosoma mansoni cercariae generate in plasma or serum an activity which contracts the isolated guinea pig ileum. This contraction is inhibited by mepiramine (an anti-histaminic drug). The activity formed by cercarial extracts produces cross-desensitization with the classical anaphylatoxin generated by agar, and induces mast cell degranulation in guinea pig mesentery as well as enhancement of vascular permeability in guinea pig skin. The generation of this anaphylatoxin was blocked by the previous heating of the serum at 56 °C for 30 minutes and by the presence in mixture of 1 × 10−2M Na33HEDTA. The possible role of this pharmacological active material in the process of skin penetration by cercariae is discussed.

INFECTION of mice with cercariae of Schistosoma mansoni produces a disease with immunopathology similar to human bilharziasis1, the chronic hepatosplenomegaly being probably secondary to granulomatous reactions to eggs deposited in the tissues2. It has been shown that thymus-derived lymphocytes have a major role in the granulomatous reaction3,4, but the reaction of the T cells to earlier stages of the parasite has not hitherto been studied. We have investigated one such reaction—the helper T-cell response to the schistosomule—by using schistosomules as carriers for a hapten and measuring the anti-hapten response in normal and infected mice. In analogous experiments using protein carriers, the anti-hapten response has been shown to be due to cooperation between T cells and B cells5. We have found evidence for a specific T-dependent helper response which is greatly increased during the first 2 weeks after infection and subsequently diminishes, and we suggest that the diminution may partly represent a specific suppression of helper T-cell function.

Butanol powder from lyophilized cercariae (Schistosoma mansoni) was extracted either with 0.9 NaCI or 0.1 M borate buffer pH 8.0, precipitated at pH 4.7 and the supernatant (total extract) fractionated by column chromatography (DEAE-Sephadex). The fractions obtained were assayed for protein and polysaccharide and skin tested in patients with active schistosomiasis mansoni. Several fractions were found to elicit a skin response. One of them (fraction D, in which no polysaccharide was detected) was at least five times more active than the total extract and contained only 20% of the absorbing material at 280 mu. The skin test is a useful and practical tool for diagnosis and epidemiological surveys of schistosomiasis (Kagan and Pellegrino, 1961). However, only crude extracts or incompletely purified antigens from cercariae or adult schistosomes have been used for this test. Maximum advantage would be realized with the use of purified and chemically defined antigens. In the course of a study aiming at the purification and characterization of antigenic, enzymatic, and biologically active components extracted from cercariae of S. mansoni, several fractions were obtained. This paper presents the results of skin tests performed with fractions obtained from column chromatography on DEAE-Sephadex. MATERIAL AND METHODS Cercarial extracts from lyophilized cercariae Large numbers of Australorbis glabratus infected with S. mansoni (200 to 500 snails) were isolated in small volumes of water under conditions favoring the emergence of cercariae. The heavy cercarial suspension was sedimented overnight in conical flasks in the cold room at 5 C. The supernatant was carefully aspirated and the packed cercariae washed twice with distilled water, and then transferred to glass ampules and shell frozen at -70 C. Lyophilized cercariae were extracted with dry n-butanol at 0 C for about 20 min with continuous stirring (5 ml of butanol/100 mg of cercariae), and the extract separated by centrifugation. The residual material was reextracted with dry butanol Received for publication 1 March 1965. * This work was supported by grants from the Research Office of the U. S. Army, the WHO, the Rockefeller Foundation, and the Conselho Nacional de Pesquisas. 753 as before and with dry acetone at 15 C. The organic solvents of the precipitated material were evaporated under vacuum over CaC12. The butanol powder was then extracted twice either with 0.9% NaCl or 0.1 M sodium borate, pH 8.0, for periods of 2 hr with continuous stirring at 0 C. Some extracts were precipitated at pH 4.7 with 0.2 M HC1 according to Melcher (1943). The 10,000-g supernatant (total extract) was dialyzed against 0.02 M EDTA buffered at pH 7.0 before being applied to the chromatography column. Fractionation of the extract The fractionation was carried out in an 0.8 by 27-cm column of DEAE Sephadex A-50, medium (Pharmacia Fine Chemicals, Sweden), previously equilibrated with 0.02 M EDTA (Kent, 1963). The elution was performed with an NaCl gradient up to 1 M NaCl, obtained by interposing in the line between the reservoir and the column, a 50-ml suction flask containing a magnetic stirring bar. The gradient was started as soon as the extract entered into the resin. The flow rate was 2 to 3 ml/hr and fractions of approximately 1 ml were collected by means of an automatic fraction collector and read with Beckman DU spectrophotometer at 280 m,i. Protein and polysaccharide determination The protein content of the samples was determined by the phenol-biuret reagent of Lowry et al. (1951). The polysaccharide was assayed by the anthrone reagent (Seifter et al., 1950). A glucose standard was always included and the results expressed as glucose.