Schistosoma mansoni: Acquired resistance of developing schistosomula to immune attack in vitro

Schistosomula, of Schistosoma mansoni transformed by skin penetration or by mechanical means, have been compared in terms of their susceptibility to in vitro cytotoxic mechanisms, both at 3 hr of age and after culture in the presence or absence of host molecules. Three-hour skin-penetrated schistosomula exhibited a significant level of protection not shown by mechanically transformed individuals. This protection may be correlated with a decreased ability to bind anti-schistosome antibody to their surfaces and to generate C3b molecules as a result of complement activation. Skin worms cultured in the presence of human serum for up to 48 hr showed a significant enhancement of resistance, but slight or no further protection was gained from culture in the absence of host molecules. Mechanically transformed schistosomula cultured for 48 hr in the presence of serum also achieved a significant level of protection but this did not approach that exhibited by the corresponding skin worms; they gained no protection whatsoever from culture in the absence of serum. There are several mechanisms possibly responsible for conferring resistance.     

Schistosoma mansoni: Ultrastructural damages due to complement on schistosomula in vitro

The ultrastructural damage induced by complement in vitro on the schistosomula of Schistosoma mansoni was studied using transmission and scanning electron microscopy. The sequence of events which leads to the killing of the schistosomula is as follows: (a) the lytic activity starts at the anterior end of the schistosomula; (b) lesions progress simultaneously in two distinct directions: from the anterior to the posterior end, and from the outer membrane to the muscle layer; (c) “bubbles” appear around parasite which might correspond to increased membrane permeability; (d) the lytic activity of the late complement components occur in the syncytium; (e) the schistosomula lose their tegument completely, exposing the muscle layer. These findings and our previous work suggest that the activation of the alternate pathway and late complement components is sufficient, without antibodies or cells, to kill schistosomula in vitro.     

Monoclonal IgE-dependent eosinophil cytotoxicity to haptenated schistosomula of Schistosoma japonicum: enhancement of the cytotoxicity and expression of Fc receptors for IgE by Nippostrongylus brasiliensis infection

To obtain direct evidence for the involvement of IgE antibodies in eosinophil-mediated killing of schistosomula of S. japonicum, dinitrophenylated (DNP) schistosomula pretreated with mouse monoclonal IgE antibodies were co-cultured with purified rat peritoneal eosinophils. It was found that the eosinophil-mediated adherence and damage to haptenated schistosomula were dependent on a monoclonal anti-DNP IgE antibody, but not on monoclonal anti-ovalbumin IgE antibody. Moreover, eosinophils from N. brasiliensis-infected rats demonstrated an enhanced ability in the IgE-dependent damage to DNP-schistosomula as compared with the cells from normal rats. The enhancement was associated with an increase in the proportion of eosinophils expressing Fc receptors for IgE.     

In vitro killing of S. mansoni schistosomula by eosinophils from infected rats: role of cytophilic antibodies.

The cytotoxic effect of peritoneal cells from Schistosoma mansoni-infected rats against antibody-opsonized or nonopsonized schistosomula in vitro has been studied during the course of infection. Eosinophil-enriched cell preparations were shown to have a high cytotoxic effect on schistosomula in the absence of antibody. The killer cells were identified as eosinophils. As in the ADCC mechanism previously described, mast cell-eosinophil interaction was required for eosinophil cytotoxicity. Rosette formation using S. mansoni antigen-coated erythrocytes was used to demonstrate the presence of anti-S. mansoni IgG2a antibody at the surface of infected eosinophils. Passive sensitization of normal eosinophils with ultracentrifugation pellets of immune rat serum resulted in a significant cytotoxicity of sensitized eosinophils. A close relationship was found between the cytotoxic activity of infected cells and the ability of the corresponding infected serum to arm normal eosinophils. At certain periods after infection, eosinophils from infected rats were less effective than normal eosinophils on antibody-coated schistosomula. EA- (rat) rosetting assay and blockade experiments with homologous immune complexes have revealed in a kinetic study that the blocking of cytotoxic activity of infected eosinophils was related to heat-stable circulating immune complexes. The possible role of immune complexes either in arming or inhibiting effector cells is suggested.     

Schistosoma mansoni: Inhibition in vitro of granulocyte cytotoxicity against schistosomula by autologous mononuclear cells from patients

Granulocytes and mononuclear cells from normal subjects are able to kill Schistosoma mansoni schistosomula in the presence of human complement in vitro. In contrast, mononuclear cells from chronic schistosomiasis patients failed to kill the parasites. The relative efficiencies expressed in terms of the mean percentage killing of schistosomula for 15 experiments were 50.5 ± 3.2 and 49.7 ± 3.5 for granulocytes from normal and infected patients, and 50.8 ± 2.8 against 23.0 ± 3.2 for mononuclear cells from normal and infected patients, respectively. The killing effect of granulocytes dropped from 48.7 ± 2.8 to 22.1 ± 2.2 when autologous mononuclear cells from chronically infected patients were added to the system. Similar inhibitory effect of granulocyte function was obtained when these cells were incubated with normal mononuclear cells precultured with concanavalin A. Extracts prepared from mononuclear cells obtained from infected patients had the same inhibitory effect of intact cells on the complement-dependent granulocyte cytotoxicity.     

Schistosoma mansoni: killing of transformed schistosomula by the alternative pathway of human complement

The interaction of mechanically transformed schistosomula of Schistosoma mansoni with the alternative pathway of human complement was studied in vitro. To detect early changes in transformation, the schistosomula were prepared at a low temperature and used immediately. As shown previously, freshly transformed schistosomula were highly susceptible to killing by normal human serum and by C4-depleted normal human serum. This serum activity was concentration dependent and was markedly reduced on a twofold serum dilution. Upon incubation at 37 C in defined synthetic medium, schistosomula rapidly became refractory to killing by the alternative pathway of complement. After 1 hr of incubation at 37 C, the percentage of schistosomula which were resistant to killing increased from 16 to 85. This conversion was accompanied by a fivefold decrease in deposition of C3b on schistosomula which had been exposed to 37 C for 1 hr and then further incubated with C4-depleted normal human serum. The following events occurred concomitantly during incubation of freshly transformed schistosomula at 37 C with a half-life of 30-60 min: (1) Decrease in activation and consumption of the alternative pathway of complement by schistosomula; (2) appearance of a strong complement consuming activity in the supernatant of incubating schistosomula; and (3) shedding of protein- and carbohydrate-containing substances from the surface of schistosomula into the supernatant. Isolated external membranes of freshly transformed schistosomula consumed the alternative pathway of complement to a greater extent than membranes of schistosomula preincubated in medium at 37 C. The results demonstrate that transformed schistosomula acquire resistance to complement killing via the alternative pathway by shedding complement-activating substances.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new method for the purification of human eosinophils and neutrophils, and a comparison of the ability of these cells to damage schistosomula of Schistosoma mansoni.

Centrifugation of human white blood cells over either Ficoll-Hypaque or slightly hypertonic Metrizamide discontinuous gradients reliably produces separate fractions that are enriched for either neutrophilic or eosinophilic granulocytes. This single step purification routinely yields 90 to 100% pure neutrophils and 85 to 100% pure eosinophils. Metrizamide gradients, in particular, reproducibly provide high yields of 90 to 100% pure eosinophils from normal subjects with 2 to 3% eosinophils in their peripheral blood. The method does not damage cells as judged by morphologic or functional criteria. The purified cell populations were tested for their ability to damage antibody-coated schistosomula either by the measurement of 51Cr release from labeled organisms, or by direct morphologic assessment. Neutrophils were superior in their ability to release 51Cr from labeled organisms, but eosinophils adhered to the organisms to a greater extent and induced microscopically detectable damage.     

The lysis of Trypanosoma cruzi epimastigotes by eosinophils and neutrophils

López A.F., Bunn Moreno M.M. and Sanderson C.J. 1978. The lysis of Trypanosoma cruzi epimastigotes by eosinophils and neutrophils. International Journal for Parasitology8: 485–489. Antibody-dependent cell-mediated cytotoxicity of T. cruzi epimastigotes has been studied by means of an isotopic assay for parasite lysis, in which the release of labelled RNA is measured. It is shown that rat eosinophils and neutrophils have approximately equal activity against the parasite in the presence of antibody. Antibody enhances the activity of neutrophils about 8-fold, whereas eosinophils have no detectable activity in the absence of antibody.Attention is drawn to the tendency of eosinophils to be inactivated by in vitro manipulation, especially adherence techniques used for removing macrophages and neutrophils.     

Eosinophil-mediated killing of schistosomula of Schistosoma mansoni: Oxidative requirement for enhancement by eosinophil colony stimulating factor (CSF-α) and supernatants with eosinophil cytotoxicity enhancing activity (E-CEA)

Factors which enhance eosinophil-mediated killing of antibody-coated schistosomula of Schistosoma mansoni include semipurified eosinophil colony stimulating factor (CSF-α) and eosinophil cytotoxicity enhancing activity (E-CEA) present in supernatants from cultured mononuclear cells. We have examined the mechanism of enhancement. Both actions require oxygen in order to enhance killing and do not enhance killing under anaerobic conditions (P ? 0.005). E-CEA had no detectable effect upon oxidative metabolism. In contrast to CSF-α which, in our previous studies, increased Superoxide anion productions and quantitative leukocyte iodination, E-CEA had no detectable effect upon oxidative metabolism. In order to test whether active oxygen products might mediate enhancement of killing, the effects of the addition of Superoxide dismutase and catalase were tested. Neither enzyme showed inhibition of CSF-α or E-CEA enhancement of eosinophil-mediated killing. The effects of CSF-α and E-CEA were not additive. These studies suggest that both CSF-α and E-CEA exert enhancement of killing by means of an as yet unidentified oxygen requiring process.     

Complement consumption by Photobacterium damselae subsp. piscicida in seabream, red porgy and seabass normal and immune serum. Effect of the capsule on the bactericidal effect

A virulent strain of Photobacterium damselae subsp. piscicida (Pdp) was grown without (C form) or with (C+ form) glucose supplementation, the latter to enhance capsule formation. Both forms were resistant to killing by normal serum of seabream, red porgy and seabass. However, the C form was killed by immune serum of all three fish species while the C+ form was killed only by seabream and red porgy sera and to a lesser extent than the C form. Both C and C+ forms consumed complement in normal serum and this consumption was enhanced by precoating the bacteria in specific fish antibody. Complement consumption was greatest in seabass serum, especially with antibody-coated C+ form yet in this case the bacteria were not killed. The killing of the C form in immune serum of all three fish species was completely inhibited by EGTA/Mg(2+), indicating that the mechanism of complement activation leading to killing of the bacteria was by the classical pathway. The results suggest that immune serum killing by the classical complement pathway may provide some degree of protection against pasteurellosis, but enhanced expression of the capsule by Pdp in vivo may restrict complement-mediated killing, especially in immunised seabass.     

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.     

Functional role of human IgG subclasses in eosinophil-mediated killing of schistosomula of Schistosoma mansoni

Although IgG antibodies and eosinophils have been shown to kill schistosomula of Schistosoma mansoni in vitro, very little data exist that describe the role of each IgG antibody isotype in this event. This study was designed to test the role of each IgG subclass in the eosinophil-dependent killing reaction. IgG antibodies purified by protein G or protein A affinity chromatography demonstrated a killing effect only in the presence of eosinophils activated in vivo or normal eosinophils activated in vitro by eosinophil activating factor. Purification of each IgG isotype allowed confirmation of these results and demonstrated that the killing effect was associated with IgG1 and IgG3 antibodies. IgG2 antibodies expressed a dual function: 1) an effector function with activated eosinophils and 2) a blocking function with normal eosinophils. IgG4 antibodies, whatever the source of eosinophils, blocked the killing mediated by IgG effector antibodies. These findings are discussed in relation to immunity and susceptibility to reinfection in human schistosomiasis.     

Clq enhancement of IgG-dependent eosinophil-mediated killing of schistosomula in vitro

Antibody-dependent eosinophil-mediated cytotoxicity plays a role in host protection against metazoan parasite invasion. We examined a possible role for Clq in eosinophil-mediated cytotoxicity by using a Schistosoma mansoni schistosomula killing system in vitro. The addition of monomeric purified human Clq enhanced IgG-dependent human eosinophil-mediated killing from 1.4-fold to 2.3-fold (mean percent killing 12% +/- 4 vs 21% +/- 4, p less than 0.005) when the immune IgG concentration was low. In contrast, there was no significant enhancement of neutrophil-mediated killing. When the IgG concentration was increased fourfold Clq did not cause enhancement of eosinophil-mediated killing (35% +/- 9 vs 37% +/- 5). Preincubation of eosinophils with type 1 collagen abrogated Clq enhancement of killing, raising the possibility of a receptor-mediated process, which depends upon cellular binding of Clq via the collagenous portion of the molecule. Eosinophils and neutrophils were examined for the presence of Clq receptors by using 125I labeled Clq. Clq binding to both cell types was saturable, reversible, and specific, indicating that binding is through specific receptors. Type 1 collagen inhibited binding of Clq to cells, suggesting that Clq binding is via the collagenous stalk of Clq. The number of receptors was approximately twice as high for eosinophils as compared with neutrophils (1.9 X 10(7) vs 1.1 X 10(7), p less than 0.025). Affinity constants for the two cell types were similar (1.5 X 10(7) vs 1.3 X 10(7). These findings suggest that Clq and receptors for Clq on eosinophils may be important for eosinophil-mediated schistosomula killing.     

Schistosoma mansoni: Effect of insulin and a low-molecular-weight fraction of serum on schistosomula in chemically defined media

Improved chemically defined media for the in vitro maintenance of Schistosoma mansoni schistosomula are described. Artificially transformed schistosomula could be maintained for 7–13 days in a mixture of equal volumes of RPMI 1640 and F-12 supplemented with 30 nM sodium selenite (DSM). Addition of 50 μg/ml insulin increased the survival time to 13–22 days. Insulin at concentrations lower than 25 μg/ml was not effective. Other proteins like hemoglobin, bovine serum albumin, human serum albumin, and lysozyme were also ineffective. A low-molecular-weight fraction from human serum that passes through an Amicon PM 10 filter (10K fraction) increased the survival time to 19–30 days. The schistosomula maintained under these conditions were actively motile for the above periods but did not grow to a significant extent and did not reach the closed-gut stage. However schistosomula maintained for 7 days in DSM or in DSM containing 50 μg/ml insulin and then transferred into DSM-serum (1:1) developed normally after an adaptation period. Insulin greatly increased the initial rate of development and the resistance of mechanically transformed schistosomula to antibodies and complement. Thus, in chemically defined synthetic medium (DSM) in the presence of 50 μg/ml insulin, schistosomula developed a resistance similar to that reached in the presence of 50% serum, but at a somewhat slower rate. On the other hand, in synthetic medium alone without insulin, both the development rate and the extent of resistance were much lower.     

The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro

Clegg J. A. and Smithers S. R., 1972. The effect of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. International journal for Parasitology2: 79–98. The sera of rhesus monkeys hyperimmunized by 2–4 exposures to S. mansoni cercariae contain an antibody lethal to schistosomula cultivated in vitro. The antibody (IgG) is dependent on labile factors in fresh monkey serum. It can be absorbed by adult worms cultivated in vitro and it is not the antibody responsible for CHR or COP reactions. A titre of lethal antibody sufficient to kill all schistosomula in vitro is maintained for 2–3 months following challenge: it then falls to a moderate level which may be retained for several years. After inactivation, hyperimmune serum inhibits the growth of cultured schistosomula but does not kill them. Following a small primary infection rhesus serum develops a marked growth-inhibiting property and a low titre of lethal antibody at about 4 months, i.e. the time when resistance to reinfection can first be reliably demonstrated.     

Schistosoma mansoni: identification and characterization of schistosomula polypeptides

Schistosomula proteins separated by a two-dimensional (NEPHGE) gel system identify 94 major silver-stained polypeptides. When compared to polypeptides similarly separated from cercariae and adult worms; cercariae share the same polypeptides as schistosomula, adult worms share ca. 60% of the polypeptides. A group of five schistosomula polypeptides 15-31 kDa (apparent pI 8.2-8.9) was not found in adult worm extracts. To identify which polypeptides were immunogens, Western blots of the NEPHGE gels were probed with sera either from humans with chronic schistosomiasis or from mice vaccinated with irradiated cercariae. For characterization studies, polyclonal antibodies were made against the five schistosomula-specific and selected immunogenic polypeptides by immunizing mice with silver-stained spots removed from NEPHGE gels. We show that the polyclonal serum against a polypeptide of 12.5 kDa and an apparent pI of 6.70 mediated complement and eosinophil-dependent killing of schistosomula in an in vitro assay. Epitopes recognized by antibody against the 12.5-kDa polypeptide show a diffuse distribution and are found on flame cells of the excretory system of the schistosomula.     

Schistosoma mansoni: complement and cercarial tail loss during in vitro transformation.

When Schistosoma mansoni cercariae are incubated at 37 C in media containing serum, the organisms lose their tails and change into viable, infective schistosomula. Tail loss does not occur in the absence of serum, or when the serum is heat inactivated. In the present studies, tail loss during in vitro conversion was shown to be complement dependent. The capacity of fresh serum to promote tail loss was markedly suppressed or abolished by cobra venom factor, zymosan, Sepharose CL-4B AND anti-C3 antibody. The alternative rather than the classic complement pathway appeared to be responsible since (1) binding of anti-C3 to cercariae required magnesium, but not calcium; (2) both C4-deficient serum and C2-deficient serum supported tail loss; but (3) human serum heated to 50 C for 20 min to inactivate Factor B did not support tail loss. Cercarial tail loss also required the terminal complement components C5 through C8. The extent and rate of tail loss was normal in agammaglobulinemic sera indicating that the complement effect was not antibody dependent.     

Surface antigens on cercariae,schistosomula and adult worms of Schistosoma mansoni

Shah J. and Ramasamy R. 1982. Surface antigens on cercariae, schistosomula and adult worms of Schistosoma mansoni. International Journal for Parasitology12: 451–461. The surface protein antigens of Schistosoma mansoni were radiolabelled by lactoperoxidase catalysed I¹²⁵-iodination and analysed by immune-precipitation and polyacrylamide gel electrophoresis. The results showed that regularly labelled surface antigens of mol. wts >150,000, 78,000, 45,000 and 22,000 were present on adult worms. Common surface antigens were observed on the cercariae, schistosomula and adult worms. It is suggested that surface antigens released from living adult worms can sensitise a host to react against the invading schistosomula of a secondary infection. However, the failure to vaccinate mice using material containing adult worm surface antigens suggests that the induction of protective immunity is a complex phenomenon.     

Association of eNOS polymorphisms (-786T>C, 4a4b, 894G>T) with colorectal cancer susceptibility in the Korean population

Background

Polymorphisms of endothelial nitric oxide synthases (eNOS) have been shown to be associated with cancer susceptibility. However, the results of such studies are conflicting to date. We investigated whether polymorphisms of the eNOS gene correlated with patients with colorectal cancer (CRC), relative to healthy individuals.

Patients and methods

In the present study, we analyzed three polymorphisms of eNOS (-786T>C, 4a4b, and 894G>T) in 509 healthy controls and 528 patients with CRC. The genotyping of eNOS polymorphisms was performed using polymerase chain reaction or polymerase chain reaction–restriction fragment length polymorphism assays.

Results

We found that the TC+CC genotype of the -786T>C polymorphism was significantly associated with an increased risk of CRC compared with the TT genotype. Similarly, the GT+TT genotype of the 894G>T polymorphism was associated with an increased susceptibility to CRC. However, no evidence was found for any association between the 4a4b polymorphism and CRC risk. In addition, the C/4b/G (-786T>C/4a4b/894G>T) haplotype was significantly associated with increased risk of CRC and C/4b/T (-786T>C/4a4b/894G>T) haplotype was only detected in CRC patients.

Conclusions

Our study suggests that the eNOS -786T>C and 894G>T polymorphisms may be associated with the development of CRC in the Korean population.