Effects on in Vitro Growth of Babesia Microti by Cells and Serum from B. Microti and Schistosoma Mansoni Infected Mice

The effect of peritoneal macrophages and serum from mice infected with Babesia microti, Schistosoma mansoni and B. microti plus S. mansoni on the growth of B. microti was assessed in short term in vitro cultures using the criterium of rate of incorporation of (3H)Hypoxan-thine. In the absence of serum, macrophages and supernatants from macrophage cultures failed to affect the in vitro growth of B. microti. In contrast, in the absen-de of macrophages, serum from mice infected with B. microti and with B. microti plus S. mansoni induced a marked inhibition of the in vitro growth of B. Microti. The level of inhibition induced by serum from mice infected with both S. mansoni and B. microti exceeded consistently that induced by serum from mice infected with B. microti only. Serum from mice only infected with S. mansoni induced a marked increase in the in vitro growth of B. microti. These findings suggest a suppression of B. microti in concurrently S. mansoni-infected mice induced by an immunological specific anti-2?. microti factor potentiated by the concurrent S. mansoni infection. The results do not indicate that activation of the mononuclear phagocytic system is of primary importance in suppression of B. microti in-concurrently S. mansoni infected mice.     

Lack of effect of unmated schistosomes on the fecundity of mated worm pairs

An imbalance between the numbers of male and female worms had no significant effect upon the fecundity of Schistosoma mansoni in rhesus monkeys or upon the number of S. mansoni eggs in the tissues of infected persons or mice. The number of S. japonicum eggs in the tissues of infected rabbits was similarly unaffected by disproportion between worm genders.     

Schistosome fecundity: influence of host genotype and intensity of infection

The host genetic influence on the fecundity of Schistosoma mansoni was studied by measuring egg excretion and accumulation of eggs in the tissues of two inbred strains of mice. The two strains, NIH/Ola and CBA/Ca, differed in both parameters. Egg excretion after infection in the NIH/Ola reached a maximum and declined earlier than was the case for the CBA/Ca mice. More eggs accumulated in the gut and lungs of CBA/Ca, while the NIH/Ola had more eggs in the liver by 100 days post-infection. Statistical analysis of both tissue eggs and faecal eggs, using a robust, non-parametric method, indicated that there is significant evidence for a density dependent reduction in fecundity of worms in more heavily infected animals. We conclude that both the genetic constitution of the murine host and the intensity of infection affect the fecundity of Schistosoma mansoni worms.     

Effect of Schistosoma mansoni infection on fecundity and perivitelline fluid composition in Biomphalaria glabrata

Egg production in the snail, Biomphalaria glabrata, infected with Schistosoma mansoni declined on day 23 postinfection, and was significantly lower than uninfected control snails by day 28 and thereafter. Protein and galactogen content of eggs produced by infected snails did not change during the period of reduced fecundity. This suggests that decreased hemolymph nutrient levels (rather than depleted albumen gland reserves) are responsible for inhibition of snail egg production. Growth rates of infected and uninfected snails were indistinguishable from days 14 through 35 postinfection. The hatching success of eggs produced by infected snails decreased slightly beginning at day 21 postinfection.     

Schistosoma mansoni: control of hepatotoxicity and egg excretion by immune serum in infected immunosuppressed mice is schistosome species-specific, but not S. mansoni strain-specific.

Immunosuppressed mice infected with Schistosoma mansoni suffer from an acute hepatotoxicity reaction, and they fail to excrete as many parasite eggs as comparably infected immunologically intact control animals. The hepatotoxicity was shown here to be preventable, and egg excretion rates were enhanced, by transfer of serum from donors with chronic S. mansoni infections, but not by serum from donors with heterologous infections of Schistosoma haematobium, Schistosoma bovis, or Schistosoma japonicum. The effects of the transferred sera are considered to be due to specific antibody, but the possibility of cytokine involvement is discussed. A high degree of serological cross-reactivity was found between sera from mice infected with the different schistosome species and unfractionated egg homogenate (SEA) in ELISA. Cross-reactivity of the heterologous sera was, however, reduced against CEF6, a partially purified fraction of S. mansoni eggs that contains the putative hepatotoxin and has serodiagnostic potential. S. mansoni isolates from Puerto Rico, Brazil, Egypt, and Kenya shared similar characteristics with respect to the immune dependence of egg excretion and hepatotoxicity in immunosuppressed mice. The S. mansoni geographic isolates were also indistinguishable serologically, in terms of both the capacity of respective infection sera to neutralize hepatotoxicity and in their capacity to promote egg excretion of the other isolates in vivo. Complete immunological cross-reactivity of the geographically distinct isolates was also observed in ELISA with both CEF6 and SEA. Utilization of CEF6 for serodiagnosis of schistosomiasis mansoni is therefore unlikely to be restricted by geographical considerations.     

Soluble proteins from Schistosoma mansoni and japonicum: a comparative biochemical and immunological analysis

1. Soluble proteins were recovered from male Schistosoma mansoni after homogenization in Tris-HCl buffer containing 0.6 M KCl and 1.0% Triton X-100 followed by preparative electrophoresis on SDS-gel. 2. Polyclonal antibodies produced in mice against the soluble fraction were used in comparative analysis of S. mansoni and S. japonicum using immunoblots and immunoprecipitation of in vitro translated polypeptides. 3. Small molecular weight polypeptide (20-22 kdalton), identified by infected mouse serum (IMS) on immunoblots, was predominant in females and was not cross-reactive with heterologous IMS. 4. A 41-43 kdalton polypeptide which appeared as a doublet on immunoblots performed with polyclonal antiserum 4M, was predominant in males of both species although the polypeptides of S. mansoni showed slower electrophoretic mobility, and therefore the larger size (43 kdalton), than that of S. japonicum. 5. Comparison of fluorograms of the immunoprecipitates of in vitro translated polypeptides indicated that IMS of S. mansoni precipitated two, 30 and 94 kdalton, polypeptides while the IMS of S. japonicum identified at 72 kdalton polypeptide. Antisera 1M, 2M and 4M also showed similarities and differences in polypeptides of in vitro translation products of the two species of Schistosoma.     

Antigenicity of a proteolytic enzyme of Fasciola hepatica

The possibility was examined of using a haemoglobinase released during in vitro incubation of adult Fasciola hepatica for immunodiagnosis of fascioliasis. By SDS gel electrophoresis the enzyme appeared as two closely migrating bands with a molecular weight of approximately 27,000 daltons. After Western blotting the bands reacted with serum from rats infected with F. hepatica and mice infected with Schistosoma mansoni. The enzyme was therefore not a good antigen for immunodiagnosis.     

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.     

Failure of anti-Fasciola antisera to induce antibody-dependent, eosinophil-mediated killing of Schistosoma mansoni schistosomula

Sera from rabbits or humans infected with Fasciola hepatica were tested for their ability to kill Schistosoma mansoni schistosomula in an antibody-dependent, eosinophil-mediated in vitro assay. In addition, anti-F. hepatica antisera raised in rabbits or calves, including one to a Fasciola/Schistosoma cross-reactive, cross-protective defined immunity antigen, were also tested for their killing ability. None of these antisera induced damage to S. mansoni schistosomula in vitro, even when enhanced with mononuclear cell supernatants containing eosinophil-activating factor. Serum from humans with S. mansoni did induce schistosomulum killing in vitro when tested under these same conditions. These results suggest that the mechanism of immunity to schistosomes induced by Fasciola antigens at the level of the schistosomula is mediated by factors other than eosinophils.     

Echinostoma paraensei: hemocytes of Biomphalaria glabrata as targets of echinostome mediated interference with host snail resistance to Schistosoma mansoni

Earlier in vivo work by Lie et al. (1977) indicated that the innate resistance of the 10R2 strain of Biomphalaria glabrata to PR1 Schistosoma mansoni could be interfered with if the snails were infected previously with another trematode, Echinostoma paraensei. We have studied this interference phenomenon using in vitro methods in an attempt to understand its mechanistic basis. Hemolymph, derived from 10R2 snails infected with E. paraensei for 14-28 days, killed 25% of S. mansoni sporocysts in vitro, significantly less (P less than 0.001) than the 90% killing rate observed with hemolymph from uninfected, control 10R2 snails. Hemolymph from the infected 10R2 snails and from schistosome susceptible M line snails did not differ significantly (P greater than 0.1) in their relative inability to kill S. mansoni sporocysts in vitro. The defect in sporocyst killing exhibited by echinostome infected 10R2 snails was traced to the cellular, rather than the humoral, component of the hemolymph. Preparations containing uninfected 10R2 snail hemolymph and echinostome daughter rediae exhibited significantly less (P less than 0.001) killing of S. mansoni sporocysts than did controls containing only 10R2 hemolymph and S. mansoni sporocysts. Our results suggest that echinostome larvae release factors that interfere with the ability of B. glabrata hemocytes to kill S. mansoni sporocysts.     

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 adult microsomal antigens, a serologic reagent. II. Specificity of antibody responses to the S. mansoni microsomal antigen (MAMA)

The purified Schistosoma mansoni adult microsomal antigen, MAMA, was used in the quantitative single-tube kinetic dependent enzyme-linked immunosorbent assay (k-ELISA) to measure antibody levels of various human patient sera. The 511 serum specimens tested were from patients with both homologous and heterologous infections. Sera from U.S., Egyptian, Brazilian, and Puerto Rican patients infected with S. mansoni reacted strongly with MAMA. Chinese patients infected with S. japonicum, and Nigerians or Egyptians infected with S. haematobium produced much lower responses to this antigen than those infected with S. mansoni. Sera from patients with echinococcosis, filariasis, paragonimiasis, clonorchiasis, trichinosis, amebiasis, and hepatitis and from healthy uninfected control individuals generally contained no detectable antibodies against this antigen. The S. mansoni adult microsomal antigen, MAMA, therefore, appears to be a highly potent and specific reagent for the serodiagnosis of S. mansoni infections.     

Isolation and characterization of surface antigens from Schistosoma mansoni. II. Antigenicity of radiolabeled proteins from adult worms.

Adult Schistosoma mansoni were radiolabeled in vitro with 125I Bolton-Hunter reagent. Surface membrane antigens were solubilized with non-ionic detergent, then reacted with infection or normal serum. The antigen-antibody complexes were then precipitated with staphylococcal protein A immunoadsorbent, eluted with urea and SDS, and fractionated by SDS-PAGE. The results indicated the presence of 6 to 8 tegument antigens, depending on the type of antisera used. Human antisera to S. japonicum and S. haematobium reacted with some but not all of the antigens identified with human S. mansoni infection serum; this implies the presence of species-specific tegument antigens. The molecular weights of the radiolabeled antigens ranged from 10,000 to 100,000. A large (greater than 100,000) molecular weight glycoprotein and an uncharacterized lipid fraction appeared to be precipitated nonspecifically. Immunoprecipitation methods with anti-mouse IgG and anti-mouse whole serum failed to detect the presence of hostlike antigens in the labeled extracts. Several of the labeled proteins from S. mansoni were found to react with serum from patients infected with either S. haematobium or with S. japonicum.     

A monoclonal antibody from infected mice to a Schistosoma mansoni egg proteinase

A number of monoclonal antibodies were obtained by fusion of SP2/0 myeloma cells and spleen lymphocytes from mice infected with Schistosoma mansoni. These antibodies were tested for their ability to inhibit acidic, thiol-dependent proteinases previously isolated from Schistosoma mansoni eggs and adult worms. One of the monoclonal antibodies isolated inhibits egg proteinase activity measured in vitro with the use of a low m.w. synthetic substrate. This antibody, which is an IgG1 isotype, does not appreciably inhibit an acidic, thiol-dependent proteinase obtained from the adult stage of Schistosoma mansoni. Immunocytochemical methods with the monoclonal antibody have been used to localize the egg proteinase within a set of "penetration" glands in the unhatched miracidium.     

Acquisition of host proteins by the tegument of Schistosoma mansoni recovered from rats

Four-week-old Schistosoma mansoni collected from experimentally infected albino rats were shown by a fluorescent antibody technique to possess several serum proteins of host origin, including IgM, IgG, complement and red blood cell components. The capacity of these rat-derived worms to acquire and shed host proteins was also demonstrated in vitro with fluoresceinated antibody-labelled worms in the presence of xenogeneic molecules. The implications of these results are discussed in relation to the mechanisms of parasite evasion and "self-cure" phenomenon.     

Isolation and partial characterization of shared antigens of Biomphalaria glabrata and Schistosoma mansoni and their evaluation by the ELISA and the EITB

Serum from humans infected with Schistosoma mansoni, when reacted with a Biomphalaria glabrata soluble hepatopancreas antigen extract (BgSHA) yields 2 lines of precipitation by gel diffusion and 1 by immunoelectrophoresis. The IgG from the serum of a human infected with S. mansoni was coupled to CNBr-activated Sepharose 4B. BgSHA (8.0 mg) was then filtered through the gel and the bound antigens, denoted BgSm, eluted with HCl-glycine, pH 2.6. These bound antigens comprised 2.8% of the total BgSHA. BgSm was then applied to an anti-Fasciola hepatica column as above. The drop through in PBS (38% yield) and containing the BgSm antigens depleted of cross-reactivity with F. hepatica was then tested by the ELISA to evaluate its serodiagnostic potential. These antigens detected a primary S. mansoni infection by 4 wk but were less sensitive than SmSEA in the detection of a primary infection with S. mansoni. However, the BgSm-specific antigens were more specific than SmSEA and showed less cross-reactivity with the serum of mice infected with F. hepatica. At least 16 peptides were seen by silver staining following SDS-PAGE with 5-20% gradient gels. The 2 more prominent bands obtained were estimated to have molecular weights of 62 and 66 kd. Nitrocellulose strips blotted with BgSHA were incubated with the serum of mice infected with S. mansoni for 12 wk and developed 6 bands with molecular weights of 66, 57, 55, 50, 48 and 32 kd.(ABSTRACT TRUNCATED AT 250 WORDS)     

Schistosoma mansoni: the effect of adrenalectomy on the murine model

Adrenal steroid hormones have been implicated, among others, as one of the most important host factors controlling the onset, establishment, and pathogenesis of schistosomiasis. They appear to inhibit oviposition by Schistosoma mansoni both in vitro and in vivo, and their effect is greatly enhanced when administered in combination with a schistosomicidal drug. Therefore, we hypothesized that adrenalectomy would greatly affect the course of the murine schistosomiasis infection. To test this hypothesis, adrenalectomized mice (Adx) infected with S. mansoni were compared with intact infected and sham-infected controls concerning their mortality rate, numbers of male and female worms, number of eggs, and liver pathology. Compared with controls, Adx infected mice showed an increase of 50% in the mortality rate, as well as 1.7-3 times as many adult worms and twice as many ova per worm pair in their liver. Thus, for the first time, there is evidence that lack of adrenal steroids mediate an increment of the adult worm burden and promote worm fecundity in vivo. The present work was done to test the hypothesis that lack of adrenal steroids enhances adult worm attrition, possibly by their direct effect on the parasite, and by upregulating or downregulating innate and adaptive immune responses.     

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.     

In vitro delayed hypersensitivity granuloma formation: development of an antigen-coated bead model

Previously, we have described an in vitro model of granulomatous hypersensitivity around Schistosoma mansoni eggs in both the murine model of schistosomiasis and in human schistosomiasis. These studies describe a new model of in vitro granuloma formation that complexes soluble egg antigen from S. mansoni eggs, a partially purified protein derivative of Mycobacterium tuberculosis (PPD), or bovine serum albumin to carrier beads. Ultrastructural and morphologic evaluations demonstrate that there are initial macrophage interactions, followed by the recruitment of antigen-specific T cells that interact with and recruit macrophages, lymphocytes, granulocytes, and fibroblasts. Finally, there is a stage of granulomatous organization involving fibroblast proliferation and collagen deposition. The in vitro reactivity, defined by a quantitative granuloma index, correlates with in vivo granulomas around S. mansoni eggs in the livers of infected cell donor animals. In vitro granuloma formation against PPD-coated beads correlated with delayed cutaneous hypersensitivity against PPD, which was judged by footpad swelling. The reactions demonstrate antigenic specificity and were intrinsically modulated in a manner that is analogous to that previously shown with the in vitro egg granuloma model. This model of in vitro granuloma formation promises to be a useful tool for elucidating mechanisms of cellular immunity and regulation.     

Schistosoma species: Transmission electron microscopy of the circumoval immune precipitin reaction on eggs

Eggs of Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, and Schistosoma mekongi incubated in serum from infected animals or humans had characteristic circumoval precipitin reaction products. When studied by transmission electron microscopy, these reaction products were seen in all species associated with egg shell pores from which antigen emerges. Reaction products were of variable density and sometimes were similar to long “septate” reaction products seen with light microscopy. No limiting membrane was seen around the reaction products. No true septa were seen. It is probable that the reaction product emerged from shell pores and was added to emerging material in spurts giving the septate appearance. No control eggs fixed immediately or first incubated in either saline or serum of uninfected controls showed characteristic reaction products.