Ron B. Podesta

Characterization of Ca2+-dependent neutral protease (calpain) from human blood flukes, Schistosoma mansoni

Calcium-dependent, neutral cysteine-proteases (calpain) were purified from human blood flukes, Schistosoma mansoni. The electrophoretic mobilities, Western blot analyses and high specificity to peptide inhibitors confirmed the presence of both calpain I and II in the purified preparation. The schistosome calpains were localized in the surface syncytial epithelium and underlying musculature. Using peptide inhibitors, calpain was shown to function as a mediator of the surface membrane synthetic process. Since there was also no immunological cross-reactivity between vertebrate and schistosome calpains using antibodies affinity-purified from native and recombinant schistosome calpains, this protease may be usefully investigated as forming the basis of a molecular vaccine against schistosomiasis.

Enhancement of Sm-p80 (large subunit of calpain) induced protective immunity against Schistosoma mansoni through co-delivery of interleukin-2 and interleukin-12 in a DNA vaccine formulation

Schistosomiasis afflicts an estimated 200 million people in 76 countries and an additional 600 million people are at risk of acquiring this infection. Even though effective anthelmintic treatment and snail eradication control programs exist, the discovery of an effective vaccine still remains the most potentially powerful means of control for this disease. We have concentrated on a vaccine candidate (large subunit of calpain or Sm-p80) because of its potential in conferring protection against challenge infection and its pivotal role in surface membrane biogenesis of schistosomes. Since surface membrane renewal is a major phenomenon employed by hemohelminths to evade host immune system; an immune response directed against Sm-p80 should make the parasite prone to immune clearance from the host by both providing a well-targeted attack and by potentially inhibiting the surface membrane biogenesis process. In the present study, we have utilized DNA immunization protocols using Sm-p80 with plasmids encoding interleukin-2 (IL-2) and interleukin-12 (IL-12). Sm-p80 by itself provided a 39% protection (P</=0.0001) against challenge infection in C57BL/6 mice. This protection was increased to 57% (P</=0.0001) when plasmid encoding IL-2 was co-administered with Sm-p80 DNA. Co-injection of plasmid DNA encoding IL-12 with Sm-p80 DNA yielded a protection level of 45% (P</=0.0001). Statistically, the protection conferred by including IL-2 and IL-12 was significantly greater than when only the Sm-p80 was used. Sm-p80 DNA by itself elicited strong responses that includes IgG(2A) and IgG(2B) antibody isotypes. The introduction of IL-2 DNA with Sm-p80 DNA led to an increase in total IgG and IgG(2A) and IgG(2B) titres. Whereas co-administration of IL-12 DNA with Sm-p80 DNA resulted in the augmentation of only total IgG and IgG(2A). This data reinforces the potential of Sm-p80 as an excellent candidate for a schistosomiasis vaccine.

Induction of protective immunity against Schistosoma mansoni via DNA priming and boosting with the large subunit of calpain (Sm-p80): Adjuvant effects of granulocyte-macrophage colony-stimulating factor and interleukin-4

ABSTRACT Considerable morbidity and mortality result from schistosomiasis, an affliction affecting an estimated 200 million people. Although schistosomicidal drugs and other control measures (including public hygiene and snail control) exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. We have targeted a vaccine candidate (large subunit of calpain, Sm-p80) because of its consistent immunogenicity, protective potential, and integral role in surface membrane biogenesis of schistosomes. Since surface membrane renewal appears to be one of the major phenomena employed by schistosomes to evade the hosts immune system; an immune response directed against Sm-p80 should render the parasite susceptible to immune clearance from the host by both providing a focus of attack and by potentially impairing the membrane repair process. In the present study, we have employed DNA immunization protocols using Sm-p80 with plasmids encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Sm-p80 by itself provided 39% protection (P = ≤0.0001) against challenge infection in C57BL/6 mice. This protection was increased to 44% (P = ≤0.0001) when the plasmid encoding GM-CSF was coadministered with Sm-p80 DNA. Coinjection of plasmid DNA encoding IL-4 with Sm-p80 DNA yielded a protection level of 42% (P = ≤0.0001). Statistically, the protection conferred by including GM-CSF, but not IL-4, was significantly greater than that when only Sm-p80 was used. Sm-p80 DNA by itself elicited strong responses that include IgG2A and IgG2B antibody isotypes. The introduction of GM-CSF DNA with Sm-p80 DNA led to distinct increases in total IgG and IgG1 titers, whereas the coadministration of IL-4 DNA with Sm-p80 DNA resulted in a slight elevation of IgG1 and IgG3 titers and in some reduction of IgG2A and IgG2B titers. Our data again indicate that Sm-p80 can be an excellent candidate for a schistosomiasis vaccine.

Effect of 5-hydroxytryptamine (5-HT; serotonin) on in vitro glucose uptake and glycogen reserves in Hymenolepis diminuta

Glucose uptake by Hymenolepis diminuta was linear for up to 60 min following in vitro incubation in 10 mM glucose. Following the addition of 1 mM 5-HT, with a 95% O2/5% CO2, gas phase, glucose uptake by H. diminuta was significantly (P less than 0.001) enhanced, remaining linear and parallel to that of control groups between 15 and 60 min incubation. Under air, the rates of glucose uptake were higher, but were only significantly increased by 5-HT during the first 30 min of incubation. In further experiments, in the absence of glucose in the incubation media worm glycogen reserves decreased by over 50% after 60 min. With the addition of 1 mM 5-HT, the reduction in glycogen content was significantly (P less than 0.025) greater, exceeding 65% after 60min. When glucose was added to the incubation media, worm glycogen reserves were not significantly depleted irrespective of the presence or absence of 5-HT. Incubations under a 95% O2/5% CO2 gas phase did not significantly influence glycogen content compared to corresponding groups incubated in air. The results suggest that 5-HT stimulates glycolysis in H. diminuta through increased glucose uptake by the worm or by a reduction in glycogen reserves in the absence of external glucose.

Effects of serotonin (5HT) and complement C3 on the synthesis of the surface membrane precursors of adult Schistosoma mansoni

The syncytial surface epithelium of Schistosoma mansoni plays an important role in immune evasion. This syncytium is covered by an unusual double-membrane complex consisting of an apical plasma membrane (APM) and an overlying envelope (En) that have been shown to have different rates of synthesis and turnover. It has been suggested that discoid bodies (DBs) and multilamellar bodies (MLBs), the major syncytial inclusion bodies of schistosomes, may be the precursors of the APM and En, respectively. In this ultrastructural study, we examined the effects of serotonin (5HT) and complement C3, which have been shown to stimulate synthesis and turnover of the APM and En, respectively, on the synthesis of DBs and MLBs in vitro. With short-time incubations (20 or 40 min), 5HT stimulated the synthesis of the DBs by 2-fold, whereas C3 accelerated synthesis of the MLBs by 2-fold. Furthermore, when microtubules within the cytoplasmic connections between the syncytium and the underlying cell bodies (the site of membrane synthesis) were disrupted with colchicine, the DBs and MLBs synthesized in response to 5HT or C3 accumulated in the cell bodies. This suggests that the transport of the organelles to the syncytium is dependent upon the microtubules but not the signaling mechanism in response to 5HT or C3. These observations also support the suggestion that the DBs and MLBs are synthesized in subsyncytial cell bodies and serve as precursors of the APM and En, respectively. The rapid synthetic response to 5HT and C3 is also consistent with rapid synthesis and turnover of the APM/En, as suggested by previous studies.

Major phospholipids and phosphatidylcholine synthesis in adult Schistosoma mansoni

The major phospholipids present in the phospholipid extract of Schistosoma Mansoni were phosphatidylcholine (28%), phosphatidylethanolamine 925%), phosphatidylserine (15%) and phosphatidylglycerol (8%). The synthesis of phosphatidylcholine in S. mansoni adults occurred by the choline to phosphatidylcholine of worm slices appeared linear over time with no demonstrable sex differences in choline incorporation. A slight difference in the incorporation of CDPcholine by separate sexes was evident. Methylation phosphatidylethanolamine to phosphatidylcholine could not be demonstrated.

Complement and 5-HT increase phosphatidylcholine incorporation into the outer bilayers of Schistosoma mansoni.

In previous studies (Young and Podesta, 1984, Journal of Parasitology 70: 879-885, 931-936), 5-HT (5-hydroxytryptamine), and the C3b component of complement were shown to increase the synthesis of phosphatidylcholine (PC) from choline in adult Schistosoma mansoni. Since the authors were primarily interested in surface membrane synthesis (the major phospholipid of which is PC), the present series of experiments were conducted to examine the effect of C3b and 5-HT on surface signal transduction, leading to the synthesis of the apical plasma membrane and overlying envelope of S. mansoni. Cercariae of S. mansoni were shed from infected Biomphalaria glabrata obtained from the University of Lowell Research Foundation, Lowell, Massachusetts. The cercariae were injected into Syrian hamsters (Charles River, St. Constant, Quebec, Canada; 2,000 cercariae per animal) or were transformed into schistosomula by penetration through excised hamster skin (Clegg and Smithers, 1972, International Journal for Parasitology 2: 79-98). Infected hamsters were necropsied at 45 days post-infection and were perfused with ice cold Krebs Ringer Phosphate Buffer (KRP pH 7.4) to remove the adult worms. Approximately 1-2,000 adult worms or 10,000 schistosomula were incubated for 1 hr at 37 C in Eagles Minimal Essential Medium (MEM) containing 10 itCi/ml [methyl14C] choline chloride (sp. act. 55 mCi/mmol, New England Nuclear) with the experimental incubations containing 520 nM 5-HT (Sigma, St. Louis, Missouri) or complement components consisting of 125 Ag C3 and 30 ,tg C4 (Kallestad, Montreal, Quebec, Canada). Schistosomula were washed briefly in MEM, homogenized in KRP, and protein was determined by the method of Bradford (1976, Analytical Biochemistry 72: 248-254). The apical plasma membrane and outer bilayer of the apical bilayer complex of the adult parasites were separated by the method of McDiarmid et al. (1982, Molecular and Biochemical Parasitology 7: 141-157). Lipids were extracted, identified, quantified and the amount of labelled choline incorporated determined as described previously (Young and Podesta, 1982, Molecular and Biochemical Parasitology 5: 165-172). The data are presented as the means ? SEM. The major phospholipids of the apical bilayer complex are phosphatidylcholine, 1.1 + 0.1 (n = 3) mg/mg protein in the plasma membrane and 2.6 ? 0.2 (n = 3) mg/mg protein in the outer bilayer; phosphatidylethanolamine, 0.4 + 0.2 (n = 3) mg/mg protein in the plasma membrane and a trace in the outer bilayer; total phospholipid, 1.7 ? 0.3 (n = 7) mg/mg protein in the plasma membrane and 2.8 ? 0.3 (n = 7) in the outer bilayer. Cholesterol was 0.6 ? 0.1 (n = 3) mg/mg protein in the plasma membrane and 1.3 ? 0.6 (n = 3) in the outer bilayer. In schistosomula, 5-HT increased choline incorporation into phosphatidylcholine from 4,470 + 80 (n = 4) to 40,800 ? 1,100 (n = 3; significantly increased P < 0.05) pmols phosphatidylcholine/mg protein/hr. The addition of C3 to the incubation medium did not affect choline incorporation into phosphatidylcholine in schistosomula (4,650 ? 70 (n = 3) pmols phosphatidylcholine/mg protein/hr). The addition of both 5-HT and C3/C4 to the adult incubations increased choline incorporation into phosphatidylcholine in the apical bilayer complex (Table I). Choline incorporation into the plasma membrane was increased almost 6-fold by 5-HT, but was unaffected by C3/C4. Choline incorporation into phosphatidylcholine in the outer bilayer was unaffected by 5-HT but was increased 4-fold in the presence of C3/C4 (Table I). It is interesting that C3/C4 does not affect choline incorporation in schistosomula as it would be available in vivo to both the schistosomulum and the adult parasite. The failure of schistosomula to respond to C3/C4 and the absence of the

Sequential removal of outer bilayer and apical plasma membrane from the surface epithelial syncytium of Schistosoma mansoni

The outer and inner bilayers of the apical membrane complex of Schistosoma mansoni were sequentially stripped from adult worms by two incubations in 0.1% digitonin solutions. Membrane removal was evaluated by electron microscopy of worms and bilayer material, using Con A-ferritin as a marker for the outer bilayer. Amounts of Con A removed by the digests were measured with a tritiated Con A marker. To measure the purity of the fractions membrane markers were characterised and quantitated for both bilayers. In the absence of the usual enzymatic markers for plasma membrane diazotised [125I]-iodosulfanilic acid was used as a marker for the outer bilayer. Alkaline phosphatase and a Na+, Mg2+-ATPase were localised to the inner bilayer. From these results we can deduce that the inner bilayer is analogous to the typical, apical plasma membrane of other animal epithelia. The outer bilayer does not share these enzymatic similarities. The integrity of the syncytium after removal of the outer bilayer and the increased levels of lactate dehydrogenase in the supernatant after removal of the inner bilayer suggests that the outer bilayer is secondary in maintaining the permeability barrier of the apical membrane complex, with respect to soluble proteins. The possible significance of these results in terms of the destructive action of complement on the parasite are discussed.

Schistosoma mansoni: Characterization and identification of calcium-binding proteins associated with the apical plasma membrane and envelope

Calcium-binding proteins (CaBPs) of Schistosoma mansoni were purified by hydrophobic affinity chromatography. Metabolically labeled CaBPs were characterized using SDS-polyacrylamide gel electrophoresis followed by fluorography. A number of CaBPs were detected in total tissue extracts, apical plasma membrane, and soluble fractions of the apical bilayer complex, ranging from 15 to 205 kDa in their molecular masses. No CaBPs were discerned in the envelope of the apical bilayer complex. Two CaBPs were positively identified as calmodulin and gelsolin via immunoblot analyses. The possible role of CaBPs in surface signal transduction mechanisms has also been briefly discussed.

Evidence for the activation of the endogenous opiate system in hamsters infected with human blood flukes, Schistosoma mansoni.

Nociceptive thresholds were investigated in golden hamsters infected with the human blood fluke, Schistosoma mansoni. Increases in thermal thresholds suggestive of analgesia were evident by 20-25 days of infection. These increased further during a 40-42 day period. The altered responses were suppressed by the opioid antagonist naloxone. Non-invasive inhibition of the activity of the pineal gland by exposure to light also reduced nocturnal analgesia in schistosome infected animals. Naloxone antagonism and pineal inhibition of morphine- induced analgesia was obtained similarly in control, uninfected animals. Taken together, these findings suggest strongly that infection with S. mansoni results in a chronic activation of the endogenous opiate system.