Impact of HepG2 Cells Glutathione Depletion on Neutral Sphingomyelinases mRNA Levels and Activity.

Liver cancer is a prevalent form of cancer worldwide. While research has shown that increasing sphingomyelin (SM) hydrolysis by activating the cell surface membrane-associated neutral sphingomyelinase 2 (nSMase2) can control cell proliferation and apoptosis, the role of total glutathione depletion in inducing tumor cell apoptosis via nSMase2 activation is still under investigation. Conversely, glutathione-mediated inhibition of reactive oxygen species (ROS) accumulation is necessary for the enzymatic activity of nSMase1 and nSMase3, increased ceramide levels, and cell apoptosis. This study evaluated the effects of depleting total glutathione in HepG2 cells using buthionine sulfoximine (BSO). The study assessed nSMases RNA levels and activities, intracellular ceramide levels, and cell proliferation using RT-qPCR, Amplex red neutral sphingomyelinase fluorescence assay, and colorimetric assays, respectively. The results indicated a lack of nSMase2 mRNA expression in treated and untreated HepG2 cells. Depletion of total glutathione resulted in a significant increase in mRNA levels but a dramatic reduction in the enzymatic activity of nSMase1 and nSMase3, a rise in ROS levels, a decrease in intracellular levels of ceramide, and an increase in cell proliferation. These findings suggest that total glutathione depletion may exacerbate liver cancer (HCC) and not support using total glutathione-depleting agents in HCC management. It is important to note that these results are limited to HepG2 cells, and further studies are necessary to determine if these effects will also occur in other cell lines. Additional research is necessary to explore the role of total glutathione depletion in inducing tumor cell apoptosis.

Mechanisms of Arachidonic Acid In Vitro Tumoricidal Impact.

To promote the potential of arachidonic acid (ARA) for cancer prevention and management, experiments were implemented to disclose the mechanisms of its tumoricidal action. Hepatocellular, lung, and breast carcinoma and normal hepatocytes cell lines were exposed to 0 or 50 µM ARA for 30 min and then assessed for proliferative capacity, surface membrane-associated sphingomyelin (SM) content, neutral sphingomyelinase (nSMase) activity, beta 2 microglobulin (ß2 m) expression, and ceramide (Cer) levels. Reactive oxygen species (ROS) content and caspase 3/7 activity were evaluated. Exposure to ARA for 30 min led to impairment of the tumor cells' proliferative capacity and revealed that the different cell lines display remarkably similar surface membrane SM content but diverse responses to ARA treatment. Arachidonic acid tumoricidal impact was shown to be associated with nSMase activation, exposure of cell surface membrane ß2 m to antibody binding, and hydrolysis of SM to Cer, which accumulated on the cell surface and in the cytosol. The ARA and Cer-mediated inhibition of tumor cell viability appeared to be independent of ROS generation or caspase 3/7 activation. The data were compared and contrasted to findings reported in the literature on ARA tumoricidal mechanisms.

Anti-Cancer Peptides: Status and Future Prospects.

The dramatic rise in cancer incidence, alongside treatment deficiencies, has elevated cancer to the second-leading cause of death globally. The increasing morbidity and mortality of this disease can be traced back to a number of causes, including treatment-related side effects, drug resistance, inadequate curative treatment and tumor relapse. Recently, anti-cancer bioactive peptides (ACPs) have emerged as a potential therapeutic choice within the pharmaceutical arsenal due to their high penetration, specificity and fewer side effects. In this contribution, we present a general overview of the literature concerning the conformational structures, modes of action and membrane interaction mechanisms of ACPs, as well as provide recent examples of their successful employment as targeting ligands in cancer treatment. The use of ACPs as a diagnostic tool is summarized, and their advantages in these applications are highlighted. This review expounds on the main approaches for peptide synthesis along with their reconstruction and modification needed to enhance their therapeutic effect. Computational approaches that could predict therapeutic efficacy and suggest ACP candidates for experimental studies are discussed. Future research prospects in this rapidly expanding area are also offered.

Cell surface sphingomyelin: key role in cancer initiation, progression, and immune evasion.

Cell surface biochemical changes, notably excessive increase in outer leaflet sphingomyelin (SM) content, are important in cancer initiation, growth, and immune evasion. Innumerable reports describe methods to initiate, promote, or enhance immunotherapy of clinically detected cancer, notwithstanding the challenges, if not impossibility, of identification of tumor-specific, or associated antigens, the lack of tumor cell surface membrane expression of major histocompatibility complex (MHC) class I alpha and ß2 microglobulin chains, and lack of expression or accessibility of Fas and other natural killer cell immune checkpoint molecules. Conversely, SM synthesis and hydrolysis are increasingly implicated in initiation of carcinogenesis and promotion of metastasis. Surface membrane SM readily forms inter- and intra- molecular hydrogen bond network, which excessive tightness would impair cell-cell contact inhibition, inter- and intra-cellular signals, metabolic pathways, and susceptibility to host immune cells and mediators. The present review aims at clarifying the tumor immune escape mechanisms, which face common immunotherapeutic approaches, and attracting attention to an entirely different, neglected, key aspect of tumorigenesis associated with biochemical changes in the cell surface that lead to failure of contact inhibition, an instrumental tumorigenesis mechanism. Additionally, the review aims to provide evidence for surface membrane SM levels and roles in cells resistance to death, failure to respond to growth suppressor signals, and immune escape, and to suggest possible novel approaches to cancer control and cure.

Biochemical and biophysical methodologies open the road for effective schistosomiasis therapy and vaccination.

BACKGROUND: Schistosomiasis caused by blood-dwelling flukes, namely Schistosoma mansoni and Schistosoma haematobium is a severe debilitating disease, widespread in sub-Saharan Africa, the Middle East, and South America. Developing and adult worms are unscathed by the surrounding immune effectors and antibodies because the parasite is protected by a double lipid bilayer armor which allows access of nutrients, while binding of specific antibodies is denied. SCOPE OF REVIEW: Fluorescence recovery after bleaching, extraction of surface membrane cholesterol by methyl-ß-cyclodextrin, inhibition and activation of sphingomyelin biosynthesis and hydrolysis, and elastic incoherent and quasi-elastic neutron scattering approaches have helped to clarify the basic mechanism of this immune evasion, and showed that sphingomyelin (SM) molecules in the worm apical lipid bilayer form with surrounding water molecules a tight hydrogen bond barrier. Viability of the parasite and permeability of the outer shield are controlled by equilibrium between SM biosynthesis and activity of a tegument-associated neutral sphingomyelinase (nSMase). MAJOR CONCLUSIONS: Excessive nSMase activation by polyunsaturated fatty acids (PUFA), such as arachidonic acid (ARA) leads to disruption of the SM molecules and associated hydrogen bond network, with subsequent access of host antibodies and immune effectors to the outer membrane and eventual parasite death. GENERAL SIGNIFICANCE: ARA was predicted and shown to be a potent schistosomicide in vitro and in vivo in experimental animals and in children. Additionally, it was advocated that schistosomiasis vaccine candidates should be selected uniquely among excretory-secretory products of developing worms, as contrary to cytosolic and surface membrane antigens, they are able to activate the effector functions of the host antibodies and toxic molecules. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo".

Rigidity and resistance of larval- and adult schistosomes-medium interface.

Schistosomiasis is second only to malaria in prevalence and severity, and is still a major health problem in many tropical countries worldwide with about 200-300 million cases and with more than 800 million people at risk of infection. Based on these data, the World Health Organization recommends fostering research efforts for understanding at any level the mechanisms of the infection and then decreasing the social and economical impact of schistosomiasis. A key role is played by the parasite apical lipid membrane, which is entirely impervious to the surrounding elements of the immune system. We have previously demonstrated that the interaction between schistosomes and surrounding medium is governed by a parasite surface membrane sphingomyelin-based hydrogen barrier. In the present article, the elastic contribution to the total motion as a function of the exchanged wave-vector Q and the mean square displacement values for Schistosoma mansoni larvae and worms and Schistosomahaematobium worms have been evaluated by quasi elastic neutron scattering (QENS). The results point out that S. mansoni larvae show a smaller mean square displacement in comparison to S. mansoni and S. haematobium worms. These values increased by repeating the measurements after one day. These differences, which are analogous to those observed for the diffusion coefficient we previously evaluated, are interpreted in terms of rigidity of the parasite-medium interaction. S. mansoni larvae are the most rigid systems, while S. haematobium worms are the most flexible. In addition, temperature and hypoxia induce a weakening of the schistosome-medium interaction. These evidences are related to the strength of the hydrogen-bonded interaction between parasites and environment that we previously determined. We have shown that S. mansoni worms are characterized by a weakened interaction in respect to the larvae, while the S. haematobium worms more weakly interact with the surrounding medium than S. mansoni. The present QENS analysis allowed us to characterize the rigidity of larval- and adult S. mansoni and S. haematobium-host interface and to relate it to the parasite resistance to the hostile elements of the surrounding medium and to the immune effectors attack.

Mechanisms of Arachidonic Acid In Vitro Schistosomicidal Potential.

Arachidonic acid (ARA) was shown to possess safe and effective schistosomicidal impact on larval and adult Schistosoma mansoni and Schistosoma hematobium in vitro and in vivo in laboratory rodents and in children residing in low and high endemicity regions. We herein examine mechanisms underlying ARA schistosomicidal potential over two experiments, using in each pool a minimum of 50 adult male, female, or mixed-sex freshly recovered, ex vivo S. mansoni. Worms incubated in fetal calf serum-free medium were exposed to 0 or 10 mM ARA for 1 h at 37 °C and immediately processed for preparation of surface membrane and whole worm body homogenate extracts. Mixed-sex worms were additionally used for evaluating the impact of ARA exposure on the visualization of outer membrane cholesterol, sphingomyelin (SM), and ceramide in immunofluorescence assays. Following assessment of protein content, extracts of intact and ARA-treated worms were examined and compared for SM content, neutral sphingomyelinase activity, reactive oxygen species levels, and caspase 3/7 activity. Arachidonic acid principally led to perturbation of the organization, integrity, and SM content of the outer membrane of male and female worms and additionally impacted female parasites via stimulating neutral sphingomyelinase activity and oxidative stress. Arachidonic powerful action on female worms combined with its previously documented ovocidal activities supports its use as safe and effective therapy against schistosomiasis, provided implementation of the sorely needed and long waited-for chemical synthesis.

Differential protective impact of peptide vaccine formulae targeting the lung- and liver-stage of challenge Schistosoma mansoni infection in mice.

The study aimed to elicit protective immune responses against murine schistosomiasis mansoni at the parasite lung- and liver stage. Two peptides showing amino acid sequence similarity to gut cysteine peptidases, which induce strong memory immune effectors in the liver, were combined with a peptide based on S. mansoni thioredoxin peroxidase (TPX), a prominent lung-stage schistosomula excretory-secretory product, and alum as adjuvant. Only one of the 2 cysteine peptidases-based peptides in a multiple antigenic peptide construct (MAP-3 and MAP-4) appeared to adjuvant protective immune responses induced by the TPX peptide in a MAP form. Production of TPX MAP-specific IgG1 serum antibodies, and increase in lung interleukin-1 (IL-1), uric acid, and reactive oxygen species (ROS) content were associated with significant (P < 0.05) 50 % reduction in recovery of lung-stage larvae. Increase in lung triglycerides and cholesterol levels appeared to provide the surviving worms with nutrients necessary for a stout double lipid bilayer barrier at the parasite-host interface. Surviving worms-released products elicited memory responses to the MAP-3 immunogen, including production of specific IgG1 antibodies and increase in liver IL-33 and ROS. Reduction in challenge worm burden recorded 45 days post infection did not exceed 48 % associated with no differences in parasite egg counts in the host liver and small intestine compared to unimmunized adjuvant control mice. Alum adjuvant assisted the second peptide, MAP-4, in production of IgG1, IgG2a, IgG2b and IgA specific antibodies and increase in liver ROS, but with no protective potential, raising doubt about the necessity of adjuvant addition. Accordingly, different vaccine formulas containing TPX MAP and 1, 2 or 3 cysteine peptidases-derived peptides with or without alum were used to immunize parallel groups of mice. Compared to unimmunized control mice, significant (P < 0.05 to < 0.005) 22 to 54 % reduction in worm burden was recorded in the different groups associated with insignificant changes in parasite egg output. The results together indicated that a schistosomiasis vaccine able to entirely prevent disease and halt its transmission still remains elusive.

Novel Approaches to Mortierella alpina Identification and Arachidonic Acid Production Optimization.

Arachidonic acid (ARA) is an integral constituent of cell structures and is instrumental for the nervous, muscular, and immune systems' functions. The sore need for this nutrient may be fulfilled via production based on the fungus Mortierella alpina. The identity of the M. alpina culture obtained from Assiut University, Egypt, was confirmed based on internal transcribed spacer DNA barcoding and elongation enzyme RNA sequencing. Liquid media glucose and peptone as carbon and nitrogen sources, respectively, and diverse micronutritional factors were adjusted for optimal biomass and ARA production. Shake flask cultivation at 25 °C for 7 days produced around 0.570 g of ARA per liter of culture. M. alpina treatment using mutagen 5-fluorouracil and octyl gallate-supplemented glucose-yeast-agar screening plates and shake-flask incubation at 25 °C, then at 20 °C, followed by aging at 10 °C, led to >3 g ARA/liter culture, a yield considered suitable for potential commercial production.

Increased hepatic interleukin-1, arachidonic acid, and reactive oxygen species mediate the protective potential of peptides shared by gut cysteine peptidases against Schistosoma mansoni infection in mice.

BACKGROUND: Multiple antigen peptide (MAP) construct of peptide with high homology to Schistosoma mansoni cathepsin B1, MAP-1, and to cathepsins of the L family, MAP-2, consistently induced significant (P < 0.05) reduction in challenge S. mansoni worm burden. It was, however, necessary to modify the vaccine formula to counteract the MAP impact on the parasite egg counts and vitality, and discover the mechanisms underlying the vaccine protective potential. METHODOLOGY: Outbred mice were immunized with MAP-2 in combination with alum and/or MAP-1. Challenge infection was performed three weeks (wks) after the second injection. Blood and liver pieces were obtained on an individual mouse basis, 23 days post-infection (PI), a time of S. mansoni development and feeding in the liver before mating. Serum samples were examined for the levels of circulating antibodies and cytokines. Liver homogenates were used for assessment of liver cytokines, uric acid, arachidonic acid (ARA), and reactive oxygen species (ROS) content. Parasitological parameters were evaluated 7 wks PI. PRINCIPAL FINDINGS: Immunization of outbred mice with MAP-2 in combination with alum and/or MAP-1 elicited highly significant (P < 0.005) reduction of around 60% in challenge S. mansoni worm burden and no increase in worm eggs' loads or vitality, compared to unimmunized or alum pre-treated control mice. Host memory responses to the immunogens are expected to be expressed in the liver stage when worm feeding and cysteine peptidases release start to be active. Serum antibody and cytokine levels were not significantly different between control and vaccinated mouse groups. Highly significant (P < 0.05 - <0.0001) increase in liver interleukin-1, ARA, and ROS content was recorded in MAP-immunized compared to control mice. CONCLUSION/SIGNIFICANCE: The findings provided an explanation for the gut cysteine peptidases vaccine-mediated reduction in challenge worm burden and increase in egg counts.

Protective immune potential of multiple antigenic peptide (MAP) constructs comprising peptides that are shared by several cysteine peptidases against Schistosoma mansoni infection in mice.

In vaccine trials, Schistosoma mansoni cathepsin B1 (SmCB1), helminth cathepsins of the L family (e.g., SmCL3), and papain consistently induce highly significant reductions in challenge worm burden and egg viability, but generated no additive protective effects when used in combination. The protective capacity of the cysteine peptidases is associated with modest (SmCB1) and poor (cathepsins L) production of cytokines and antibodies, essentially of the type 2 axis, and is only marginally reduced upon use of proteolytically inactive enzymes. In this work, peptides shared by SmCB1, cathepsins of the L family, papain and other allergens were selected, synthesized as tetrabranched multiple antigen peptide constructs (MAP-1 and MAP-2), and used in two independent experiments to immunize outbred mice, in parallel with papain. The two peptides elicited significant (P < 0.05) reduction in challenge worm burden when compared to unimmunized mice, albeit lower than that achieved by papain. Protection was associated with modest serum type 2 cytokines and antibody levels in MAP-, and papain-immunized mice. Immunization with papain also elicited a reduction in parasite egg load, viability, and granuloma numbers in liver and intestine. MAP-1 and MAP-2 immunogens displayed some opposite effects- MAP-1 leading to higher egg numbers with poor vitality, whereas MAP-2 immunization yielded fewer eggs. Cysteine peptidase thus appear to carry peptides that elicit opposing outcomes, highlighting the difficulty of reaching fully fledged protection, unless a vaccine is based on carefully selected peptides and combined with an effective adjuvant.

Clarification of Arachidonic Acid Metabolic Pathway Intricacies.

Surrounding inflammation activates phospholipase A2, which cleaves and releases arachidonic acid (ARA) from cell membranes. The four cis double bonds are instrumental in ARA susceptibility to oxidation, resulting in the generation of numerous bioactive metabolites of critical importance for the immune system, namely inflammation in response to pathogens, resolution of inflammation, wound healing, and mood and energy balance. The ARA metabolism steps are replete with intricacies, deterring researchers from identifying targets, which could be useful in modulating the synthesis of ARA metabolites toward exclusive protection of the host from pathogens, endogenous excessive danger signals, pain, inflammation, stress, and anxiety disorders. While ARA metabolic pathways are reasonably defined, it was deemed mandatory to fully clarify the flow and direction of protons, electrons, and oxygen atoms and the intricacies behind formation and breakage of double bonds and cyclic structures. This in-depth novel information will perfect the development of strategies and drugs aimed at counteracting inflammation and promoting healing.

In vitro and in vivo activities of arachidonic acid against Schistosoma mansoni and Schistosoma haematobium.

The development of arachidonic acid (ARA) for treatment of schistosomiasis is an entirely novel approach based on a breakthrough discovery in schistosome biology revealing that activation of parasite tegument-bound neutral sphingomyelinase (nSMase) by unsaturated fatty acids, such as ARA, induces exposure of parasite surface membrane antigens to antibody binding and eventual attrition of developing schistosomula and adult worms. Here, we demonstrate that 5 mM ARA leads to irreversible killing of ex vivo 1-, 3-, 4-, 5-, and 6-week-old Schistosoma mansoni and 9-, 10-, and 12-week-old Schistosoma haematobium worms within 3 to 4 h, depending on the parasite age, even when the worms were maintained in up to 50% fetal calf serum. ARA-mediated worm attrition was prevented by nSMase inhibitors, such as CaCl(2) and GW4869. Scanning and transmission electron microscopy revealed that ARA-mediated worm killing was associated with spine destruction, membrane blebbing, and disorganization of the apical membrane structure. ARA-mediated S. mansoni and S. haematobium worm attrition was reproduced in vivo in a series of 6 independent experiments using BALB/c or C57BL/6 mice, indicating that ARA in a pure form (Sigma) or included in infant formula (Nestle) consistently led to 40 to 80% decrease in the total worm burden. Arachidonic acid is already marketed for human use in the United States and Canada for proper development of newborns and muscle growth of athletes; thus, ARA has potential as a safe and cost-effective addition to antischistosomal therapy.

Immunofluorescent Localization of Proteins in Schistosoma mansoni.

Immunofluorescence allows the detection, visualization, and localization of proteins by using the ability of antibodies to firmly bind to specific antigens. Proteins must be accessible to thorough interaction with the specific antibodies. Different immune evasion mechanisms of parasites are directed to hamper or prevent access of antibodies to critical proteins or virulence factors. The blood fluke Schistosoma mansoni would not survive a day in the host blood capillaries if antibodies were able to readily bind to proteins located at the surface and mediate its attrition and demise by the complement system and/or the FcγR- or FcαR-bearing leukocytes. The worm surface is the area of parasite-host interaction and the route to critical nutrients, but is selectively permeable, allowing access of nutrient molecules but not host antibodies. Gentle procedures, which, however, are not commonly in use in vivo, are required to increase the permeability of the parasite outer membrane shield to just allow access of specific antibodies and identify and localize the proteins at the apical surface. Robust methods involving acetone, methanol, and Triton X-100 treatment lead to disintegration of the dual lipid bilayer cover with exposure of the proteins located in the tegument beneath. Internal proteins may not be accessed except following cryostat or paraffin sectioning. Accordingly, vaccine-induced specific antibodies to the apical surface or tegument proteins are unable to harm intact parasites. Specific antibodies to surface membrane proteins may only add to the action of administered or endo schistosomicides via acceleration of killing and interference with repair of severely and lightly impacted parasites, respectively. Therefore, careful immunofluorescent localization of S. mansoni proteins is important for devising the different control strategies against infection.

Arachidonic Acid Is a Safe and Efficacious Schistosomicide, and an Endoschistosomicide in Natural and Experimental Infections, and Cysteine Peptidase Vaccinated Hosts.

Blood flukes of the genus Schistosoma are covered by a protective heptalaminated, double lipid bilayer surface membrane. Large amounts of sphingomyelin (SM) in the outer leaflet form with surrounding water molecules a tight hydrogen bond barrier, which allows entry of nutrients and prevents access of host immune effectors. Excessive hydrolysis of SM to phosphoryl choline and ceramide via activation of the parasite tegument-associated neutral sphingomyelinase (nSMase) with the polyunsaturated fatty acid, arachidonic acid (ARA) leads to parasite death, via allowing exposure of apical membrane antigens to antibody-dependent cell-mediated cytotoxicity (ADCC), and accumulation of the pro-apoptotic ceramide. Surface membrane nSMase represents, thus, a worm Achilles heel, and ARA a valid schistosomicide. Several experiments conducted in vitro using larval, juvenile, and adult Schistosoma mansoni and Schistosoma haematobium documented ARA schistosomicidal potential. Arachidonic acid schistosomicidal action was shown to be safe and efficacious in mice and hamsters infected with S. mansoni and S. haematobium, respectively, and in children with light S. mansoni infection. A combination of praziquantel and ARA led to outstanding cure rates in children with heavy S. mansoni infection. Additionally, ample evidence was obtained for the powerful ARA ovocidal potential in vivo and in vitro against S. mansoni and S. haematobium liver and intestine eggs. Studies documented ARA as an endogenous schistosomicide in the final mammalian and intermediate snail hosts, and in mice and hamsters, immunized with the cysteine peptidase-based vaccine. These findings together support our advocating the nutrient ARA as the safe and efficacious schistosomicide of the future.

Specific Antibodies and Arachidonic Acid Mediate the Protection Induced by the Schistosoma mansoni Cysteine Peptidase-Based Vaccine in Mice.

Several reports have documented the reproducible and considerable efficacy of the cysteine peptidase-based schistosomiasis vaccine in the protection of mice and hamsters against infection with Schistosoma mansoni and Schistosomahaematobium, respectively. Here, we attempt to identify and define the protection mechanism(s) of the vaccine in the outbred CD-1 mice-S. mansoni model. Mice were percutaneously exposed to S. mansoni cercariae following immunization twice with 0 or 10 µg S. mansoni recombinant cathepsin B1 (SmCB1) or L3 (SmCL3). They were examined at specified intervals post infection (pi) for the level of serum antibodies, uric acid, which amplifies type 2 immune responses and is an anti-oxidant, lipids, in particular, arachidonic acid (ARA), which is an endoschistosomicide and ovocide, as well as uric acid and ARA in the lung and liver. Memory IgG1, IgG2a, and IgG2b antibodies to the cysteine peptidase immunogen were detectable at and following day 17 pi. Serum, lung, and liver uric acid levels in immunized mice were higher than in naïve and unimmunized mice, likely as a consequence of cysteine peptidase-mediated catabolic activity. Increased circulating uric acid in cysteine peptidase-immunized mice was associated with elevation in the amount of ARA in lung and liver at every test interval, and in serum starting at day 17 pi. Together, the results suggest the collaboration of humoral antibodies and ARA schistosomicidal potential in the attrition of challenge S. mansoni (p < 0.0005) at the liver stage, and ARA direct parasite egg killing (p < 0.005). The anti-oxidant and reactive oxygen species-scavenger properties of uric acid may be responsible for the cysteine peptidase vaccine protection ceiling. This article represents a step towards clarifying the protection mechanism of the cysteine peptidase-based schistosomiasis vaccine.

Transforming growth factor-beta and Th17 responses in resistance to primary murine schistosomiasis mansoni.

Discovery of the T-helper (Th) 17 cell lineage and functions in immune responses of mouse and man prompted us to investigate the role of transforming growth factor-beta (TGF-beta) and interleukin (IL)-17 in innate resistance to murine schistosomiasis mansoni. Schistosoma mansoni-infected BALB/c and C57BL/6 mice were administered with recombinant TGF-beta or mouse monoclonal antibody to TGF-beta to evaluate the impact of this cytokine on host immune responses against lung-stage schistosomula, and subsequent effects on adult worm parameters. Developing schistosomula elicited increase in peripheral blood mononuclear cells (PBMC) mRNA expression and/or plasma levels of IL-4, IL-17, and interferon-gamma (IFN-gamma), cytokines known to antagonize each other, resulting in impaired Th1/Th2, and Th17 immune responses and parasite evasion. Mice treated with TGF-beta showed elevated PBMC mRNA expression of IL-6, IL-17, TGF-beta, and TNF-alpha mRNA and increased IL-23 and IL-17 or TGF-beta plasma levels, associated with significantly (P<0.02-<0.0001) lower S. mansoni adult worm burden compared to controls in both mouse strains, thus suggesting that TGF-beta led to heightened Th17 responses that mediated resistance to the infection. Mice treated with antibody to TGF-beta showed increase in PBMC mRNA expression and plasma levels of IL-4, IL-12p70, and IFN-gamma, and significantly (P<0.02 and <0.0001) reduced worm burden and liver worm egg counts than untreated mice, indicating that Th1/Th2 immune responses were potentiated, resulting in significant innate resistance to schistosomiasis. The implications of these observations for schistosome immune evasion and vaccination were discussed.

Role of T lymphocytes and papain enzymatic activity in the protection induced by the cysteine protease against Schistosoma mansoni in mice.

Papain, an experimental model protease, was used to decipher the protective mechanism(s) of the cysteine peptidase-based schistosomiasis vaccine. To examine the role of T lymphocytes, athymic nude (nu/nu) and immunocompetent haired (nu/+) mice were subcutaneously (sc) injected with 50 µg active papain two days before percutaneous exposure to 100 cercariae of Schistosoma mansoni. Highly significant (P < 0.005) reductions in worm burden required competent T lymphocytes, while significant increases (P < 0.05) of >80% in dead parasite ova in the small intestine were independent of T cell activity and likely relied on the innate immune axis. To investigate the role of enzymatic activity, immunocompetent mice were sc injected with 50 µg active or E-64-inactivated papain two days before exposure to cercariae. The reductions in worm burden were highly significant (P < 0.0001), reaching >65% and 40% in active and inactivated papain-treated mice, respectively. Similar highly significant (P < 0.0001) decreases of 85% in the viability of parasite ova in the small intestine occurred in both active and inactivated papain-treated mice. These findings indicated that immune responses elicited by one or more papain structural motifs are necessary and sufficient for induction of considerable parasite and egg attrition. Correlates of protection included IgG1-dominated antibody responses and increases in the levels of uric acid and arachidonic acid in the lung and liver upon parasite migration in these sites. Identification of the shared patterns or motifs in cysteine peptidases and evaluation of their immune protective potential will pave the way to the development of a safe, efficacious, storage-stable, and cost-effective schistosomiasis vaccine.

Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase is a lung-stage schistosomula surface membrane antigen.

We have previously reported that Schistosoma mansoni larvae emerging from host lung at pH 7.5-7.8 and then fixed with diluted formaldehyde (HCHO) readily bind radiation-attenuated cercariae (RA) vaccine serum antibodies, as assessed by indirect membrane immunofluorescence (IF). Here we show that S. mansoni schistosomula emerging from lung pieces under 5% CO2 (pH < or = 7.3) readily bind RA vaccine serum antibodies, provided they have been incubated for 12 h at pH 7.5-7.8 in foetal calf serum-free RPMI medium, and fixed with diluted HCHO. Ex vivo larvae exposed during incubation to GW4869, a specific inhibitor of tegument-bound, neutral sphingomyelinase (nSMase) displayed significantly diminished binding of RA vaccine serum antibodies, thus suggesting that nSMase activity at pH > or = 7.5 leads to exposure of lung-stage larvae surface membrane antigens to specific antibody detection. More importantly, ex vivo larvae readily bound antibodies directed to dipeptidic multiple antigen peptide constructs, based on S. mansoni-specific sequences in S. mansoni glyceraldehyde 3-phosphate dehydrogenase (SG3PDH). Lung-stage schistosomula IF reactivity was diminished following antiserum absorption with recombinant SG3PDH. The data together indicate that intact ex vivo, as well as, 5-day-old in vitro-grown larvae express SG3PDH on their surface membrane. The findings are discussed in relation to the importance of surface membrane proteins as candidate vaccine antigens.