The expression of prosaposin and its receptors, GRP37 and GPR37L1, are increased in the developing dorsal root ganglion.

Prosaposin (PSAP), a highly conserved glycoprotein, is a precursor of saposins A-D. Accumulating evidence suggests that PSAP is a neurotrophic factor, as well as a regulator of lysosomal enzymes. Recently, the orphan G-protein-coupled receptors GPR37 and GPR37L1 were recognized as PSAP receptors, but their functions have not yet been clarified. In this study, we examined the distribution of PSAP and its receptors in the dorsal root ganglion (DRG) during development using specific antibodies, and showed that PSAP accumulates primarily in lysosomes and is dispersed throughout the cytoplasm of satellite cells. Later, PSAP colocalized with two receptors in satellite cells, and formed a characteristic ring shape approximately 8 weeks after birth, during a period of rapid DRG development. This ring shape, which was only observed around larger neurons, is evidence that several satellite cells are synchronously activated. We found that sortilin, a transporter of a wide variety of intracellular proteins containing PSAP, is strongly localized to the inner side of satellite cells, which contact the neuronal surface. These findings suggest that PSAP and GPR37/GPR37L1 play a role in activating both satellite and nerve cells.

High-resolution Crystal Structure of Human pERp1, A Saposin-like Protein Involved in IgA, IgM and Integrin Maturation in the Endoplasmic Reticulum.

The folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein folding factors, some of which are protein-specific. The ER resident saposin-like protein pERp1 (MZB1, CNPY5) is crucial for the correct folding of IgA, IgM and integrins. pERp1 also plays a role in ER calcium homeostasis and plasma cell mobility. As an important factor for proper IgM maturation and hence immune function, pERp1 is upregulated in many auto-immune diseases. This makes it a potential therapeutic target. pERp1 belongs to the CNPY family of ER resident saposin-like proteins. To date, five of these proteins have been identified. All are implicated in protein folding and all contain a saposin-like domain. All previously structurally characterized saposins are involved in lipid binding. However, there are no reports of CNPY family members interacting with lipids, suggesting a novel function for the saposin fold. However, the molecular mechanisms of their function remain elusive. To date, no structure of any CNPY protein has been reported. Here, we present the high-resolution (1.4 Å) crystal structure of human pERp1 and confirm that it has a saposin-fold with unique structural elements not present in other saposin-fold structures. The implications for the role of CNPY proteins in protein folding in the ER are discussed.

Two short antimicrobial peptides derived from prosaposin-like proteins in the starry flounder (Platichthys stellatus).

Prosaposin (PSAP) is a precursor of saposin (SAP), which is present in lysosomal and secreted proteins. PSAP is a member of the SAP-like protein families, which comprise multifunctional proteins. In particular, their antimicrobial activity has been reported. We identified PSAP-like (PsPSAPL) sequences from starry flounder and analysed their expression and antimicrobial activity based on cDNA and amino acid sequences. PsPSAPL showed conservation of three saposin B type domains at high levels, and PsPSAPL mRNA was relatively abundantly distributed in the brain and gills of healthy starry founders. PsPSAPL mRNA showed significant expression changes in response to viral haemorrhagic septicaemia virus and Streptococcus parauberis. Synthetic peptides (PsPSAPL-1 and -2), prepared based on amino acid sequences, were used to confirm as well as analyse the antimicrobial activity against bacteria and parasites. Consequently, PsPSAPL-1 and -2 were found to significantly inhibit the growth of various bacteria and kill the Miamiensis avidus. In addition, bacterial biofilm formation was significantly inhibited. Safety was also confirmed by analysing cell haemolysis. These results indicate the immunological function of PsPSAP and the potential antimicrobial activity of the AMPs PsPSAPL-1 and -2.

The Saposin-Like Protein AplD Displays Pore-Forming Activity and Participates in Defense Against Bacterial Infection During a Multicellular Stage of Dictyostelium discoideum.

Due to their archaic life style and microbivor behavior, amoebae may represent a source of antimicrobial peptides and proteins. The amoebic protozoon Dictyostelium discoideum has been a model organism in cell biology for decades and has recently also been used for research on host-pathogen interactions and the evolution of innate immunity. In the genome of D. discoideum, genes can be identified that potentially allow the synthesis of a variety of antimicrobial proteins. However, at the protein level only very few antimicrobial proteins have been characterized that may interact directly with bacteria and help in fighting infection of D. discoideum with potential pathogens. Here, we focus on a large group of gene products that structurally belong to the saposin-like protein (SAPLIP) family and which members we named provisionally Apls (amoebapore-like peptides) according to their similarity to a comprehensively studied antimicrobial and cytotoxic pore-forming protein of the protozoan parasite Entamoeba histolytica. We focused on AplD because it is the only Apl gene that is reported to be primarily transcribed further during the multicellular stages such as the mobile slug stage. Upon knock-out (KO) of the gene, aplD- slugs became highly vulnerable to virulent Klebsiella pneumoniae. AplD- slugs harbored bacterial clumps in their interior and were unable to slough off the pathogen in their slime sheath. Re-expression of AplD in aplD- slugs rescued the susceptibility toward K. pneumoniae. The purified recombinant protein rAplD formed pores in liposomes and was also capable of permeabilizing the membrane of live Bacillus megaterium. We propose that the multifarious Apl family of D. discoideum comprises antimicrobial effector polypeptides that are instrumental to interact with bacteria and their phospholipid membranes. The variety of its members would allow a complementary and synergistic action against a variety of microbes, which the amoeba encounters in its environment.

A Comparative Study of Human Saposins.

Saposins are small proteins implicated in trafficking and loading of lipids onto Cluster of Differentiation 1 (CD1) receptor proteins that in turn present lipid antigens to T cells and a variety of T-cell receptors, thus playing a crucial role in innate and adaptive immune responses in humans. Despite their low sequence identity, the four types of human saposins share a similar folding pattern consisting of four helices linked by three conserved disulfide bridges. However, their lipid-binding abilities as well as their activities in extracting, transporting and loading onto CD1 molecules a variety of sphingo- and phospholipids in biological membranes display two striking characteristics: a strong pH-dependence and a structural change between a compact, closed conformation and an open conformation. In this work, we present a comparative computational study of structural, electrostatic, and dynamic features of human saposins based upon their available experimental structures. By means of structural alignments, surface analyses, calculation of pH-dependent protonation states, Poisson-Boltzmann electrostatic potentials, and molecular dynamics simulations at three pH values representative of biological media where saposins fulfill their function, our results shed light into their intrinsic features. The similarities and differences in this class of proteins depend on tiny variations of local structural details that allow saposins to be key players in triggering responses in the human immune system.

Performance of different prostate specific antibodies in the cytological diagnosis of metastatic prostate adenocarcinoma.

BACKGROUND: Diagnosis of metastatic prostate adenocarcinoma (metPA) in cytology specimens can be challenging and frequently requires the use of immunohistochemistry (IHC). Prostate specific membrane antigen (PSMA) and NKX3.1 have emerged as promising IHC markers to determine prostatic origin of metPA in surgical specimens. Our goal is to evaluate the performance of PSMA and NKX3.1 and compare them with those of prostate-specific antigen (PSA) and prostate specific alkaline phosphatase (PSAP) in the cytological diagnosis of metPA MATERIALS: Cytology specimens from patients with a history of prostate adenocarcinoma at our institution between January 01, 2005 and December 31, 2015 were retrieved. IHC stains were performed on the cell blocks. In addition to the staining pattern and intensity, the sensitivity, and specificity of PSMA and NKX3.1 were assessed and compared to those of PSA and PSAP markers. RESULTS: A total of 56 cytology cases were retrieved with the following diagnoses: 13 metPA, 37 metastatic carcinomas from other origins, 4 rare atypical cells, and 2 benign. Additional 9 cases were re-classified as metPA based on positive PSMA and/or NKX3.1 immunostains. In our cohort of 22 cases of metPA, 18 were positive for PSMA (82%), 15 for NKX3.1 (68%), 9 for PSA (41%), and 9 for PSAP (41%). PSMA and NKX3.1 were negative in all 6 cases of metastatic carcinoma of nonprostate origin (specificity 100%). PSMA demonstrated strong membranous staining pattern, and NKX3.1 exhibited moderate nuclear staining pattern. CONCLUSION: Because of their higher sensitivity and specificity, PSMA and NKX3.1 are valuable surrogate markers for metPA in cytology specimens, when compared with PSA and PSAP markers.

Expression, purification and in vitro refolding of the recombinant truncated Saposin-like protein 2 antigen for development of diagnosis of human fascioliasis.

Early diagnosis of fascioliasis is critical in prevention of injury to the liver and bile ducts. Saposin-like protein (FhSAP-2) is probably the most ideal antigen of Fasciola hepatica for development of ELISA kits. SAP-2 has a conserved tertiary structure containing three disulfide bonds and conformational epitopes. Therefore, antigenicity of SAP-2 is greatly depends on disulfide bond formation and proper folding. We produced the recombinant truncated SAP-2 (rtSAP-2) in the SHuffle® T7 and Rosetta strain of Escherichia coli, in soluble and insoluble forms, respectively and purified by immobilized metal affinity chromatography (IMAC). The refolding process of denatured rtSAP-2 was performed using dialysis and dilution methods in the presence of chemical additives, along with reduced/oxidized glutathione (in vitro). Physicochemical studies, including non-reducing gel electrophoresis, Ellman's assay, Western blotting and ELISA showed the most antigenicity and likely correct folding of rtSAP-2, which was obtained by dialysis method. An IgG ELISA test was developed using rtSAP-2 refolded by dialysis and compared with excretory/secretory products of parasite with 52 positive fascioliasis samples, 79 other parasitic samples and 70 negative controls samples. The results exhibited 100% sensitivity and 98% specificity for rtSAP-2, also, 100% and 95.3% for excretory/secretory (E/S) antigen, respectively. In conclusion, it is suggested that rtSAP-2 with the correct folding could be used as a candidate antigen for detection of human fascioliasis.

Saposin-like Proteins, a Multigene Family of Schistosoma Species, are Biomarkers for the Immunodiagnosis of Schistosomiasis Japonica.

BACKGROUND: One major obstacle to schistosomiasis prevention and control is the lack of accurate and sensitive diagnostic approaches, which are essential for planning, targeting, and evaluating disease control efforts. METHODS: Based on bioinformatics analysis, we identified a multigene family of saposin-like protein (SAPLP) in the schistosome genomes. Schistosoma japonicum SAPLPs (SjSAPLPs), including recently reported promising biomarker SjSP-13, were systematically and comparatively assessed as immunodiagnostic antigens for schistosomiasis japonica. RESULTS: Two novel antigens (SjSAPLP4 and SjSAPLP5) could specifically react to serum samples from both S. japonicum-infected laboratory animals and patients. The sensitivities of SjSAPLP4, SjSAPLP5, and SjSP-13 for immunodiagnosis were 98% (95% confidence interval, 88.0%-99.9%), 96% (85.1%-99.3%), and 88% (75.0%-95.0%), respectively, and 100% (91.1%-100%) specificity was observed for the 3 antigens with enzyme-linked immunosorbent assay; there was no cross-reaction with clonorchiosis (0 of 19 patients), echinococcosis (0 of 20 patients), or trichinellosis (0 of 18 patients) for the 3 antigens. Antibodies to the 3 antigens could be detected in the serum samples of rabbits infected with 1000 cercariae as early as 3-4 weeks after infection. CONCLUSIONS: These results suggest that SjSAPLP4 and SjSAPLP5 could serve as novel biomarkers for the immunodiagnosis of schistosomiasis japonica, which will further improve diagnostic sensitivity and specificity.

Saposin-like protein 2 has an immunodiagnostic potential for detecting Fasciolosis gigantica.

Saposin-like protein 2 (SAP-2) plays an important role in the digestive process of Fasciola gigantica (Fg). It is one of the major proteins synthesized by the caecal epithelial cells and released into fluke's excretion-secretion. Therefore, FgSAP-2 is a plausible target for detecting fasciolosis. A polyclonal antibody (PoAb) against recombinant FgSAP-2 was produced by immunizing rabbits with the recombinant protein (rFgSAP-2), and used in sandwich ELISA assay to detect the circulating FgSAP-2 in sera of mice experimentally infected with F. gigantica metacercariae. The assay could detect rFgSAP-2 and the native FgSAP-2 in the excretory-secretory (ES) and whole body (WB) fractions of adult F. gigantica at the concentrations as low as 38 pg/ml, 24 ng/ml, and 102 ng/ml, respectively. As well, the sera from mice experimentally infected with F. gigantica were tested positive by this sandwich ELISA, which exhibited sensitivity, specificity, false positive rate, false negative rate and accuracy at 99.99, 98.67, 1.33, 0.01 and 99.32%, respectively. Therefore, this assay could be used for diagnosis of fasciolosis by F. gigantica.

Molecular characterization and identification of Th1 epitopes of a Schistosoma japonicum protein similar to prosaposin.

The tegument of schistosomula contains T cell antigens that might simulate the protective mechanisms of the radiation-attenuated vaccine in a mouse model of schistosomiasis. Immune mechanisms mediated by the CD4+ Th1 response are important in the RAV model. To rapidly identify Th1 epitopes in molecules from the Schistosoma japonicum schistosomula tegument, this study analyzed S. japonicum proteomics data. Preliminary experiments identified a protein similar to prosaposin (SjPSAP) from the tegument of schistosomula. We confirmed that SjPSAP was present in the tegument of the parasite using an indirect immunofluorescence assay. We then identified Th cell epitopes in SjPSAP using in silico prediction combined with experimental validation. From the SjPSAP sequence, we used several algorithms to predict 11 promiscuous Th cell epitopes that might bind to both murine and human MHC class II molecules. To validate the in silico predictions, proliferation and cytokine production profiles of spleen lymphocytes from BALB/c mice immunized with the 11 predicted peptides were measured in vitro using a modified methyl thiazolyl tetrazolium assay and flow cytometry. The results showed that 4 of the 11 predicted peptides induced a recall CD4+ Th1 response in vitro. We measured direct binding of the four peptides predicted to induce a response to antigen-presenting cells from BALB/c mice using a fluorometric method and found that the peptides bound to both I-Ad and I-Ed mouse molecules. These results demonstrated that potentially protective Th1-type epitopes in SjPSAP molecules could be identified rapidly by combining in silico prediction with experimental validation. This strategy could be a fast method for identifying Th1 epitopes in a schistosoma antigen with features such as large size or poor expression of recombinant antigens.

Vaccine potential of recombinant saposin-like protein 2 against Fasciolosis gigantica in mice.

Saposin-like protein 2 (SAP-2) is a protein that adult of Fasciola spp. use to lyse plasma membrane of red blood cells, so that their contents can be digested by proteases for the parasites' nutrients. Thus SAP-2 is a plausible target for vaccination against these parasites. Recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was expressed in Escherichia coli BL21 (DE3). A vaccination was performed in ICR mice (n=10) by subcutaneous injection with 50µg of rFgSAP-2 combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 30 F. gigantica metacercariae by oral route. The percentages of protection of rFgSAP-2 vaccine against F. gigantica were estimated to be 76.4-78.5% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. The antibodies in immune sera of vaccinated mice were shown by immuno-blotting to react with native FgSAP-2 in the extract of 2- and 4-week-old juvenile parasites. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune responses, it was found that both Th1 and Th2 humoral immune response were significantly increased in rFgSAP-2 immunized group compared with the control groups, with higher levels of Th2 (IgG1) than Th1 (IgG2a). The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) in rFgSAP-2-immunized group showed no significant difference from those of the non-immunized and infected group, indicating that early juvenile parasites induced liver parenchyma damage, even though the numbers of worm recoveries were significantly different. This study indicates that rFgSAP-2 has a high potential as a vaccine candidate against F. gigantica in mice, and this potential will be tested in larger economic animals.

Production and characterization of a monoclonal antibody against recombinant saposin-like protein 2 of Fasciola gigantica.

A monoclonal antibody (MoAb) against recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with rFgSAP-2. This MoAb is an IgG1, κ light chain isotype. By immunoblotting and indirect ELISA, the MoAb reacted specifically with rFgSAP-2, the natural FgSAP-2 at 10kDa in whole body (WB) and excretory-secretory (ES) fractions of F. gigantica. It did not cross react with antigens in WB fractions from other parasites, including Opisthorchis viverrini, Schistosoma mansoni which are human parasites, Haemonchus placei, Setaria labiato-papillosa, Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gigantocotyle explanatum, Gastrothylax crumenifer, and Paramphistomum cervi which are ruminant parasites. By immunohistochemistry, the FgSAP-2 protein was localized only in the cytoplasm of caecal epithelial cells of 4-week-old juvenile and adult stages, but not in metacercariae, newly excysted juvenile (NEJ), 2- and 3-week-old juveniles. This finding indicated that FgSAP-2 is an abundantly expressed parasite protein that is released into the ES, hence SAP-2 and its MoAb may be used for immunodiagnosis of ruminant and human fasciolosis.

Distribution and characteristics of ABFs, cecropins, nemapores, and lysozymes in nematodes.

Several groups of antimicrobial effector molecules have been identified in nematodes, but most studies have been limited to Caenorhabditis elegans and, to a lesser extent, Ascaris suum. Although these two species are not closely related, they are not representative of overall nematode diversity. This study utilized available sequence information to investigate whether four groups of antimicrobial effectors (defensin-like antibacterial factors [ABFs], cecropins, saposin domain-containing proteins, and lysozymes) are components of an archetypal nematode immune system or more narrowly restricted. Saposin domain-containing proteins (caenopores in C. elegans) and lysozymes were widely distributed and found in most taxa, but likely have digestive as well as defensive functions. ABFs were widely distributed in fewer taxa, suggesting selective loss in some lineages. In contrast, cecropins were identified in only three related species, suggesting acquisition of this effector molecule in their common ancestor.

Toll-like receptor 4 can recognize SapC-DOPS to stimulate macrophages to express several cytokines.

OBJECTIVE AND DESIGN: SapC-DOPS is a newly combined compound consisting of saposin C and dioleoylphosphatidylserine (DOPS). Our recent study showed that SapC-DOPS exhibits anti-tumor activity. However, SapC-DOPS has recognition elements of Toll-like receptor (TLR) 2 and TLR4; therefore, we want to know whether SapC-DOPS can induce abnormal immunoreaction via identification TLRs. METHODS: We investigated the capacity of SapC-DOPS to induce cytokines in vivo and in vitro and analyzed the involvement of TLR and NF-kB in these cytokines production. RESULTS: SapC-DOPS could activate the cytokine production by peripheral macrophages, enhance the expressions of TLR4 and stimulate the NF-κB nuclear translocation. PDTC, an NF-κB inhibitor, could decrease the SapC-DOPS inducible TNF-α and IL-1ß production. CONCLUSIONS: SapC-DOPS was similar to LPS in the immune response and may induce the production of cytokines in macrophages via the TLR4 signaling pathway and, at least in part, the alteration of the NF-κB pathway.

Partial immunity to Fasciola hepatica in mice after vaccination with FhSAP2 delivered as recombinant protein or DNA construct.

INTRODUCTION: We recently reported the biochemical characterization of a novel Fasciola hepatica recombinant antigen termed FhSAP2, which has previously shown to elicit protection to F. hepatica infection in rabbits. Further we reported that intramuscular (IM) injections of BALB/c mice with a pFLAG-CMV(-2) vector carrying cDNA encoding for FhSAP2 (cDNA-FhSAP2) induce high levels of immune response. The aim of the present study is to ascertain whether the immune response induced by this DNA construct may induce protection in mice against subsequent infection with F. hepatica metacercariae (mc). In addition, protection following subcutaneous (SC) injections with recombinant FhSAP2 was evaluated. METHODS: Mice received three IM injections with 100 microg of cDNA-FhSAP2 or three SC injections with 20 microg of FhSAP2. Four weeks after the last vaccination mice were challenged orally with 5 F. hepatica me and euthanized 45 days after challenge. RESULTS: Mean worm burdens found in mice vaccinated with cDNA-FhSAP2 was reduced by 83.3% and the mean worm burdens found in mice vaccinated with the recombinant protein was reduced by 60% when compared with controls. All vaccinated animals had less liver damage than challenge controls. Vaccination with cDNA-FhSAP2 seems to favor a mixed Th1/Th2-antibody dependent with higher predominance of Th1-regulated antibody response. CONCLUSIONS: The vaccination with cDNA-FhSAP2 or recombinant FhSAP2 may protect hosts against F. hepatica infections. The vaccination with cDNA form of FhSAP2 appeared to be a little more efficient preventingthe infection. The predominance of Th1-dependent antibodies in the vaccinated animals may be responsible for the protection but this should be confirmed by Th1-cytokines determinations.

Regional expression of prosaposin in the wild-type and saposin D-deficient mouse brain detected by an anti-mouse prosaposin-specific antibody.

Prosaposin is a precursor of saposins A, B, C, and D. Saposins are indispensable for lysosomal hydrolysis of sphingolipids. The notion that prosaposin itself is likely involved in brain development led us to generate an anti-mouse prosaposin-specific antibody that do not cross-react with any of the processed saposins. We have used it to study expression of prosaposin in the brain of wild-type (WT) and saposin D knockout mice (Sap-D(-/-)). Immunoblot studies indicated that prosaposin, already abundant in the brain of WT, was dramatically increased in Sap-D(-/-). By immunohistochemistry, the brain of WT was rich in prosaposin in hippocampal CA3 pyramidal neurons, tufted cells and mitral cells in olfactory bulb, and cerebellar Purkinje cells. In Sap-D(-/-), immunoreactivity of prosaposin was increased in these neurons, most notably in the CA3 pyramidal neurons which contained prosaposin immuno-positive inclusion bodies in the endoplasmic reticulum. Further characterization of these prosaposin-rich neurons may provide new insights into the physiological functions of prosaposin in the nervous system.

Alpha anomers of iGb3 and Gb3 stimulate cytokine production by natural killer T cells.

Natural killer T cells (NKT cells) respond to presentation of specific glycolipids with release of a variety of proinflammatory and immunomodulatory cytokines. The repertoire of glycolipid antigens for these cells includes alpha-glycosylceramides, alpha-glycosyldiacylglycerols, and the triglycosylceramide iGb3. Two features of iGb3 set it apart from these other antigens: (i) three sugars are required for stimulation and (ii) the glycosidic bond between ceramide and the proximal sugar is beta in iGb3, whereas it is alpha in other antigens. We have synthesized the alpha versions of iGb3 and Gb3 and demonstrate that they are effective antigens for NKT cells and that they do not require lysosomal processing to the monoglycosylceramides for stimulation. These triglycosylceramides constitute a new class of antigen that stimulates NKT cells comparably to monoglycosylceramides.

Saposin-like proteins are expressed in the gastrodermis of Schistosoma mansoni and are immunogenic in natural infections.

BACKGROUND: Schistosomes are parasitic blood flukes that inhabit the portal blood system of humans. Ingested red cells are lysed in the gastrodermis to enable the parasites to digest hemoglobin. Saposin-like proteins (SAPLIPs) have been reported from the gastrodermis of related flukes, and at least one is hemolytic and a promising vaccine antigen. We now provide the first report of SAPLIPs from schistosomes and explore their role in host-parasite interactions. METHODS: We identified expressed sequence tags encoding a family of SAPLIPs from Schistosoma mansoni and produced one (termed Sm-SLP-1) in recombinant form using baculovirus. The anatomic site of SLP-1 expression within the worm was assessed and its recognition by sera from chronically infected humans and mice was determined. The vaccine efficacy of Sm-SLP-1 was tested in a mouse model. RESULTS: Full-length sequences were obtained for two cDNAs, Sm-slp-1 and Sm-slp-2. The Sm-slp-1 open reading frame contained a single SAPLIP domain while Sm-slp-2 had a double domain. Sm-SLP-1 was immunolocalized to the gastrodermis of adult worms, but did not confer protection in a murine vaccination model of schistosomiasis. Mice infected with S. mansoni generated a specific antibody response to Sm-SLP-1. Individuals who were infected with S. mansoni had IgG that recognized Sm-SLP-1. IgG levels were statistically higher in individuals with heavy infection. CONCLUSIONS: Sm-SLP-1 is expressed in the gastrodermis of S. mansoni. It is immunogenic in humans and mice, but is not protective as a vaccine in its current form. Schistosome SAPLIPs warrant further attention to elucidate their roles in host-parasite interactions and to further explore their potential as vaccine and diagnostic antigens.

Fasciola hepatica: identification of CD4+ T-helper epitopes from the 11.5 kDa saposin-like protein SAP-2 using synthetic peptides.

Fasciola hepatica saposin-like protein (FhSAP-2) is a novel antigen expressed at an early stage of infection and has been shown to induce in rabbits a significant protection to infection with F. hepatica. There are no studies to identify the immunologically relevant regions of FhSAP-2. In this work the amino acid sequence of FhSAP-2 was analyzed to identify potential T-cell epitopes. A predictive algorithm identified four possible sites. Experimental determination of the T-cell epitopes was achieved using a panel of overlapping peptides spanning the entire sequence of FhSAP-2, which was evaluated for their ability to induce lymphoproliferative responses of spleen cells from 8 immunized BALB/c (H-2d) mice. Five different epitopes were identified. There was minimal agreement between theoretical and experimental approaches. It was found that peptides containing amino acid residues AVTFA and IDIDLCDICT as part of their structure induce high levels of IL-2 and IFNgammain vitro and was classified as Th1 epitopes. Peptides that contain the residues ADQTV, CIEFVQQEVD and YIIDHVDQHN induced significant amount of IL-4 and IL-2 were considered as containers of Th0 epitopes. Identification of prominent T-cell epitopes from FhSAP-2 offers the possibility of understanding how the CD4+ T-cell response is involved in protection against fasciolosis and how it is implicated in susceptibility to infection.