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.

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.

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.

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.

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.

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.

Fasciola antigens as vaccines against fascioliasis and schistosomiasis.

Fascioliasis is an important trematode infection of herbivores worldwide with increasing evidence of prevalence as a disease of humans. Vaccination studies with purified native and recombinant Fasciola antigens suggest that this approach to diminished morbidity and mortality and reduced transmission is a realistic goal. Among the major potential vaccine candidates are fatty acid binding protein (FABP), cysteine (cathepsin) proteases, haemoglobulin, leucine aminopeptidase, and a saposin-like protein. In the case of Fasciola hepatica FABP, cross-reaction and cross-protection against Schistosoma mansoni is an important feature. In addition to protective effects with significant worm burden reductions, some vaccine candidates also have anti-fecundity (smaller flukes), anti-pathology (less liver lesions), and anti-embryonation effects. Optimism is tempered by the fact that fascioliasis in humans is an orphan disease and in need of governmental and foundation support.