Protective immune response in BALB/c mice induced by DNA vaccine of the ROP8 gene of Toxoplasma gondii.

Toxoplasmosis in humans and other animals is caused by the protozoan parasite Toxoplasma gondii. During the process of host cell invasion and parasitophorous vacuole formation by the tachyzoites, the parasite secretes Rhoptry protein 8 (ROP8), an apical secretory organelle. Thus, ROP8 is an important protein for the pathogenesis of T. gondii. The ROP8 DNA was constructed into a pVAX-1 vaccine vector and used for immunizing BALB/c mice. Immunized mice developed immune response characterized by significant antibody responses, antigen-specific proliferation of spleen cells, and production of high levels of IFN-γ (816 ± 26.3 pg/mL). Challenge experiments showed significant levels of increase in the survival period (29 days compared with 9 days in control) in ROP8 DNA vaccinated mice after a lethal challenge with T. gondii. Results presented in this study suggest that ROP8 DNA is a promising and potential vaccine candidate against toxoplasmosis.

Sumoylation of human translationally controlled tumor protein is important for its nuclear transport.

Translationally controlled tumor protein (TCTP) lacks nuclear bipartite localization signal sequence; yet TCTP is present abundantly in the nucleus. At present it is not known how TCTP gets transported to the nucleus. Sequence analyses showed that all TCTPs described to date have putative small ubiquitin-like modifier (SUMO) motifs. Since SUMO modification plays an important role in the nuclear transport of proteins, we evaluated whether SUMO motifs are important for transport of TCTP into the nucleus. We show that TCTP exists in sumoylated form in cytoplasm and nucleus of mammalian cells. Point mutation of lysine residue in the SUMO motif compromised the ability of TCTP to get sumoylated in vitro. When cells were transfected with FLAG-tagged mutated TCTP, nuclear transport of TCTP was inhibited confirming that sumoylation is critical for the nuclear transport of TCTP. Our previous studies demonstrated that TCTP can function as an antioxidant protein in the nucleus. When we mutated TCTP at the SUMO motif the antioxidant function of TCTP was compromised. Results presented in this study thus show that sumoylation plays an important role in the transport of TCTP into the nucleus where they function as antioxidant protein.

Cloning and characterization of high mobility group box protein 1 (HMGB1) of Wuchereria bancrofti and Brugia malayi.

A human homologue of high mobility group box 1 (HMGB1) protein was cloned and characterized from the human filarial parasites Wuchereria bancrofti and Brugia malayi. Sequence analysis showed that W. bancrofti HMGB1 (WbHMGB1) and B. malayi HMGB1 (BmHMGB1) proteins share 99 % sequence identity. Filarial HMGB1 showed typical architectural sequence characteristics of HMGB family of proteins and consisted of only a single HMG box domain that had significant sequence similarity to the pro-inflammatory B box domain of human HMGB1. When incubated with mouse peritoneal macrophages and human promyelocytic leukemia cells, rBmHMGB1 induced secretion of significant levels of pro-inflammatory cytokines such as TNF-α, GM-CSF, and IL-6. Functional analysis also showed that the filarial HMGB1 proteins can bind to supercoiled DNA similar to other HMG family of proteins. BmHMGB1 protein is expressed in the adult and microfilarial stages of the parasite and is found in the excretory secretions of the live parasites. These findings suggest that filarial HMGB1 may have a significant role in lymphatic pathology associated with lymphatic filariasis.

T-oligo induces apoptosis in advanced prostate cancer cells.

Prostate cancer is the most frequently diagnosed malignancy in men. As cancer progresses from an androgen-sensitive stage to hormone-refractory stage, it turns resistant to androgen ablation therapy. At this stage, effective newer therapies that induce apoptosis are needed for treatment of prostate cancer. DNA oligonucleotides homologous to the telomere 3' overhang (T-oligo) induce apoptosis in several human cancer cells. In the present study, we studied the effect of T-oligo on prostate cancer cells. Our studies showed that androgen-independent DU-145 cells are sensitive to T-oligo in terms of inhibition of proliferation. Moreover, T-oligo induced DU-145 cells to undergo apoptosis. Therefore, our results are encouraging for further investigation in the potential application of T-oligo as a novel therapeutic approach for prostate cancer, especially the androgen-independent.

Translationally controlled tumor protein is a novel heat shock protein with chaperone-like activity.

Translationally controlled tumor protein (TCTP) is often designated as a stress-related protein because of its highly regulated expression in stress conditions. Following a thermal shock, TCTP expression is highly upregulated in a variety of cells. However, at present it is not known whether this upregulation has any cell protective function similar to other heat shock proteins. In this study human TCTP (HuTCTP) and a TCTP homolog (SmTCTP) from Schistosoma mansoni were evaluated for heat shock protein-like function and molecular chaperone activity. Our results show that similar to other molecular chaperones, both human and parasite TCTPs can bind to a variety of denatured proteins and protect them from the harmful effects of thermal shock. An important observation was the ability of both HuTCTP and SmTCTP to bind to native protein and protect them from thermal denaturation. Over expression of TCTP in bacterial cells protected them from heat shock-induced death. These findings suggest that TCTP may belong to a novel small molecular weight heat shock protein.

Gene silencing of translationally controlled tumor protein (TCTP) by siRNA inhibits cell growth and induces apoptosis of human prostate cancer cells.

Translationally controlled tumor protein (TCTP) is a novel anti apoptotic protein which is highly expressed in several cancer cell types including prostate cancer. However, studies investigating the role of TCTP in prostate cancer are scarce. Therefore, in this study we evaluated the effect of small interference RNA (siRNA) based knocking down of TCTP gene in prostate cancer cells. Cell proliferation and apoptosis were evaluated. Our results showed that TCTP is highly expressed in LNCaP cells compared to normal prostate epithelial cells. Transfection with TCTP siRNA specifically and drastically reduced the expression of both mRNA and protein levels of TCTP in LNCaP cells. The decreased expression of TCTP was associated with decreased viability of LNCaP cells. Further analysis of the transfected LNCaP cells showed that they undergo apoptosis via caspase-8 and caspase-3 dependent pathways. Results presented herein suggest a potential therapeutic application for prostate cancer by targeting TCTP gene using an siRNA approach.

Short hairpin RNA (shRNA) constructs targeting high mobility group box-1 (HMGB1) expression leads to inhibition of prostate cancer cell survival and apoptosis.

High mobility group box protein 1 (HMGB1), transcriptional activity regulatory protein is associated with most cancers including prostate cancer. To investigate the effects of down-regulation of HMGB1 expression, we have transfected LNCaP cells with four short hairpin RNA (shRNA) targeting HMGB1 plasmid vectors. Transfection with the four shRNAs efficiently and specifically reduced the HMGB1 expression in LNCaP cells. The gene silencing effects on HMGB1 expression were subsequently confirmed by RT-PCR and immunoblotting analyses. Down-regulation of HMGB1 expression resulted in the inhibition of cell growth in LNCaP prostate cancer cells and the decreased cell number was due to transfected cells undergoing apoptosis via caspase-3-dependent pathways. These findings suggest that HMGB1 is critical for the survival of prostate cancer cells and targeted knockdown of HMGB1 mRNA can be used as a strategy to kill prostate cancer cells. Our findings may have some potential therapeutic relevance for treating prostate cancer.

Glycyrrhizin induces apoptosis in prostate cancer cell lines DU-145 and LNCaP.

Over 2 million Americans are currently living with prostate cancer. Current chemotherapeutic strategies are only partially effective in controlling the disease. There is always a need for an effective newer drug for treating prostate cancer. Use of active principles from medically important herbs has proven to be effective in treating various forms of cancers. Glycyrrhizin, a triterpene compound isolated from roots of licorice has been found to exhibit potent in vitro cytotoxic activity against several human cancer cell lines. In this study, we evaluated the effects of glycyrrhizin on the viability of two human prostate cancer cells LNCaP (hormone-dependent) and DU-145 (hormone-independent) in vitro. Cell viability assay showed that glycyrrhizin inhibited the cell proliferation of prostate cancer cells in a time- and dose-dependent manner. The decreased viability of prostate cancer cells was due to apoptosis as confirmed by Annexin-V FITC flow cytometric analyses. Glycyrrhizin also caused DNA damage in DU-145 and LNCaP cells in a time-dependent manner. Caspase-3 and -8 activities were not detected in glycyrrhizin-treated prostate cancer cells suggesting that caspase-independent pathways may be involved in the apoptotic mechanism. Collectively, these studies suggest that glycyrrhizin has therapeutic potential against prostate cancer.

A novel small heat shock protein 12.6 (HSP12.6) from Brugia malayi functions as a human IL-10 receptor binding protein.

Phage display cDNA expression library of the third stage larvae (L3) of Brugia malayi was screened for identifying target(s) that bound to the human interleukin-10 receptor (huIL10R). This iterative screening identified an insert that showed significant homology to Caenorhabditis elegans HSP12.6. The gene was designated B. malayi HSP12.6 (BmHSP12.6) and has orthologues in several gastrointestinal nematode genome (Ancylostoma caninum, Ascaris lumbricoides and Ascaris suum) but the gene or gene product has not been studied further in these parasites. Structural analyses of BmHSP12.6 showed that it has a highly conserved alpha-crystallin central domain that is characteristic of other small heat shock proteins (HSPs). BmHSP12.6 has a short N-terminal domain and an unusually small C-terminal domain flanking the crystallin domain suggesting that this protein belongs to a novel class of small HSPs. BmHSP12.6 appears to be differentially transcribed with highest expression in the vertebrate stages of the parasite (L4, adult and mf) compared to its mosquito vector stage (L3). More importantly recombinant BmHSP12.6 bound to huIL10R in a dose dependent fashion and inhibited the binding of human IL-10 (huIL10) to huIL10R in vitro. rBmHSP12.6 also enhanced the growth and proliferation of MC/9 mast cells in vitro similar to huIL10. This study thus describes a novel small HSP from B. malayi that has the capacity to bind to huIL10R, block binding of huIL10 to huIL10R and function similar to huIL10.

Identification and cloning of a novel tetraspanin (TSP) homologue from Brugia malayi.

This is the first report of a tetraspanin (TSP)-like molecule in the lymphatic filarial parasites. Expressed sequence tag (EST) database search for TSP like molecules in the filarial genome resulted in three significant EST hits (two partial ESTs from Brugia malayi and one full length EST from Wuchereria bancrofti). The full length gene cloned from B. malayi showed significant similarity to Caenorhabditis elegans TSP and human TSP and hence the gene was named B. malayi TSP (BmTSP). Subsequent Genbank analysis with the predicted ORF of BmTSP showed additional homologous genes reported from Schistosoma mansoni and Taenia solium parasites. Structural analyses showed that BmTSP has four transmembrane domains and other conserved domains such as CCG and two other critical cysteine residues present within the large extracellular loop similar to other reported TSPs. In addition, putative post-translational modifications such as N-glycosylation, protein kinase c phosphorylation, casein kinase II phosphorylation and N-myristoylation sites have been found in BmTSP sequence. Further, PCR analyses showed that BmTSP is differentially transcribed, with highest level of expression being present in the adult stages followed by L3 and mf stages. This study thus describes a novel TSP cloned from B. malayi, its putative functions in cuticle biogenesis and role in protective immunity.

Translationally controlled tumor protein of Brugia malayi functions as an antioxidant protein.

Translationally controlled tumor protein (TCTP) is one of the most abundantly expressed proteins in the filarial parasites as well as in the other organisms. Several functions have been suggested for TCTP family of proteins ranging from calcium binding to histamine release function. However, its physiological function is still a mystery. Previous studies showed that the expression of TCTP is increased several-fold during oxidative stress. In the present work, we report the putative antioxidant function of Brugia malayi TCTP (BmTCTP). When tested in vitro, rBmTCTP could be reduced by a variety of reducing agents including thioredoxin. Such reduced form of rBmTCTP was able to protect DNA from oxidative damage, suggesting that BmTCTP may have an antioxidant function in the parasite. Sequence analysis of filarial TCTPs revealed that there are three cysteine amino acids located in the central portion of the protein. Subsequent targeted residue modification studies showed that these cysteine residues in rBmTCTP are critical for its antioxidant function. To determine the significance of this finding, rBmTCTP was overexpressed in vivo in Escherichia coli and subjected to oxidative stress. These studies showed that rBmTCTP significantly protected cells form oxidative damage. Taken together, these findings suggest that BmTCTP might be functioning as a non-classical antioxidant protein in the filarial parasites.

Immune response studies with Wuchereria bancrofti vespid allergen homologue (WbVAH) in human lymphatic filariasis.

A homologue of Brugia malayi venom allergen (BmVAH) was cloned from the infective stages (L3) of Wuchereria bancrofti. Sequence analysis showed 90% sequence identity between WbVAH and BmVAH. Recombinant WbVAH was then expressed and purified. VAH from other nematode parasites is being evaluated as potential vaccine candidates. Because W. bancrofti infections are more prevalent than B. malayi, it will significantly benefit using W. bancrofti antigens for vaccine development. In this study, we have evaluated the human immune responses to rWbVAH in putatively immune individuals who live in the endemic regions (endemic normal, EN) to determine the vaccine potential of WbVAH. These responses were then compared to those in infected individuals (microfilaraemic, MF and chronic pathology, CP). Results show that EN subjects carry WbVAH-specific IgG1, IgG2, and IgG3 circulating antibodies. It is interesting to note that CP patients also carried antibodies against WbVAH that was mainly of the IgG3 isotype. Peripheral blood mononuclear cells (PBMC) from EN individuals responded strongly to rWbVAH by proliferating and secreting IFN-gamma. PBMC from MF patients also proliferated in response to rWbVAH but secreted mainly IL-10. Thus, there was a clear dichotomy in the cytokine production by infected patients vs individuals who are putatively immune (EN). Although vaccine potential of WbVAH has not been established yet, our findings suggest that WbVAH mediated immune responses in EN individuals is primarily Th1-biased. Further vaccination studies are underway in animal models to determine the role of WbVAH in protective immunity against W. bancrofti and B. malayi infections.

Brugia malayi: comparison of protective immune responses induced by Bm-alt-2 DNA, recombinant Bm-ALT-2 protein and prime-boost vaccine regimens in a jird model.

Immunization of jirds with Bm-alt-2 elicited partial protection against challenge infection with the filarial parasite Brugia malayi. In this study, we initially compared the protective immune responses elicited following immunization with recombinant Bm-ALT-2 protein regimen and Bm-alt-2 DNA regimen. These studies showed that protein vaccination conferred approximately 75% protection compared to DNA vaccination that conferred only 57% protection. Analysis of the protective immune responses showed that the protein immunization promoted a Th2-biased response with an increase in IL-4, IL-5 and IgG1 responses, whereas, the DNA vaccine promoted a Th1-biased response with profound IFN-gamma and IgG2a responses. Since protein vaccination gave better results than DNA vaccination, we then wanted to evaluate whether a prime-boost vaccination that combined DNA prime and protein boost will significantly increase the protective responses induced by the protein vaccine. Our results suggest that prime-boost vaccination had no added advantage and was comparatively less effective (64% protection) than the Bm-ALT-2 protein alone vaccination. Prime boost vaccination generated mixed Th1/Th2 responses with a slightly diminished Th2 responses compared to protein vaccination. Thus, our results suggest that Bm-ALT-2 protein vaccination regimen may be slightly better than prime-boost vaccine regimen and the mechanism of protection appears to be largely mediated by a Th2-biased response.

Cloning and characterization of a high mobility group box 1 (HMGB1) homologue protein from Schistosoma mansoni.

Mammalian homologue of high mobility group box chromatin protein (HMGB) 1 was identified and cloned from human parasites, Schistosoma mansoni and S. haematobium. Sequence analyses showed that the parasite HMGB1s has 35-40% identity to human and rodent HMGB1s, and 33% identity to Caenorhabditis elegans HMGB1. Parasite HMGB1s also contains an A box and B box domain similar to mammalian HMGB1, however, it lacks the C-terminal tail that is present in mammalian HMGB1s. Analysis of the expression of HMGB1 in various life cycle stages of S. mansoni reveal S. mansoni HMGB1 (SmHMGB1) as a stage-specific protein, expressed abundantly in egg and adult female stages and at moderate levels in skin-stage schistosomula. Significant levels of SmHMGB1 were also present in excretory secretions of egg stages. Subsequent characterization studies showed that SmHMGB1 is a potent inducer of pro-inflammatory cytokines such as TNFalpha, IL-1Ralpha, IL-2Ralpha, IL-6, IL-13, IL-13Ralpha1, IL-15 and MIP-1alpha from mouse peritoneal macrophages. Pro-inflammatory activity, especially production of TNFalpha-inducing activity, appears to be a function of the B box domain protein. This was confirmed by both real-time reverse transcription PCR and by cytokine ELISA. Thus, results presented in this study suggest that SmHMGB1 may be a key molecule in the development of host inflammatory immune responses associated with schistosomiasis.

Cloning and characterization of a novel immunogenic protein 3 (NIP3) from Brugia malayi by immuno screening of a phage-display cDNA expression library.

Iterative screening of a phage display cDNA expression library of the third-stage larvae (L3) of Brugia malayi with sera from putatively immune individuals (endemic normal, EN) identified a novel clone with insert showing significant homology to Onchocerca volvulus novel immunogenic protein-3 (Ov-NIP3) gene and Caenorhabditis elegans NIP3-like protein and hence the gene was designated Brugia malayi NIP3-like protein (BmNIP3). EST database analysis showed that ESTs from several gastrointestinal nematodes such as Ancylostoma caninum, Teladorsagia circumcincta, Haemonchus contortus and Strongyloides stercoralis has BmNIP3 homologues, but the gene has not been described from these parasites. Sequence analyses showed that BmNIP3 has three potential mucin-type O-glycosylation sites and several serine/threonine phosphorylation sites. As expected, BmNIP3 protein isolated from the parasite was serine/threonine phosphorylated. Further analyses showed that BmNIP3 is differentially transcribed, with highest level of expression present in the larval (L3 and L4) stages. Mice immunized with rBmNIP3 developed strong antibody responses predominantly of the IgG1 and IgG2a subtype. A similar analyses of the sera samples from EN individuals showed that they also carry high levels of IgG1 and IgG2 antibodies against BmNIP3, whereas, chronically infected patients carry largely IgG3 antibodies and MF individuals carry high levels of IgG1 antibodies against BmNIP3. This study thus describes a novel protein from B. malayi that appears to be highly immunogenic in both humans and mice.

Comparison of skin invasion among three major species of Schistosoma.

A comparison of the host-finding behavior, mode of skin invasion and skin-migratory patterns of the three major schistosomes of humans reveals major differences. Among the three species, Schistosoma japonicum is remarkable at conserving energy during the host-finding process, and exhibiting swift migration through the skin to reach the predilection site sooner and mature earlier compared with Schistosoma mansoni and Schistosoma haematobium. In this article, we summarize and compare the penetration and migratory behavior of schistosomula of the three major human schistosomes through mouse and human skin.

Novel phage display-based subtractive screening to identify vaccine candidates of Brugia malayi.

This study describes a novel phage display method based on an iterative subtraction strategy to identify candidate vaccine antigens of Brugia malayi. A cDNA library of the infective larval stage of B. malayi expressed on the surface of T7 phage was sequentially screened with sera samples from human subjects showing different manifestations of the disease. Antigens that selectively and specifically bind to immune sera were then enriched using a multi-step panning procedure. This strategy identified five antigens, four of which were previously reported (ALT-2, TPX-2, VAH and COX-2) and the other one was a novel cuticular collagen (Col-4). Sera from immune individuals specifically recognized all the five antigens. However, ALT-2 appeared to be the most predominantly recognized antigen by the immune sera. Therefore, it was decided to evaluate the vaccine potential of recombinant ALT-2 (rALT-2) in a mouse and jird model. The results presented show that immunization with rALT-2 conferred over 73% protection against a challenge infection in the jird model and over 64% protection in the mouse model. The present study suggests that phage display-based cDNA screening may be a powerful tool to identify candidate vaccine antigens of infectious agents.

Skin-stage schistosomula of Schistosoma mansoni produce an apoptosis-inducing factor that can cause apoptosis of T cells.

Skin-stage schistosomula of Schistosoma mansoni were found to secrete molecules that are pro-apoptotic for skin T lymphocytes as measured by annexin V staining, caspase-3 activity, caspase-8 activities, and DNA fragmentation. Caspase-8 activities in lymphocytes peaked approximately 8 h and caspase-3 activity peaked approximately 16 h after exposure to the parasite secretions. Subset analysis showed that mainly CD4(+) and CD8(+) cells (but not B cells) were susceptible to the parasite-induced pro-apoptotic effect. In situ staining confirmed the presence of apoptotic T cells around challenge parasites in the skin of naive or immunized animals. Analysis of T cells to identify the potential molecular pathway of the parasite-induced apoptosis showed increases in the expression of Fas, FasL, and the Fas-associated death domain. Blocking of FasL with a fusion protein reversed the parasite-induced apoptosis, suggesting a role for the Fas/FasL-mediated pathway in the parasite-induced T cell apoptosis. Subsequent analyses of the secretions of skin-stage schistosomula identified the pro-apoptotic activity as being associated with a protein of approximately 23 kDa. This protein was termed S. mansoni-derived apoptosis-inducing factor.

Cloning and characterization of a calcium-binding, histamine-releasing protein from Schistosoma mansoni.

A homologue of the mammalian translationally controlled tumor protein (TCTP) was cloned from the human parasite Schistosoma mansoni (SmTCTP). Sequence analysis showed that SmTCTP differed from other reported TCTPs in having only one signature sequence. Subsequently, SmTCTP was cloned in a T7 expression system and expressed as a histidine-tagged fusion protein. Recombinant SmTCTP (rSmTCTP) has a molecular mass of approximately 23 kDa with the histidine tag. Further analysis showed that SmTCTP transcripts and protein are expressed in all life cycle stages of the parasite within the vertebrate hosts. Interestingly, antibodies to SmTCTP were present in the sera of mice 9 weeks after infection with S. mansoni. Characterization studies showed that rSmTCTP is a calcium-binding protein that can cause histamine release from basophil/mast cells and induce eosinophil infiltration. These findings suggest that SmTCTP may have an important role in the development of allergic inflammatory responses associated with schistosomiasis and may be a target for new drug development.