Munirathinam Gnanasekar

Novel Phage Display-Based Subtractive Screening To Identify Vaccine Candidates of Brugia malayi

ABSTRACT 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.

Molecular characterization of a calcium binding translationally controlled tumor protein homologue from the filarial parasites Brugia malayi and Wuchereria bancrofti

We have cloned homologues of the mammalian translationally controlled tumor protein (TCTP) from the human filarial parasites Wuchereria bancrofti and Brugia malayi. TCTP genes from B. malayi and W. bancrofti were expressed in a T7 promoter vector as histidine tagged fusion proteins. Both the recombinant B. malayi TCTP (rBm-TCTP) and recombinant W. bancrofti TCTP (rWb-TCTP) have a molecular mass of approximately 28 kDa with the histidine tag. Sequence analyses showed that there is a 98% similarity between the two filarial TCTPs at amino acid levels and are immunologically cross-reactive. Analysis of soluble proteins from various lifecycle stages of B. malayi suggested that the expression of Bm-TCTP might be differentially regulated and occurs in multimeric form. Recombinant TCTP were found to form multimers in solution under non-reducing conditions. The tendency for filarial TCTPs to become multimers was predicted by the presence of the Lupas coiled coil structure in their sequence. Despite the absence of a signal sequence, Bm-TCTP is present abundantly in the excretory/secretions (ES) of microfilariae. Characterization studies showed that both Bm- and Wb-TCTPs are calcium-binding proteins and have histamine-releasing function in vitro. When injected intraperitoneally both the filarial TCTPs induced inflammatory infiltration of eosinophils into the peritoneal cavity of mice suggesting that the filarial TCTPs may have a role in the allergic inflammatory responses associated with filarial infections.

Evaluation of Wuchereria bancrofti GST as a vaccine candidate for lymphatic filariasis.

Background Lymphatic filarial parasites survive within the lymphatic vessels for years despite the complex immune environment surrounding them. Parasites possibly accomplish this by adopting various immunomodulatory strategies, which include release of glutathione-S-transferases (GSTs) that counteract the oxidative free radicals produced by the host. Since GSTs produced by parasites appear to be critical for the survival of parasites in the host, several studies evaluated the potential of parasite GSTs as vaccine candidates especially against schistosomiasis, fascioliasis and Seteria cervi. However, vaccine potential of GSTs of lymphatic filarial parasites has not been evaluated before. Methods/Principal Findings In the present study, the GST gene was cloned from the third stage larval (L3) cDNA libraries of Wuchereria bancrofti, and recombinant GST (WbGST) was expressed and purified. Serum samples from individuals living in an endemic area were analyzed for their reactivity with rWbGST. These findings showed that sera from endemic normal individuals (EN) carry significant levels of anti-WbGST IgG antibodies compared to subjects who are microfilaraemic (Mf) or show symptoms of clinical pathology (CP). Isotype analysis of the anti-WbGST IgG antibodies showed a predominance of IgG1 and IgG3 antibodies in EN individuals. Subsequent functional analysis of the rWbGST showed that the rWbGST protein retained the enzymatic activity of GST and the antibodies in EN sera could inhibit this enzymatic activity. Similar results were obtained when anti-rWbGST antibodies raised in mice were used in the neutralization assay. Brugia malayi GST and WbGST show significant sequence similarity. Therefore, to evaluate the vaccine potential of rWbGST, we used B. malayi L3 as challenge parasites. Vaccine potential of rWbGST was initially evaluated by confirming the role of human and mice WbGST antibodies in an antibody dependent cellular cytotoxicity (ADCC) assay. Subsequent vaccination studies in a jird model showed that approximately 61% protection could be achieved against a B. malayi L3 challenge infection in jirds immunized with rWbGST. Conclusions Results of this study show that rWbGST is a potential vaccine candidate against lymphatic filariasis. Nearly 61% protection can be achieved against a B. malayi challenge infection in a jird model. The study also showed that the WbGST protein retained the enzymatic activity of GST and this enzymatic activity appears to be critical for the survival of the parasite in the host.

Praziquantel Affects the Regulatory Myosin Light Chain of Schistosoma mansoni

ABSTRACT Praziquantel (PZQ) is the drug of choice for schistosomiasis and probably is the only highly effective drug currently available for treating schistosomiasis-infected individuals. The mode of action of PZQ involves increasing the calcium uptake of the parasite, resulting in tegumental damage and death of the parasite. Despite its remarkable function, the target of PZQ has not been identified yet. To begin to understand where PZQ acts, in this study we expressed the cDNA library of Schistosoma mansoni on the surface of T7 bacteriophages and screened this library with labeled PZQ. This procedure identified a clone that strongly bound to PZQ. Subsequent DNA analysis of inserts showed that the clone coded for regulatory myosin light chain protein. The gene was then cloned, and recombinant S. mansoni myosin light chain (SmMLC) was expressed. Immunoblot analysis using antibodies raised to recombinant SmMLC (rSmMLC) showed that SmMLC is abundantly expressed in schistosomula and adult stages compared to the amount in cercarial stages. In vitro analyses also confirmed that PZQ strongly binds to rSmMLC. Further, peptide mapping studies showed that PZQ binds to amino acids 46 to 76 of SmMLC. Immunoprecipitation analysis confirmed that SmMLC is phosphorylated in vivo upon exposure to PZQ. Interestingly, significant levels of anti-SmMLC antibodies were present in vaccinated mice compared to the amount in infected mice, suggesting that SmMLC may be a potential target for protective immunity in schistosomiasis. These findings suggest that PZQ affects SmMLC function, and this may have a role in PZQ action.

Targeting receptor for advanced glycation end products (RAGE) expression induces apoptosis and inhibits prostate tumor growth

Expression of receptor for advanced glycation end products (RAGE) plays a key role in the progression of prostate cancer. However, the therapeutic potential of targeting RAGE expression in prostate cancer is not yet evaluated. Therefore in this study, we have investigated the effects of silencing the expression of RAGE by RNAi approach both in vitro and in vivo. The results of this study showed that down regulation of RAGE expression by RNAi inhibited the cell proliferation of androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells. Furthermore, targeting RAGE expression resulted in apoptotic elimination of these prostate cancer cells by activation of caspase-8 and caspase-3 death signaling. Of note, the levels of prostate specific antigen (PSA) were also reduced in LNCaP cells transfected with RAGE RNAi constructs. Importantly, the RAGE RNAi constructs when administered in nude mice bearing prostate tumors, inhibited the tumor growth by targeting the expression of RAGE, and its physiological ligand, HMGB1 and by up regulating death receptors DR4 and DR5 expression. Collectively, the results of this study for the first time show that targeting RAGE by RNAi may be a promising alternative therapeutic strategy for treating prostate cancer.

18α-glycyrrhetinic acid targets prostate cancer cells by down-regulating inflammation-related genes

Glycyrrhetinic acid is an active triterpenoid metabolite of glycyrrhizin abundantly present in licorice roots. Glycyrrhetinic acid exists as α and β stereo-isomeric forms. Both stereo-isomeric forms are known to have anti-inflammatory and anticancer activity. However, the effects and anticancer mechanism of α glycyrrhetinic acid in prostate cancer cells has not yet been evaluated. Therefore, we investigated the growth inhibition, induction of apoptosis and the anticancer mechanisms of 18α-glycyrrhetinic acid (AGA), on the androgen-independent metastatic prostate cancer cell line DU-145. Our results showed that AGA inhibited proliferation and growth of these cells by inducing apoptosis as determined by Annexin V and flow cytometry analyses. Our studies also showed that HUVEC tube formation was drastically reduced when cultured in conditioned medium of AGA-treated DU-145 cells. In addition, AGA treatment prevented the invasion of DU-145 prostate cancer cells on matrigel coated transwells via down-regulation of NF-κB (p65), VEGF and MMP-9 expression. Furthermore, AGA treatment also down-regulated the expression of pro-inflammatory cytokine/growth factor genes HMGB1, IL-6 and IL-8 in DU-145 cells. Interestingly, AGA simultaneously upregulated the expression of non-steroidal anti-inflammatory gene-1 (NAG-1) in DU-145 cells suggesting its anti-inflammatory activity on prostate cancer cells. Taken together, the results of this study suggest that AGA may be a promising anticancer agent that merits further investigation for the chemoprevention and treatment of prostate cancer.

Possible role of Toxoplasma gondii in brain cancer through modulation of host microRNAs

BackgroundThe obligate intracellular protozoan parasite Toxoplasma gondii infects humans and other warm-blooded animals and establishes a chronic infection in the central nervous system after invasion. Studies showing a positive correlation between anti-Toxoplasma antibodies and incidences of brain cancer have led to the notion that Toxoplasma infections increase the risk of brain cancer. However, molecular events involved in Toxoplasma induced brain cancers are not well understood.Presentation of the hypothesisToxoplasma gains control of host cell functions including proliferation and apoptosis by channelizing parasite proteins into the cell cytoplasm and some of the proteins are targeted to the host nucleus. Recent studies have shown that Toxoplasma is capable of manipulating host micro RNAs (miRNAs), which play a central role in post-transcriptional regulation of gene expression. Therefore, we hypothesize that Toxoplasma promotes brain carcinogenesis by altering the host miRNAome using parasitic proteins and/or miRNAs.Testing the hypothesisThe miRNA expression profiles of brain cancer specimens obtained from patients infected with Toxoplasma could be analyzed and compared with that of normal tissues as well as brain cancer tissues from Toxoplasma uninfected individuals to identify dysregulated miRNAs in Toxoplasma- driven brain cancer cells. Identified miRNAs will be further confirmed by studying cancer related miRNA profiles of the different types of brain cells before and after Toxoplasma infection using cell lines and experimental animals.Expected outcomeThe miRNAs specifically associated with brain cancers that are caused by Toxoplasma infection will be identified.Implications of the hypothesisToxoplasma infection may promote initiation and progression of cancer by modifying the miRNAome in brain cells. If this hypothesis is true, the outcome of this research would lead to the development of novel biomarkers and therapeutic tools against Toxoplasma driven brain cancers.

HMGB1: A Promising Therapeutic Target for Prostate Cancer

High mobility group box 1 (HMGB1) was originally discovered as a chromatin-binding protein several decades ago. It is now increasingly evident that HMGB1 plays a major role in several disease conditions such as atherosclerosis, diabetes, arthritis, sepsis, and cancer. It is intriguing how deregulation of HMGB1 can result in a myriad of disease conditions. Interestingly, HMGB1 is involved in cell proliferation, angiogenesis, and metastasis during cancer progression. Furthermore, HMGB1 has been demonstrated to exert intracellular and extracellular functions, activating key oncogenic signaling pathways. This paper focuses on the role of HMGB1 in prostate cancer development and highlights the potential of HMGB1 to serve as a key target for prostate cancer treatment.

Vitamin K2, a naturally occurring menaquinone, exerts therapeutic effects on both hormone-dependent and hormone-independent prostate cancer cells

In recent years, several studies have shown that vitamin k2 (VK2) has anticancer activity in a variety of cancer cells. The antitumor effects of VK2 in prostate cancer are currently not known. In the present study, we sought to characterize the anticancer potential of VK2 in both androgen-dependent and -independent prostate cancer cells. Our investigations show that VK2 is able to suppress viability of androgen-dependent and androgen-independent prostate cancer cells via caspase-3 and -8 dependent apoptosis. We also show that VK2 treatment reduces androgen receptor expression and PSA secretion in androgen-dependent prostate cancer cells. Our results also implicate VK2 as a potential anti-inflammatory agent, as several inflammatory genes are downregulated in prostate cancer cells following treatment with VK2. Additionally, AKT and NF-kB levels in prostate cancer cells are reduced significantly when treated with VK2. These findings correlated with the results of the Boyden chamber and angiogenesis assay, as VK2 treatment reduced cell migration and angiogenesis potential of prostate cancer cells. Finally, in a nude mice model, VK2 administration resulted in significant inhibition of both androgen-dependent and androgen-independent tumor growth. Overall, our results suggest that VK2 may be a potential therapeutic agent in the treatment of 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.