Gnanasekar Munirathinam

Biochemical Characterization and Evaluation of a Brugia malayi Small Heat Shock Protein as a Vaccine against Lymphatic Filariasis

Filarial nematodes enjoy one of the longest life spans of any human pathogen due to effective immune evasion strategies developed by the parasite. Among the various immune evasion strategies exhibited by the parasite, Interleukin 10 (IL-10) productions and IL-10 mediated immune suppression has significant negative impact on the host immune system. Recently, we identified a small heat shock protein expressed by Brugia malayi (BmHsp12.6) that can bind to soluble human IL-10 receptor alpha (IL-10R) and activate IL-10 mediated effects in cell lines. In this study we show that the IL-10R binding region of BmHsp12.6 is localized to its N-terminal region. This region has significant sequence similarity to the receptor binding region of human IL-10. In vitro studies confirm that the N-terminal region of BmHsp12.6 (N-BmHsp12.6) has IL-10 like activity and the region containing the alpha crystalline domain and C-terminus of BmHsp12.6 (BmHsp12.6αc) has no IL-10 like activity. However, BmHsp12.6αc contains B cell, T cell and CTL epitopes. Members of the sHSP families are excellent vaccine candidates. Evaluation of sera samples from putatively immune endemic normal (EN) subjects showed IgG1 and IgG3 antibodies against BmHsp12.6αc and these antibodies were involved in the ADCC mediated protection. Subsequent vaccination trials with BmHsp12.6αc in a mouse model using a heterologous prime boost approach showed that 83% protection can be achieved against B. malayi L3 challenge. Results presented in this study thus show that the N-BmHsp12.6 subunit of BmHsp12.6 has immunoregulatory function, whereas, the BmHsp12.6αc subunit of BmHsp12.6 has significant vaccine potential.

Piperine, a Bioactive Component of Pepper Spice Exerts Therapeutic Effects on Androgen Dependent and Androgen Independent Prostate Cancer Cells.

Prostate cancer is the most common solid malignancy in men, with 32,000 deaths annually. Piperine, a major alkaloid constituent of black pepper, has previously been reported to have anti-cancer activity in variety of cancer cell lines. The effect of piperine against prostate cancer is not currently known. Therefore, in this study, we investigated the anti-tumor mechanisms of piperine on androgen dependent and androgen independent prostate cancer cells. Here, we show that piperine inhibited the proliferation of LNCaP, PC-3, 22RV1 and DU-145 prostate cancer cells in a dose dependent manner. Furthermore, Annexin-V staining demonstrated that piperine treatment induced apoptosis in hormone dependent prostate cancer cells (LNCaP). Using global caspase activation assay, we show that piperine-induced apoptosis resulted in caspase activation in LNCaP and PC-3 cells. Further studies revealed that piperine treatment resulted in the activation of caspase-3 and cleavage of PARP-1 proteins in LNCaP, PC-3 and DU-145 prostate cancer cells. Piperine treatment also disrupted androgen receptor (AR) expression in LNCaP prostate cancer cells. Our evaluations further show that there is a significant reduction of Prostate Specific Antigen (PSA) levels following piperine treatment in LNCaP cells. NF-kB and STAT-3 transcription factors have previously been shown to play a role in angiogenesis and invasion of prostate cancer cells. Interestingly, treatment of LNCaP, PC-3 and DU-145 prostate cancer cells with piperine resulted in reduced expression of phosphorylated STAT-3 and Nuclear factor-κB (NF-kB) transcription factors. These results correlated with the results of Boyden chamber assay, wherein piperine treatment reduced the cell migration of LNCaP and PC-3 cells. Finally, we show that piperine treatment significantly reduced the androgen dependent and androgen independent tumor growth in nude mice model xenotransplanted with prostate cancer cells. Taken together, these results support further investigation of piperine as a potential therapeutic agent in the treatment of prostate cancer.

Multivalent fusion protein vaccine for lymphatic filariasis

Lymphatic filariasis affects approximately 3% of the whole world population. Mass drug administration is currently the major control strategy to eradicate this infection from endemic regions by year 2020. Combination drug treatments are highly efficient in controlling the infection. However, there are no effective vaccines available for human or animal lymphatic filariasis despite the identification of several subunit vaccines. Lymphatic filariasis parasites are multicellular organisms and potentially use multiple mechanisms to survive in the host. Therefore, there is a need to combine two or more vaccine candidate antigens to achieve the desired effect. In this study we combined three well characterized vaccine antigens of Brugia malayi, heat shock protein 12.6 (HSP12.6), Abundant Larval transcript-2 (ALT-2) and tetraspanin large extra cellular loop (TSP-LEL) as a multivalent fusion vaccine. Putative immune individuals carry circulating antibodies against all three antigens. Depletion of these antigen specific antibodies from the sera samples removed the ability of the sera to participate in the killing of B. malayi L3 in an antibody dependent cellular cytotoxicity (ADCC) mechanism. Vaccination trials in mice with a bivalent [HSP12.6+ALT-2 (HA), HSP12.6+TSP-LEL (HT) or TSP-LEL+ALT-2 (TA)] or trivalent [HSP12.6+ALT-2+TSP-LEL (HAT)] vaccines using DNA, protein or heterologous prime boost regimen showed that trivalent HAT vaccine either as protein alone or as heterologous prime boost vaccine could confer significant protection (95%) against B. malayi L3 challenge. Immune correlates of protection suggest a Th1/Th2 bias. These finding suggests that the trivalent HAT fusion protein is a promising prophylactic vaccine against lymphatic filariasis infection in human.

Dexamethasone promotes tolerance in vivo by enriching CD11cloCD40lo tolerogenic macrophages

We previously showed that antigen immunization in the presence of the immunosuppressant dexamethasone (a strategy we termed “suppressed immunization”) could tolerize established recall responses of T cells. However, the mechanism by which dexamethasone acts as a tolerogenic adjuvant has remained unclear. In the present study, we show that dexamethasone enriches CD11cloCD40lo macrophages in a dose‐dependent manner in the spleen and peripheral lymph nodes of mice by depleting all other CD11c+CD40+ cells including dendritic cells. The enriched macrophages display a distinct MHC class II (MHC II)loCD86hi phenotype. Upon activation by antigen in vivo, CD11cloCD40lo macrophages upregulate IL‐10, a classic marker for tolerogenic antigen‐presenting cells, and elicit a serum IL‐10 response. When presenting antigen in vivo, these cells do not elicit recall responses from memory T cells, but rather stimulate the expansion of antigen‐specific regulatory T cells. Moreover, the depletion of CD11cloCD40lo macrophages during suppressed immunization diminishes the tolerogenic efficacy of the treatment. These results indicate that dexamethasone acts as a tolerogenic adjuvant partly by enriching the CD11cloCD40lo tolerogenic macrophages.

Protective immune responses to biolistic DNA vaccination of Brugia malayi abundant larval transcript-2.

Biolistic vaccination using gene gun is developed as a safer tool for delivery of DNA vaccines, a technique that combines high vaccine efficiency with lower antigen dosage and lower cost per vaccine dose. In this study, we compared the protective responses in mice after delivering the Brugia malayi abundant larval transcript-2 (BmALT-2) DNA vaccine using the conventional intradermal approach or with the needleless gene gun delivery approach. BmALT-2 is a leading vaccine candidate against B. malayi, a lymphatic filarial parasite of human. After optimizing the DNA dose and gene gun parameters for delivery into mouse skin, groups of mice were biolistically vaccinated with 5 μg of BmALT-2pVAX. Groups of mice vaccinated intradermally with 5 μg or 100 μg of BmALT-2pVAX was used for comparison of vaccine efficacy. Results demonstrated that gene gun vaccination with 5 μg of BmALT-2pVAX conferred significant protection against challenge infection that was comparable to the degree of protection conferred by intradermal vaccination with 100 μg of BmALT-2pVAX. This observation was further supported by an in vitro antibody dependent cellular cytotoxicity (ADCC) assay. Analysis of the immune response showed that the gene gun vaccination predominantly induced an IgG1 antibody response and significantly high Th2 cytokine response (IL-4) from spleen cells compared to intradermal BmALT-2 DNA delivery that induced predominantly an IgG2a and Th1 cytokine response (IFN-γ, IL-12 and TNF-α). These findings show that host protective responses could be achieved with 20 fold decrease in DNA dose using a gene gun and could prove to be an efficient delivery method in BmALT-2 DNA vaccination against lymphatic filariasis.

Large Extracellular Loop of Tetraspanin as a Potential Vaccine Candidate for Filariasis

Lymphatic filariasis affects nearly 120 million people worldwide and mass preventive chemotherapy is currently used as a strategy to control this infection. This has substantially reduced the incidence of the infection in several parts of the world. However, a prophylactic vaccine would be more effective in preventing future infections and will supplement the mass chemotherapy efforts. Unfortunately, there is no licensed vaccine available currently to prevent this infection. Molecules expressed on the surface of the parasite are potential candidates for vaccine development as they are exposed to the host immune system. In this study we show that the large extracellular loop of tetraspanin (TSP LEL), a protein expressed on the cuticle of Brugia malayi and Wuchereria bancrofti is a potential vaccine candidate. Our results showed that BmTSP LEL is expressed on the surface of B. malayi infective third stage larvae (L3) and sera from human subjects who are putatively immune to lymphatic filariasis carry high titer of IgG1 and IgG3 antibodies against BmTSP LEL and WbTSP LEL. We also showed that these antibodies in the sera of human subjects can participate in the killing of B. malayi L3 in an antibody dependent cell-mediated cytotoxicity mechanism. Vaccination trials in mice showed that close to 64% protection were achieved against challenge infections with B. malayi L3. Immunized animals showed high titer of anti-WbTSP LEL IgG1, IgG2a and IgG2b antibodies in the sera and IFN-γ secreting cells in the spleen. Onchocerca volvulus another filarial parasite also expresses TSP LEL. Cross-reactivity studies showed that IgG1 antibody in the sera of endemic normal subjects, recognize OvTSP LEL. Similarly, anti-OvTSP LEL antibodies in the sera of subjects who are immune to O. volvulus were also shown to cross-react with rWbTSP LEL and rBmTSP LEL. These findings thus suggested that rTSP LEL can be developed as a potential vaccine candidate against multiple filarial infections.

Photosensitizers in prostate cancer therapy

The search for new therapeutics for the treatment of prostate cancer is ongoing with a focus on the balance between the harms and benefits of treatment. New therapies are being constantly developed to offer treatments similar to radical therapies, with limited side effects. Photodynamic therapy (PDT) is a promising strategy in delivering focal treatment in primary as well as post radiotherapy prostate cancer. PDT involves activation of a photosensitizer (PS) by appropriate wavelength of light, generating transient levels of reactive oxygen species (ROS). Several photosensitizers have been developed with a focus on treating prostate cancer like mTHPC, motexafin lutetium, padoporfin and so on. This article will review newly developed photosensitizers under clinical trials for the treatment of prostate cancer, along with the potential advantages and disadvantages in delivering focal therapy.

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.

Granuloma Formation around Filarial Larvae Triggered by Host Responses to an Excretory/Secretory Antigen

ABSTRACT In previous studies using a murine model of filarial infection, granuloma formation was found to be a most important host-protective mechanism. We have also shown that in vitro cytoadherence is a surrogate for the formation of antifilarial granulomas in vivo and that it requires “alternatively activated” host cells and a source of antifilarial antibody. We show here that antibodies against L3 excretory/secretory (E/S) products can facilitate in vitro cytoadherence. We generated a set of hybridomas reactive with filarial E/S products and screened them for their ability to mediate in vitro cytoadherence. One clone (no. 1E9) was positive in this assay. We then screened a novel expression library of filarial antigens displayed on the surface of T7 bacteriophage for reactivity with 1E9. Phage expressing two filarial antigens (TCTP and BmALT-2) reacted with 1E9. Immunization of mice showed that the cohort immunized with BmALT-2 cleared a challenge infection with infective Brugia pahangi L3 in an accelerated manner, whereas cohorts immunized with TCTP cleared larvae with the same kinetics as in unimmunized mice. These data confirm that BmALT-2 is the antigenic target of granuloma-mediated killing of B. pahangi L3. Our findings also confirm previous studies that BmALT-2 is a potential vaccine candidate for filarial infection. Our data reinforce the work of others and also provide a possible mechanism by which immune responses to BmALT-2 may provide host protection.

Vitamin K and its analogs: Potential avenues for prostate cancer management

Epidemiological studies have demonstrated a relationship between cancer incidence and dietary habits. Especially intake of certain essential nutrients like vitamins has been shown to be beneficial in experimental studies and some clinical trials. Vitamin K (VK) is an essential nutrient involved in the blood clotting cascade, and there are considerable experimental data demonstrating its potential anticancer activity in several cancer types including prostate cancer. Previous in vitro and in vivo studies have focused mainly on anti-oxidative effects as the underlying anticancer mechanism of VK. However, recent studies reveal that VK inhibits the growth of cancer cells through other mechanisms, including apoptosis, cell cycle arrest, autophagy, and modulation of various transcription factors such as Myc and Fos. In the present review, we focus on the anticancer effect of dietary VK and its analogs on prostate cancer, with an emphasis on the signaling pathways that are activated following exposure to these compounds. This review also highlights the potential of VK and its derivatives as an adjuvant treatment in combination with other vitamins or with chemotherapeutic drugs. Based on our recent results and a review of the existing literature, we present evidence that VK and its derivatives can potentially be explored as cancer therapy, especially for prostate cancer.Epidemiological studies have demonstrated a relationship between cancer incidence and dietary habits. Especially intake of certain essential nutrients like vitamins has been shown to be beneficial in experimental studies and some clinical trials. Vitamin K (VK) is an essential nutrient involved in the blood clotting cascade, and there are considerable experimental data demonstrating its potential anticancer activity in several cancer types including prostate cancer. Previous in vitro and in vivo studies have focused mainly on anti-oxidative effects as the underlying anticancer mechanism of VK. However, recent studies reveal that VK inhibits the growth of cancer cells through other mechanisms, including apoptosis, cell cycle arrest, autophagy, and modulation of various transcription factors such as Myc and Fos. In the present review, we focus on the anticancer effect of dietary VK and its analogs on prostate cancer, with an emphasis on the signaling pathways that are activated following exposure to these compounds. This review also highlights the potential of VK and its derivatives as an adjuvant treatment in combination with other vitamins or with chemotherapeutic drugs. Based on our recent results and a review of the existing literature, we present evidence that VK and its derivatives can potentially be explored as cancer therapy, especially for prostate cancer.