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

Toxicity Assessment of Mesoporous Silica Nanoparticles upon Intravenous Injection in Mice: Implications for Drug Delivery

Nanoparticles are popular tools utilized to selectively deliver drugs and contrast agents for identification and treatment of disease. To determine the usefulness and translational potential of mesoporous silica nanoparticles (MSNs), further evaluations of toxicity are required. MSNs are among the most utilized nano-delivery systems due to ease of synthesis, pore structure, and functionalization. This study aims to elucidate toxicity as a result of intravenous injection of 25 nm MSNs coated with chitosan (C) or polyethylene glycol (PEG) in mice. Following acute and chronic injections, blood was evaluated for standard blood chemistry and complete blood count analyses. Blood chemistry results primarily indicated that no abnormalities were present following acute or chronic injections of MSNs, or C/PEG-coated MSNs. After four weekly administered treatments, vital organs showed minor exacerbation of pre-existing lesions in the 35KPEG-MSN and moderate exacerbation of pre-existing lesions in uncoated MSN and 2KPEG-MSN treatment groups. In contrast, C-MSN treatment groups had minimal changes compared to controls. This study suggests 25 nm MSNs coated with chitosan should elicit minimal toxicity when administered as either single or multiple intravenous injections, but MSNs coated with PEG, especially 2KPEG may exacerbate pre-existing vascular conditions. Further studies should evaluate varying sizes and types of nanoparticles to provide a better overall understanding on the relation between nanoparticles and in vivo toxicity

Cathepsin B Degradable Star-Shaped Peptidic Macromolecules for Delivery of 2‑Methoxyestradiol

2-Methoxyestradiol (2ME), a natural metabolite of estradiol, has antiproliferative and antiangiogenic activity. However, its clinical success is limited due to poor water solubility and poor pharmacokinetic parameters suggesting the need for a delivery vehicle. In this study we evaluated cathepsin B degradable star-shaped peptidic macromolecules (SPMs) that can potentially be used to create higher generation and high molecular weight peptidic polymer as delivery vehicle of 2ME. Two peptidic macromolecules having positively charged amine (ASPM) or negatively charged carboxyl surface groups (CSPM) were synthesized and evaluated for their degradation in the presence of cathepsin B and stability in the presence of neutral or acidic buffer and serum. Both ASPM and CSPM degraded rapidly in the presence of cathepsin B. Both were stable in neutral and acidic buffer whereas only CSPM exhibited substantial stability in the presence of serum. Both macromolecules were nontoxic toward breast cancer cells whereas 2ME-containing macromolecules exhibited antiproliferative activity in the micromolar range. Overall, results from the current study indicate that tetrapeptide GFLG can be used to create star-shaped macromolecules that are degraded in the presence of cathepsin B and have the potential to be developed as delivery vehicles of 2ME

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 protein12.6 (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

Applying dynamic contrast enhanced MSOT imaging to intratumoral pharmacokinetic modeling

Examining the dynamics of an agent in the tumor microenvironment can offer critical insights to the influx rate and accumulation of the agent. Intratumoral kinetic characterization in the in vivo setting can further elicudate distribution patterns and tumor microenvironment.Dynamic contrast-enhanced Multispectral Optoacoustic Tomographic imaging (DCE-MSOT) acquires serial MSOT images with the administration of an exogenous contrast agent over time. We tracked the dynamics of a tumor-targeted contrast agent, HypoxiSense 680 (HS680), in breast xenograft mouse models using MSOT. Arterial input function (AIF) approach with MSOT imaging allowed for tracking HS680 dynamics within the mouse. The optoacoustic signal for HS680 was quantified using the ROI function in the ViewMSOT software. A two-compartment pharmacokinetics (PK) model constructed in MATLAB to fit rate parameters. The contrast influx (kin) and outflux (kout) rate constants predicted are kin = 1.96 × 10−2 s-1 and kout = 9.5 × 10-3 s-1 (R = 0.9945). Keywords: Pharmacokinetic modeling, Targeted contrast agent, Intratumoral kinetics, Tumor microenvironment, Hypoxia, Multispectral optoacoustic imagin

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

Cytokine levels in human PBMC.

<p>Cytokines (pg/ml) in the culture supernatants of human PBMC were measured using an ELISA. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034077#s3" target="_blank">Results</a> show that significant level of IFN-γ is secreted by PBMC of EN individuals in response to rBmHsp12.6. Experiments were repeated two times. Each bar represents mean concentration ± S.D. * Significant (p<0.05) IFN-γ secretions compared to other two groups (CP and MF).</p

Splenocytes from vaccinated mice proliferated in response to the antigens.

<p>Single cell suspension of spleen cells (2×10⁵) from vaccinated and control mice were stimulated with respective peptides or protein for 72 hrs at 37°C. Control wells were either stimulated with Con A (positive control) or left unstimulated (negative control). Mice vaccinated with <b>A</b>) BmHsp12.6, <b>B</b>) BmHsp12.6αc or <b>C</b>) N-BmHsp12.6 using homologous DNA vaccine regimen or a heterologous prime boost approach. A non-specific recombinant protein (rSmGBF) was used as negative control. Data is presented as mean stimulation index (S.I.) of five mice ± S.D. * Significant (p<0.005) S.I. value compared to control cells.</p

Anti-rBmHsp12.6 IgG antibody levels in the sera of human.

<p>Levels of total IgG antibodies against (<b>A</b>) rBmHsp12.6 protein, (<b>B</b>) BmHsp12.6αc peptide or (<b>C</b>) N-BmHsp12.6 peptide in the sera of EN, MF, CP and NEN subjects were measured using an indirect ELISA. A total of 20 sera samples were evaluated from EN, MF, and CP and 10 samples from NEN. Each data point represents sera sample from a single individual. Horizontal lines represent geometric mean value. Data is represented as scatter plot where each dot represents absorbance of individual sera.</p