Visible-Light-Active BiOI/TiO2 Heterojunction Photocatalysts for Remediation of Crude Oil-Contaminated Water.

In this study, BiOI-sensitized TiO2 (BiOI/TiO2) nanocomposites with different levels of BiOI deposited via sequential ionic layer adsorption and reaction (SILAR) have been explored for the degradation of methyl orange, 4-chlorophenol (4-CP), and crude oil in water under visible (>400 nm) irradiation with excellent degradation performance. The reaction progress for methyl orange and 4-chlorophenol was monitored by a UV-vis spectrophotometer, and the degradation of the crude oil hydrocarbons was determined by GC-MS. The BiOI/TiO2 heterojunction improves separation of photogenerated charges, which enhances the degradation efficiency. Evaluation of the visible-light photocatalytic performance of the synthesized catalysts against methyl orange degradation confirmed that four SILAR cycles are the optimal deposition condition for the best degradation efficiency. The efficiency was further confirmed by degrading 4-CP and crude oil, achieving 38.30 and 85.62% degradation, respectively, compared with 0.0% (4-CP) and 70.56% (crude oil) achieved by TiO2. The efficiency of TiO2 in degrading crude oil was mainly due to adsorption along with photolysis. This study provides a simple and cost-effective alternative to traditional remediation methods requiring high energy consumption for remediation of crude oil-polluted water and refinery wastewater using visible-light photocatalysis along with adsorption.

EVALUATION OF IMMUNE FUNCTION IN TWO POPULATIONS OF GREEN SEA TURTLES (CHELONIA MYDAS) IN A DEGRADED VERSUS A NONDEGRADED HABITAT.

There is a strong correlation between degraded marine habitats and the prevalence of diseases such as green turtle fibropapillomatosis (GTFP) in coastal populations. In GTFP, small to large tumors grow on the turtle's soft tissues and shell, while internal nodules may also occur. The disease primarily affects juvenile green sea turtles (Chelonia mydas) that reside in nearshore waters. As a link has been shown between environmental pollution and immune suppression in a variety of animals, the objective of our research was to compare innate and adaptive immune responsiveness in green sea turtles from a severely degraded and a more pristine habitat, which differ greatly in rates of GTFP. We quantified phagocytosis by flow cytometry and performed in vitro stimulation analysis to measure activity of both the innate and adaptive immune systems in wild-caught Florida green turtles. Sea turtles from the degraded environment, both with and without visible cutaneous tumors, exhibited significantly reduced phagocytosis and stimulation indices than did those from the less polluted environment. Our results suggest that environmental factors may contribute to the development of GTFP and thus can impact the health of sea turtle populations.

Octadecyl chain-bearing PEGylated poly(propyleneimine)-based dendrimersomes: physicochemical studies, redox-responsiveness, DNA condensation, cytotoxicity and gene delivery to cancer cells.

Stimuli-responsive nanocarriers have become increasingly important for nucleic acid and drug delivery in cancer therapy. Here, we report the synthesis, characterization and evaluation of disulphide-linked, octadecyl (C18 alkyl) chain-bearing PEGylated generation 3-diaminobutyric polypropylenimine dendrimer-based vesicles (or dendrimersomes) for gene delivery. The lipid-bearing PEGylated dendrimer was successfully synthesized through in situ two-step reaction. It was able to spontaneously self-assemble into stable, cationic, nanosized vesicles, with low critical aggregation concentration value, and also showed redox-responsiveness in presence of a glutathione concentration similar to that of the cytosolic reducing environment. In addition, it was able to condense more than 70% of DNA at dendrimer: DNA weight ratios of 5 : 1 and higher. This dendriplex resulted in an enhanced cellular uptake of DNA at dendrimer: DNA weight ratios of 10 : 1 and 20 : 1, by up to 16-fold and by up to 28-fold compared with naked DNA in PC-3 and DU145 prostate cancer cell lines respectively. At a dendrimer: DNA weight ratio of 20 : 1, it led to an increase in gene expression in PC-3 and DU145 cells, compared with DAB dendriplex. These octadecyl chain-bearing, PEGylated dendrimer-based vesicles are therefore promising redox-sensitive drug and gene delivery systems for potential applications in combination cancer therapy.

Camptothecin-based dendrimersomes for gene delivery and redox-responsive drug delivery to cancer cells.

Combination therapy involving chemotherapeutic drugs and genes is emerging as a promising strategy to provide a synergistic therapeutic effect, to overcome drug resistance while reducing the severe side effects associated with conventional chemotherapeutic drugs. However, the lack of nanomedicines able to simultaneously carry anti-cancer drugs and nucleic acids limits the application of this therapeutic strategy. To overcome this issue, we proposed to synthesize a pro-drug dendrimer by conjugating the PEGylated, positively charged generation 3-diaminobutyric polypropylenimine dendrimer to the anti-cancer drug camptothecin with a redox-sensitive disulphide linkage, and evaluate its efficacy to co-deliver the complexed DNA and camptothecin to cancer cells. This PEGylated pro-drug dendrimer was found to spontaneously self-assemble into cationic (∼3-5 mV) vesicles at pH 7.4, at a critical aggregation concentration of about 200 µg mL-1. These vesicles (dendrimersomes) became smaller (150-200 nm) with increasing dendrimer concentration and remained stable over 7 days. They were able to release about 70% of the conjugated camptothecin in presence of 50 mM glutathione (equivalent to the intracellular environment of tumor tissue). They could also condense more than 85% of the DNA at dendrimer : DNA weight ratios of 5 : 1 and higher. DNA condensation occurred instantly and was found to be stable for at least 24 h. This led to an enhanced cellular uptake of DNA (by up to 1.6-fold) and increased gene transfection (by up to 2.4-fold) in prostate cancer cells in comparison with the unmodified dendrimer. These novel dendrimersomes are therefore promising for single carrier-based combination cancer therapy.

Redox-sensitive, cholesterol-bearing PEGylated poly(propylene imine)-based dendrimersomes for drug and gene delivery to cancer cells.

Stimuli-responsive nanocarriers have attracted increased attention as materials that can facilitate drug and gene delivery in cancer therapy. The present study reports the development of redox-sensitive dendrimersomes comprising disulfide-linked cholesterol-bearing PEGylated dendrimers, which can be used as drug and gene delivery systems. Two disulfide-linked cholesterol-bearing PEGylated generation 3 diaminobutyric polypropylenimine dendrimers have been successfully synthesized via an in situ two-step reaction. They were able to spontaneously self-assemble into stable, cationic, nanosized vesicles (or dendrimersomes) with lower critical aggregation concentration values for high-cholesterol-bearing vesicles. These dendrimersomes were able to entrap both hydrophilic and hydrophobic dyes, and they also showed a redox-responsive sustained release of the entrapped guests in the presence of a glutathione concentration similar to that of a cytosolic reducing environment. The high-cholesterol-bearing dendrimersomes were found to have a higher melting enthalpy, increased adsorption tendency on mica surface, entrapping ability for a larger amount of hydrophobic drugs, and increased resistance to redox-responsive environments in comparison with their low-cholesterol counterpart. In addition, both dendrimersomes were able to condense more than 85% of the DNA at all the tested ratios for the low-cholesterol vesicles, and at dendrimer : DNA weight ratios of 1 : 1 and higher for the high-cholesterol vesicles. These vesicles resulted in an enhanced cellular uptake of DNA, by up to 15-fold when compared with naked DNA with low-cholesterol vesicles. As a result, they increased the gene transfection on the PC-3 prostate cancer cell line, with the highest transfection being obtained with low-cholesterol vesicle complexes at a dendrimer : DNA weight ratio of 5 : 1 and high-cholesterol vesicle complexes at a dendrimer : DNA weight ratio of 10 : 1. These transfection levels were about 5-fold higher than those observed when treated with naked DNA. These cholesterol-bearing PEGylated dendrimer-based vesicles are, therefore, promising as redox-sensitive drugs and gene delivery systems for potential applications in combination cancer therapies.

Regression of prostate tumors after intravenous administration of lactoferrin-bearing polypropylenimine dendriplexes encoding TNF-α, TRAIL, and interleukin-12.

The possibility of using gene therapy for the treatment of prostate cancer is limited by the lack of intravenously administered delivery systems able to safely and selectively deliver therapeutic genes to tumors. Given that lactoferrin (Lf) receptors are overexpressed on prostate cancer cells, we hypothesized that the conjugation of Lf to generation 3-diaminobutyric polypropylenimine dendrimer would improve its transfection and therapeutic efficacy in prostate cancer cells. In this study, we demonstrated that the intravenous administration of Lf-bearing DAB dendriplexes encoding TNFα resulted in the complete suppression of 70% of PC-3 and 50% of DU145 tumors over one month. Treatment with DAB-Lf dendriplex encoding TRAIL led to tumor suppression of 40% of PC-3 tumors and 20% of DU145 tumors. The treatment was well tolerated by the animals. Lf-bearing generation 3-polypropylenimine dendrimer is therefore a highly promising delivery system for non-viral gene therapy of prostate cancer.

Effects of α-conotoxin ImI on TNF-α, IL-8 and TGF-ß expression by human macrophage-like cells derived from THP-1 pre-monocytic leukemic cells.

α7 nicotinic acetylcholine receptors (nAChRs) are ubiquitous in the nervous system and ensure important neurophysiological functionality for many processes. However, they are also found in cells of the immune system, where their role has been less studied. Here we report the pro-inflammatory effect of ImI, a well characterized conotoxin that inhibits α7 nAChRs, on differentiated THP-1 pre-monocyte macrophages (MDM) obtained by phorbol 12-myristate 13 acetate (PMA) treatment. Enzyme-linked immunosorbent assay (ELISA) performed on supernatant fluids of LPS challenged MDM showed ImI-mediated upregulation of pro-inflammatory cytokine TNF-α in an ImI concentration-dependent manner from 0.5 to 5.0 µmol/L and for IL-8 up to 1.0 µmol/L. Levels of anti-inflammatory cytokine TGF-ß remained practically unaffected in ImI treated MDMs. Nicotine at 10 µmol/L significantly downregulated the release of TNF-α, but showed a lesser effect on IL-8 secretion and no effect on TGF-ß. Fluorescent competitive assays involving ImI, α-bungarotoxin and nicotine using MDM and the murine macrophage RAW 264.7 suggest a common binding site in the α7 receptor. This work extends the application of conotoxins as molecular probes to non-excitatory cells, such as macrophages and supports the involvement of the α7 nAChR in regulating the inflammatory response via the cholinergic anti-inflammatory pathway (CAP).

Allergen induced pulmonary inflammation enhances mammary tumor growth and metastasis: Role of CHI3L1.

Metastasis is the primary cause of mortality in women with breast cancer. Metastasis to the lungs is greater in patients with pulmonary inflammatory illnesses. It is unknown how pre-existing pulmonary inflammation affects mammary tumor progression. We developed a novel breast cancer model in which pulmonary inflammation is induced in mice prior to tumor cell implantation. In the present study, we determined how pre-existing allergen-induced inflammation changes the pulmonary microenvironment to exacerbate tumor metastasis. We showed that pre-existing pulmonary inflammation in mammary tumor bearers is associated with: 1) an increase in growth of the primary tumor and metastasis; 2) an increase in the expression of a glycoprotein known as CHI3L1; and 3) increase in the levels of myeloid populations in their lungs. We also showed that myeloid derived cells from the lungs of allergic tumor bearers produce higher amounts of CHI3L1 than the saline controls. We previously showed that CHI3L1 induces the expression of proinflammatory and protumorigenic molecules. In this study, we show that CHI3L1 knockout tumor bearers with pre-existing allergic pulmonary inflammation had decreased levels of myeloid-derived cells, decreased levels of proinflammatory mediators, and a significant reduction in tumor volume and metastasis compared with the wild-type controls. Pre-existing inflammation and CHI3L1 might be driving the establishment of a premetastatic milieu in the lungs and aiding in the support of metastatic foci. Understanding the role of allergen-induced CHI3L1 and inflammation in tumor bearers and its effects on the pulmonary microenvironment could result in targeted therapies for breast cancer.

Semaphorin7A promotes tumor growth and exerts a pro-angiogenic effect in macrophages of mammary tumor-bearing mice.

Semaphorins are a large family of molecules involved in axonal guidance during the development of the nervous system and have been recently shown to have both angiogenic and anti-angiogenic properties. Specifically, semaphorin 7A (SEMA7A) has been reported to have a chemotactic activity in neurogenesis and to be an immune modulator through α1ß1integrins. SEMA7A has been shown to promote monocyte chemotaxis and induce them to produce proinflammatory mediators. In this study we explored the role of SEMA7A in a murine model of breast cancer. We show that SEMA7A is highly expressed by DA-3 murine mammary tumor cells in comparison to normal mammary cells (EpH4), and that peritoneal elicited macrophages from mammary tumor-bearing mice also express SEMA7A at higher levels compared to those derived from normal mice. We also show that murine macrophages treated with recombinant murine SEMA7A significantly increased their expression of proangiogenic molecule CXCL2/MIP-2. Gene silencing of SEMA7A in peritoneal elicited macrophages from DA-3 tumor-bearing mice resulted in decreased CXCL2/MIP-2 expression. Mice implanted with SEMA7A silenced tumor cells showed decreased angiogenesis in the tumors compared to the wild type tumors. Furthermore, peritoneal elicited macrophages from mice bearing SEMA7A-silenced tumors produce significantly (p < 0.01) lower levels of angiogenic proteins, such as CXCL2/MIP-2, CXCL1, and MMP-9, compared to those from control DA-3 mammary tumors. We postulate that SEMA7A in mammary carcinomas may skew monocytes into a pro-tumorigenic phenotype to support tumor growth. SEMA7A could prove to be valuable in establishing new research avenues toward unraveling important tumor-host immune interactions in breast cancer patients.

Production of functional native human interleukin-2 in tobacco chloroplasts.

Interleukin-2 (IL-2) is an important T lymphocyte-derived cytokine in the mammalian immune system. Non-native, recombinant IL-2 derived from Escherichia coli is used widely in both medical research and treatment of diseases. Recombinant human IL-2 gene has been expressed in plant nuclear genomes, therefore it can be spread to the environment through pollen. Furthermore, all the plant-produced IL-2 reported thus far had been attached with artificial tags or fusion proteins, which may trigger unintended immunological responses and therefore compromise its full utility as a medicine. To expand the potential of using plant chloroplasts to produce functional native human therapeutic proteins, we inserted an engineered human interleukin-2 (hIL-2)-coding gene, without any tags, into the chloroplast genome of tobacco (Nicotiana tabacum L.). Partially purified hIL-2 protein from the leaves of the transplastomic plants induced in vitro proliferation of IL-2-dependent murine T lymphocytes. Our study demonstrates that plant chloroplasts can serve as a bio-factory for production of an active native human interleukin in a self-contained and therefore environmentally safe manner.

Exploring the role of CHI3L1 in "pre-metastatic" lungs of mammary tumor-bearing mice.

Elevated levels of chitinase-3-like-1 (CHI3L1) are associated with poor prognosis, shorter recurrence-free intervals and low survival in breast cancer patients. Breast cancer often metastasizes to the lung. We hypothesized that molecules expressed in the "pre-metastatic" lung microenvironment could support the newly immigrant tumor cells by providing growth and angiogenic factors. Macrophages are known to play an important role in tumor growth by releasing pro-angiogenic molecules. Using mouse mammary tumor models, we have previously shown that during neoplastic progression both the mammary tumor cells and splenic macrophages from tumor-bearing mice express higher levels of CHI3L1 compared to normal control mice. However, the role of CHI3L1 in inducing angiogenesis by macrophages at the pulmonary microenvironment to support newly arriving breast cancer cells is not yet known. In this study, we determined the expression of CHI3L1 in bronchoalveolar lavage macrophages and interstitial macrophages in regulating angiogenesis that could support the growth of newly immigrant mammary tumor cells into the lung. Here we show that in vitro treatment of pulmonary macrophages with recombinant murine CHI3L1 resulted in enhanced expression of pro-angiogenic molecules including CCL2, CXCL2, and MMP-9. We and others have previously shown that inhibition of CHI3L1 decreases the production of angiogenic molecules. In this study, we explored if in vivo administration of chitin microparticles has an effect on the expression of CHI3L1 and pro-angiogenic molecules in the lungs of mammary tumor-bearing mice. We show that treatment with chitin microparticles decreases the expression of CHI3L1 and pro-angiogenic molecules in the "metastatic" lung. These studies suggest that targeting CHI3L1 may serve as a potential therapeutic agent to inhibit angiogenesis and thus possibly tumor growth and metastasis.

NK cells in the CD19- B220+ bone marrow fraction are increased in senescence and reduce E2A and surrogate light chain proteins in B cell precursors.

E2A encoded proteins, key transcriptional regulators in B lineage specification and commitment, have been shown to decrease in B cell precursors in old age. E2A regulates genes encoding the surrogate light chain proteins lambda5 and VpreB. In old age, B cell precursors express less surrogate light chain and this results in compromised pre-B cell receptor function and diminished expansion of new pre-B cells in senescence. Herein, we show that aged bone marrow has increased Hardy Fraction A (CD19(-) B220(+)) cells, including NK cells, that can inhibit both E47 (E2A) protein and surrogate light chain protein expression in B cell precursors. In vitro, NK-associated inhibition of E47 protein is contact-independent and partially reversed by neutralization of TNFalpha. In vivo, depletion of NK cells in aged mice by treatment with anti-asialo GM1 antibody led to restoration of surrogate light chain protein levels to that typical of young B cell precursors. These studies suggest that NK cells, within the CD19(-) B220(+) bone marrow cell fraction, may contribute to a bone marrow microenvironment that has the potential to negatively regulate E47 (E2A) as well as surrogate light chain levels in B cell precursors in old age.

Inhibition of Single Minded 2 gene expression mediates tumor-selective apoptosis and differentiation in human colon cancer cells.

A Down's syndrome associated gene, Single Minded 2 gene short form (SIM2-s), is specifically expressed in colon tumors but not in the normal colon. Antisense inhibition of SIM2-s in a RKO-derived colon carcinoma cell line causes growth inhibition, apoptosis, and inhibition of tumor growth in a nude mouse tumoriginicity model. The mechanism of cell death in tumor cells is unclear. In the present study, we investigated the pathways underlying apoptosis. Apoptosis was seen in a tumor cell-specific manner in RKO cells but not in normal renal epithelial cells, despite inhibition of SIM2-s expression in both of these cells by the antisense. Apoptosis was depended on WT p53 status and was caspase-dependent; it was inhibited by a pharmacological inhibitor of mitogen-activated protein kinase activity. Expression of a key stress response gene, growth arrest and DNA damage gene (GADD)45alpha, was up-regulated in antisense-treated tumor cells but not in normal cells. In an isogenic RKO cell line expressing stable antisense RNA to GADD45alpha, a significant protection of the antisense-induced apoptosis was seen. Whereas antisense-treated RKO cells did not undergo cell cycle arrest, several markers of differentiation were deregulated, including alkaline phosphatase activity, a marker of terminal differentiation. Protection of apoptosis and block of differentiation showed a correlation in the RKO model. Our results support the tumor cell-selective nature of SIM2-s gene function, provide a direct link between SIM2-s and differentiation, and may provide a model to identify SIM2-s targets.