Unprecedented enhancement of recombinant protein production in sugarcane culms using a combinatorial promoter stacking system.

Plants represent a safe and cost-effective platform for producing high-value proteins with pharmaceutical properties; however, the ability to accumulate these in commercially viable quantities is challenging. Ideal crops to serve as biofactories would include low-input, fast-growing, high-biomass species such as sugarcane. The objective of this study was to develop an efficient expression system to enable large-scale production of high-value recombinant proteins in sugarcane culms. Bovine lysozyme (BvLz) is a potent broad-spectrum antimicrobial enzyme used in the food, cosmetics and agricultural industries. Here, we report a novel strategy to achieve high-level expression of recombinant proteins using a combinatorial stacked promoter system. We demonstrate this by co-expressing BvLz under the control of multiple constitutive and culm-regulated promoters on separate expression vectors and combinatorial plant transformation. BvLz accumulation reached 1.4% of total soluble protein (TSP) (10.0 mg BvLz/kg culm mass) in stacked multiple promoter:BvLz lines, compared to 0.07% of TSP (0.56 mg/kg) in single promoter:BvLz lines. BvLz accumulation was further boosted to 11.5% of TSP (82.5 mg/kg) through event stacking by re-transforming the stacked promoter:BvLz lines with additional BvLz expression vectors. The protein accumulation achieved with the combinatorial promoter stacking expression system was stable in multiple vegetative propagations, demonstrating the feasibility of using sugarcane as a biofactory for producing high-value proteins and bioproducts.

Transdermal and transbuccal drug delivery systems: enhancement using iontophoretic and chemical approaches.

We investigated the enhancement effect of chemical enhancers and iontophoresis on the in vitro transdermal and transbuccal delivery of lidocaine HCl (LHCl), nicotine hydrogen tartrate (NHT), and diltiazem HCl (DHCl) using porcine skin and buccal tissues. Dodecyl 2-(N,N-dimethylamino) propionate (DDAIP), dodecyl-2-(N,N-dimethylamino) propionate hydrochloride (DDAIP HCl), N-(4-bromobenzoyl)-S,S-dimethyliminosulfurane (Br-iminosulfurane), and azone (laurocapram) were used as chemical enhancers. The study results showed that the application of iontophoresis at either 0.1 mA or 0.3 mA significantly enhanced transdermal and transmucosal delivery of LHCl, NHT and DHCl. It was also demonstrated that iontophoresis had a more pronounced enhancement effect on transdermal delivery than on transbuccal delivery of LHCl, NHT and DHCl. In addition, DDAIP HCl was found to be the most effective enhancer for transbuccal delivery of LHCl and NHT.

A6 peptide activates CD44 adhesive activity, induces FAK and MEK phosphorylation, and inhibits the migration and metastasis of CD44-expressing cells.

The A6 peptide (acetyl-KPSSPPEE-amino) has antitumor activity in the absence of significant adverse events in murine tumor models and clinical trials. A6 shares sequence homology with CD44, an adhesion receptor involved in metastasis that is also a marker of cancer stem cells and drug-resistant phenotypes. We investigated the mechanism of action of A6 by examining its effects on CD44 activity, cell migration, and metastasis. A6 inhibited the migration of a subset of ovarian and breast cancer cell lines, exhibiting IC(50) values of 5 to 110 nmol/L. The ability of A6 to inhibit migration in vitro correlated with CD44 expression. Immunopreciptation studies showed that CD44 binds A6 and that biotin-tagged A6 can be cross-linked to CD44. The binding of A6 altered the structure of CD44 such that it was no longer recognized by a monoclonal antibody to a specific epitope. Importantly, A6 potentiated the CD44-dependent adhesion of cancer cells to hyaluronic acid and activated CD44-mediated signaling, as evidenced by focal adhesion kinase and MAP/ERK kinase phosphorylation. In vivo, A6 (100 mg/kg delivered s.c. twice daily) reduced the number of lung foci generated by the i.v. injection of B16-F10 melanoma cells by 50% (P = 0.029 in an unpaired t test). We conclude that A6 potently blocks the migration of CD44-positive cells in vitro through an interaction with CD44 that alters its structure and activates CD44 to enhance ligand binding and downstream signaling. The concurrent ability of A6 to agonize the CD44 receptor suggests that CD44 activation may represent a novel strategy for inhibiting metastatic disease.

Enhanced in vitro transbuccal drug delivery of ondansetron HCl.

The effect of chemical enhancers and iontophoresis on the in vitro transbuccal delivery of 0.5% ondansetron HCl (ODAN HCl) was investigated using porcine buccal tissue. The chemical enhancers used were dodecyl 2-(N,N-dimethyl amino) propionate (DDAIP), its HCl salt dodecyl-2-(N,N-dimethylamino) propionate hydrochloride (DDAIP HCl), N-(4-bromobenzoyl)-S,S-dimethyliminosulfurane (Br-iminosulfurane), and azone. This study demonstrated that anodal iontophoresis at 0.1, 0.2 and 0.3 mA current intensity significantly increased transbuccal delivery of ODAN HCl 3.3-fold, 5.2-fold and 7.1-fold respectively, compared to control. DDAIP HCl provided significantly higher transbuccal delivery of ODAN HCl than did DDAIP, azone and Br-iminosulfurane. It was found that DDAIP HCl in water significantly enhanced drug permeability (920 µg/cm(2)) compared to DDAIP HCl in propylene glycol (PG) (490 µg/cm(2)) during 24h. It was also found that 5% (w/v) DDAIP HCl in water alone provided higher permeation flux (29.3 µg/cm(2)/h) than iontophoresis alone at 0.3 mA (22.8 µg/cm(2)/h) during the same 8h treatment. A light microscopy study showed that treatment with chemical enhancers and iontophoresis did not cause major morphological changes in the buccal tissue. EpiOral™ MTS cytotoxicity studies demonstrated that DDAIP HCl at less than 5% (w/v) in water did not have significant detrimental effects on the cells.

Splenic natural killer cell activity in two models of experimental neurodegenerative diseases.

Natural killer (NK) cells are antitumour/anti-viral effectors and play important roles in shaping the immune system, but their role in neurodegenerative diseases is not clear. Here, we investigated the fate of these cells in two neurodegenerative diseases. In the first model, the activity of NK cells was examined in mice with experimental autoimmune encephalomyelitis (EAE) treated with glatiramer acetate (GA or Copaxone), a drug used to treat EAE in animals and multiple sclerosis in human. The second disease model is twitcher (Galc(twi)/Galc(twi)) mice, which represents an authentic model of human Krabbe's disease. Administration of GA ameliorated EAE in SJL mice corroborated with isolating NK cells that expressed higher killing than cells isolated from vehicle-dosed animals against immature or mature dendritic cells (DCs). However, this drug showed no effect on the numbers of NK cells or the expression of CD69 molecule. On the other hand, NK cells either disappeared from the spleens or were present in low numbers in the white pulp areas of Galc(twi)/Galc(twi) mice, which have increased D-galactosyl-beta1-1'-sphingosine (GalSph) levels. Analysis by confocal microscopy shows that NK cells found in the spleens of Galc(twi)/Galc(twi) mice were apoptotic. Incubating NK cells in vitro with GalSph induced the apoptosis in these cells, confirming the results of twitcher mice. Our results provide the first evidence showing that amelioration of EAE in mice is corroborated with NK cell lysis of antigen-presenting DCs, whereas NK cell distribution into the spleen is altered in a devastating lipid disorder corroborated with induction of their apoptosis.

Oxazolone-induced delayed type hypersensitivity reaction in the adult yucatan pigs. A useful model for drug development and validation.

The purpose of this study was to establish a model of delayed type hypersensitivity (DTH) reaction in the ear skin of large animals such as adult Yucatan pigs, which may aid in evaluating the efficacy of therapeutic modalities of newly developed anti-inflammatory drugs. The pigs were sensitized with oxazolone, re-challenged with the same irritant six days later, and dosed with either vehicle or with cyclosporine A (CsA) before and after challenge. CsA reduced the redness, inhibited the accumulation of ear fluid and inflammatory cells, as well as the release of the inflammatory mediators. Further, CsA inhibited the proliferation of T cells collected from the spleens or PBMCs of CsA-treated pigs when these cells were stimulated in vitro with PMA plus Ionomycin. These results indicate that pig skin can be used to evaluate modalities for the purpose of developing drugs that may be used to treat DTH in humans.

Functional expression of H4 histamine receptor in human natural killer cells, monocytes, and dendritic cells.

We describe here the protein expression of H4 histamine receptor in cells of the innate immune system, which include NK cells, monocytes, and dendritic cells (DCs). Anti-H4R specifically stained permeabilized NK cells, THP-1 clone 15 monocytes, and DCs. This binding was inhibited by incubating anti-H4R Ab with its corresponding peptide. Histamine induced NK cells, THP-1 clone 15 cells, and DCs chemotaxis with high affinity. The ED(50) chemotactic effect was 5 nM, 6.8 nM, and 2.7 nM for NK cells, THP-1 clone 15 cells, and DCs, respectively. Thioperamide, an H3R/H4R antagonist, inhibited histamine-induced chemotaxis in all these cells. However, histamine failed to induce the mobilization of [Ca(2+)](i) in NK cells and THP-1 clone 15 cells, but it induced calcium fluxes in DCs. Using a new method of detecting NK cell-mediated cytolysis, it was observed that NK cells efficiently lysed K562 target cells and that histamine did not affect this NK cell activity. In summary, this is the first demonstration of the protein expression of H4 receptor in NK cells. Also, the results of the chemotactic effects of histamine on NK cells and THP-1 cells are novel. These results may shed some light on the colocalization of cells of innate immune arm at sites of inflammation. They are also important for developing drugs that target H4R for the treatment of various disorders, such as autoimmune and immunodeficient diseases.

IL-6 and IL-8 release is mediated via multiple signaling pathways after stimulating dendritic cells with lysophospholipids.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are bioactive lipid mediators, which are known to play major roles in allergic reactions as well as in tumor pathogenesis. Here, the biological activities and signal pathways of these lysophospholipids (LPLs) in dendritic cells (DCs) were characterized further. Flow cytometric and immunoblot analyses indicate that immature as well as mature DCs express the LPL receptors S1P1, S1P3, S1P5, and LPA2, but not S1P2, S1P4, LPA1, or LPA3. Moreover, enzyme-linked immunosorbent assay experiments demonstrate that simultaneous addition of these LPLs to immature DCs in the presence of lipopolysaccharide enhanced the secretion of the inflammatory cytokines interleukin (IL)-6 and IL-8 in maturing DCs. In contrast, no modification of IL-6 or IL-8 release was observed after exposure of mature DCs to LPLs alone. In addition, studies with pertussis toxin and mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 suggested that Gi proteins and MAPK pathway are involved in these LPL-induced cell responses. Corroborating these findings, we observed that LPLs induce the phosphorylation of extracellular signal-regulated kinase 1/2 in immature DCs but not in mature DCs. Further analyses show that inhibitors of phosholipase D, Rho, and protein kinase C also inhibited the LPL-induced release of IL-6 and IL-8. Therefore, our findings suggest that lipopolysaccharide in DCs uncouples LPL receptors from the signal-transducing machinery during maturation and that exposure of LPLs at early time-points to maturing DCs modifies the proinflammatory capacity of mature DCs.

Human resting CD16-, CD16+ and IL-2-, IL-12-, IL-15- or IFN-alpha-activated natural killer cells differentially respond to sphingosylphosphorylcholine, lysophosphatidylcholine and platelet-activating factor.

The phosphorylcholine-containing lipid lysophosphatidylcholine (LPC) is abundant in the bloodstream, whereas sphingosylphosphorylcholine (SPC) and platelet-activating factor (PAF) highly accumulate at inflamed sites. Utilizing RT-PCR, flow cytometry and immunoblot analyses, we show for the first time that ovarian cancer G protein-coupled receptor 1, the receptor for SPC, is expressed in IL-2-, IL-12- and IL-15-activated but not in resting CD16-, resting CD16+ or IFN-alpha-activated NK cells. Similarly, G2 accumulation and PAF receptor are variably expressed in these subsets of NK cells. SPC, LPC and PAF differentially induce the chemotaxis of resting and activated NK cells. In the chemotaxis assay, it is observed that resting CD16-CD56bright and CD16+CD56dim cells predominantly respond to LPC, whereas activated NK cells, regardless of the sort of stimulus, robustly respond to PAF. SPC is also a potent chemoattractant for IL-2-, IL-12- and IL-15- but not for IFN-alpha-activated NK cells. Further analysis shows that, depending on the cytokine pattern of NK cell activation, phosphorylcholine-containing lipids differentially affect IFN-gamma secretion by these cells. Our results provide one possible explanation for the tissue compartmentation of NK cells and their ability to secrete IFN-gamma. Furthermore, these results may provide novel information regarding NK cell regulation during inflammation.