Multifunctional elastin-like polypeptide nanocarriers for efficient miRNA delivery in cancer therapy.

BACKGROUND: The exogenous delivery of miRNA to mimic and restore miRNA-34a activity in various cancer models holds significant promise in cancer treatment. Nevertheless, its effectiveness is often impeded by challenges, including a short half-life, propensity for off-target accumulation, susceptibility to inactivation by blood-based enzymes, concerns regarding patient safety, and the substantial cost associated with scaling up. As a means of overcoming these barriers, we propose the development of miRNA-loaded Tat-A86 nanoparticles by virtue of Tat-A86's ability to shield the loaded agent from external environmental factors, reducing degradation and inactivation, while enhancing circulation time and targeted accumulation. RESULTS: Genetically engineered Tat-A86, featuring 16 copies of the interleukin-4 receptor (IL-4R)-binding peptide (AP1), Tat for tumor penetration, and an elastin-like polypeptide (ELP) for presenting target ligands and ensuring stability, served as the basis for this delivery system. Comparative groups, including Tat-E60 and A86, were employed to discern differences in binding and penetration. The designed ELP-based nanoparticle Tat-A86 effectively condensed miRNA, forming stable nanocomplexes under physiological conditions. The miRNA/Tat-A86 formulation bound specifically to tumor cells and facilitated stable miRNA delivery into them, effectively inhibiting tumor growth. The efficacy of miRNA/Tat-A86 was further evaluated using three-dimensional spheroids of lewis lung carcinoma (LLC) as in vitro model and LLC tumor-bearing mice as an in vivo model. It was found that miRNA/Tat-A86 facilitates effective cell killing by markedly improving miRNA penetration, leading to a substantial reduction in the size of LLC spheroids. Compared to other controls, Tat-A86 demonstrated superior efficacy in suppressing the growth of 3D cellular aggregates. Moreover, at equivalent doses, miRNA-34a delivered by Tat-A86 inhibited the growth of LLC cells in allograft mice. CONCLUSIONS: Overall, these studies demonstrate that Tat-A86 nanoparticles can deliver miRNA systemically, overcoming the basic hurdles impeding miRNA delivery by facilitating both miRNA uptake and stability, ultimately leading to improved therapeutic effects.

The macrophage odorant receptor Olfr78 mediates the lactate-induced M2 phenotype of tumor-associated macrophages.

Expression and function of odorant receptors (ORs), which account for more than 50% of G protein-coupled receptors, are being increasingly reported in nonolfactory sites. However, ORs that can be targeted by drugs to treat diseases remain poorly identified. Tumor-derived lactate plays a crucial role in multiple signaling pathways leading to generation of tumor-associated macrophages (TAMs). In this study, we hypothesized that the macrophage OR Olfr78 functions as a lactate sensor and shapes the macrophage-tumor axis. Using Olfr78+/+ and Olfr78-/- bone marrow-derived macrophages with or without exogenous Olfr78 expression, we demonstrated that Olfr78 sensed tumor-derived lactate, which was the main factor in tumor-conditioned media responsible for generation of protumoral M2-TAMs. Olfr78 functioned together with Gpr132 to mediate lactate-induced generation of protumoral M2-TAMs. In addition, syngeneic Olfr78-deficient mice exhibited reduced tumor progression and metastasis together with an increased anti- versus protumoral immune cell population. We propose that the Olfr78-lactate interaction is a therapeutic target to reduce and prevent tumor progression and metastasis.

Inhibition of Tumor Growth against Chemoresistant Cholangiocarcinoma by a Proapoptotic Peptide Targeting Interleukin-4 Receptor.

Cholangiocarcinoma (CCA) has a poor prognosis and high chemoresistance. Interleukin-4 receptor (IL-4R) is overexpressed in several cancer cells and plays a crucial role in tumor progression and drug resistance. IL4RPep-1, an IL-4R-binding peptide, has been identified by phage display and used for tumor targeting. In this study, we exploited IL4RPep-1 to guide the tumor-specific delivery of a proapoptotic peptide to chemoresistant CCA, thereby inhibiting tumor growth. Immunohistochemistry of human primary CCA tissues showed that IL-4R levels were upregulated in moderately to poorly differentiated types, and higher levels of IL-4R are correlated with lower survival rates in patients with CCA. IL4RPep-1 was observed to preferentially bind with high IL-4R-expressing KKU-213 human CCA cells, whereas it barely bound with low IL-4R-expressing KKU-055 cells. A hybrid of IL4RPep-1 and a proapoptotic peptide (KLAKLAK)2 (named as IL4RPep-1-KLA) induced cytotoxicity and apoptosis in KKU-213 cells and increased those levels induced by 5-fluorouracil (5-FU). IL4RPep-1-KLA was internalized in the cells and colocalized with mitochondria. Whole-body fluorescence imaging and immunohistochemical analysis of tumor tissues showed the homing of IL4RPep-1-KLA as well as IL4RPep-1 to KKU-213 tumor in mice. Systemic administration of IL4RPep-1-KLA efficiently inhibited KKU-213 tumor growth, whereas treatment with 5-FU alone did not significantly inhibit tumor growth in mice. No significant systemic side effects including liver toxicity and immunotoxicity were observed in mice during peptide treatments. These findings suggest that IL4RPep-1-KLA holds potential as a targeted therapeutic agent against chemoresistant CCA.

Imaging of bioluminescent Acinetobacter baumannii in a mouse pneumonia model.

Bioluminescence imaging is a non-invasive tool for in vivo real-time monitoring of infectious disease progression in animal models. However, no bioluminescence imaging assay has been developed to monitor Acinetobacter baumannii infections. In the current study, bioluminescent strains of A. baumannii ATCC 17978 and its isogenic ΔompA mutant were constructed by integrating the promoter of the ompA gene and the luxCDABE luciferase gene into the bacterial chromosome. In an acute murine pneumonia model, bioluminescence of the two reporter strains was clearly visible in the lungs and the bioluminescent signal increased over time. Bioluminescence was correlated with bacterial burden and histopathology in reporter strain-infected mice, suggesting that bioluminescent bacteria are useful for monitoring A. baumannii infections in animal models.

A targeted ferritin-microplasmin based thrombolytic nanocage selectively dissolves blood clots.

The use of thrombolytic therapies is limited by an increased risk of systemic hemorrhage due to lysis of hemostatic clots. We sought to develop a plasmin-based thrombolytic nanocage that efficiently dissolves the clot without causing systemic fibrinolysis or disrupting hemostatic clots. Here, we generated a double chambered short-length ferritin (sFt) construct that has an N-terminal region fused to multivalent clot targeting peptides (CLT: CNAGESSKNC) and a C-terminal end fused to a microplasmin (µPn); CLT recognizes fibrin-fibronectin complexes in clots, µPn efficiently dissolves clots, and the assembly of double chambered sFt (CLT-sFt-µPn) into nanocage structure protects the activated-µPn from its circulating inhibitors. Importantly, activated CLT-sFt-µPn thrombolytic nanocage showed a prolonged circulatory life over activated-µPn and efficiently lysed the preexisting clots in both arterial and venous thromboses models. Thus, CLT-sFt-µPn thrombolytic nanocage platform represents the prototype of a targeted clot-busting agent with high efficacy and safety over existing thrombolytic therapies.

Elevated Interleukin-13 Receptor Alpha 1 Expression in Tumor Cells Is Associated with Poor Prognosis in Patients with Invasive Breast Cancer.

BACKGROUND: Interleukin (IL)-13 is an immunoregulatory, anti-inflammatory cytokine that is produced by numerous immune cells, and plasma membrane receptor for IL-13 (IL-13R) is known to be expressed in various human malignancies and in immune cells. METHODS: The authors evaluated the expression of IL-13R alpha 1 (IL-13Rα1, an IL-13R subtype) by immunohistochemistry in tissue microarrays of 1213 invasive breast cancer (IBC) samples to determine the prognostic value of IL-13Rα1 expression. RESULTS: High IL-13Rα1 expression was observed in 619 (51%) cases and was found to be associated with an older (≥50 years) age (p = 0.022), lymph node metastasis (p = 0.015), ductal and micropapillary histologic subtypes (p < 0.001), lymphovascular invasion (p = 0.012), HER2 positivity (p < 0.001), and a high (>20%) Ki-67 index (p = 0.039). No significant correlation was found between IL-13Rα1 expression and clinicopathological variables, including tumor size, histological grade, hormone receptor expressions, and tumor-infiltrating lymphocyte levels. Patients with high IL-13Rα1 expression showed poorer overall survival (p = 0.044) and disease-free survival (DFS, p = 0.001) than those with low/negative expression. Subgroup analysis revealed an association between IL-13Rα1 expression and survival for HER2-negative, but not for HER2-positive tumors. Multivariate analysis showed high IL-13Rα1 expression was an independent negative prognostic factor of DFS (p = 0.019). CONCLUSIONS: The results of this study suggest the IL-13 and IL-13Rα1 interaction promotes cancer cell growth and metastasis, and IL-13Rα1 expression is a potential prognostic marker in IBC.

2-Hydroxy-3-methoxybenzoic acid attenuates mast cell-mediated allergic reaction in mice via modulation of the FcεRI signaling pathway.

Mast cells are important effector cells in immunoglobulin (Ig) E-mediated allergic reactions such as asthma, atopic dermatitis and rhinitis. Vanillic acid, a natural product, has shown anti-oxidant and anti-inflammatory activities. In the present study, we investigated the anti-allergic inflammatory effects of ortho-vanillic acid (2-hydroxy-3-methoxybenzoic acid, o-VA) that was a derivative of vanillic acid isolated from Amomum xanthioides. In mouse anaphylaxis models, oral administration of o-VA (2, 10, 50 mg/kg) dose-dependently attenuated ovalbumin-induced active systemic anaphylaxis and IgE-mediated cutaneous allergic reactions such as hypothermia, histamine release, IgE production and vasodilation; administration of o-VA also suppressed the mast cell degranulator compound 48/80-induced anaphylaxis. In cultured mast cell line RBL-2H3 and isolated rat peritoneal mast cells in vitro, pretreatment with o-VA (1-100 µmol/L) dose-dependently inhibited DNP-HSA-induced degranulation of mast cells by decreasing the intracellular free calcium level, and suppressed the expression of pro-inflammatory cytokines TNF-α and IL-4. Pretreatment of RBL-2H3 cells with o-VA suppressed DNP-HSA-induced phosphorylation of Lyn, Syk, Akt, and the nuclear translocation of nuclear factor-κB. In conclusion, o-VA suppresses the mast cell-mediated allergic inflammatory response by blocking the signaling pathways downstream of high affinity IgE receptor (FcεRI) on the surface of mast cells.

Association between perfluorooctanoic acid exposure and degranulation of mast cells in allergic inflammation.

Perfluorooctanoic acid (PFOA) has wide applications, including as a raw material for converted paper and packaging products. With the widespread use of PFOA, concerns regarding its potential environmental and health impacts have increased. In spite of the known hepatotoxicity and genotoxicity of PFOA, correlation with PFOA and allergic inflammation is not well known. In this study, the effect of PFOA on the degranulation of mast cells and mast cell-mediated allergic inflammation in the presence of FcεRI cross-linking was evaluated. In immunoglobulin (Ig) E-stimulated mast cells, PFOA increased the release of histamine and ß-hexosaminidase by the up-regulation of intracellular calcium levels. PFOA enhanced gene expression of several pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-8 by the activation of nuclear factor (NF)-κB in IgE-stimulated mast cells. Also, PFOA exacerbated allergic symptoms via hypothermia, and an increase of serum histamine, TNF-α, IgE and IgG1 in the ovalbumin-induced systemic anaphylaxis. The present data indicate that PFOA aggravated FcɛRI-mediated mast cell degranulation and allergic symptoms. Copyright © 2016 John Wiley & Sons, Ltd.

1,2,4,5-Tetramethoxybenzene Suppresses House Dust Mite-Induced Allergic Inflammation in BALB/c Mice.

BACKGROUND: Atopic dermatitis (AD) is the most common allergic inflammatory skin disease. The activation of innate immunity by house dust mite (Dermatophagoides farinae extract, DFE) allergen plays an important role in the pathogenesis of AD. We previously showed the inhibitory effect of an extract of Amomum xanthioides on allergic diseases, and isolated 1,2,4,5-tetramethoxybenzene (TMB) as a major active component. In this study, we investigated whether TMB relieves DFE-induced allergic inflammation symptoms. METHODS: We established a DFE-induced allergic inflammation model in BALB/c mice by repeated skin exposure to DFE. To define the underlying mechanisms of action, we used a tumor necrosis factor-α and interferon-x03B3;-activated human keratinocytes (HaCaT cell line) and mouse keratinocytes (3PC cell line) cell line model. RESULTS: Oral administration of TMB suppressed allergic inflammation symptoms, such as histopathological analysis and ear thickness, in addition to serum IgE, DFE-specific IgE and IgG2a levels. TMB decreased the serum histamine levels and tissue infiltration of inflammatory cells, including mast cells and eosinophils. TMB also inhibited CD4+IFN-x03B3;+, CD4+IL-4+, and CD4+IL-17A+ lymphocyte expansion in the draining lymph nodes and expression of the Th2 cytokines in the ear tissue. TMB significantly inhibited the expression of cytokines and chemokines by the downregulation of the mitogen-activated protein kinases and nuclear factor of activated cytoplasmic T cells in HaCaT cells. CONCLUSIONS: TMB improved DFE-induced allergic inflammation by suppressing the production of proinflammatory cytokines and chemokines. Our results suggest that TMB might be a potential therapeutic agent for AD.

Double-Chambered Ferritin Platform: Dual-Function Payloads of Cytotoxic Peptides and Fluorescent Protein.

Ferritin cage nanoparticles are promising platforms for targeted delivery of imaging and therapeutic agents. One of the main advantages of cage nanoparticles is the ability to display multiple functionalities through genetic modification so as to achieve desired therapeutic or diagnostic functions. Ferritin complexes formed from short ferritin (sFt) lacking the fifth helix can accommodate dual peptide and protein functionalities on N- and C-terminal sites in sFt monomers. The resulting double-chambered NanoCage (DCNC) offers the potential of dual activities; these activities are augmented by the avidity of the ligands, which do not impede each other's function. Here we demonstrated proof-of-concept of DCNCs, loading the tumor-targeting proapoptotic peptide CGKRK(KLAKLAK)2 onto the N-terminal chamber and green fluorescent protein (GFP) onto the C-terminal chamber. The resulting KLAK-sFt-GFP DCNCs were internalized into the human breast adenocarcinoma cell line MDA-MB-231 and induced apoptosis. These findings suggest that DCNCs containing various combinations of peptides and proteins could be applied as therapeutics in different diseases.

Designing Peptide Bunches on Nanocage for Bispecific or Superaffinity Targeting.

Ferritin cage nanoparticles are promising platforms for targeted delivery of imaging and therapeutic agents because their cage structure can accommodate small molecules and their surfaces can be decorated with multiple functionalities. However, selective targeting is still a challenge for translating ferritin-based nanomedicines into the clinic, especially for heterogeneous diseases such as cancer. Targeting peptides can be genetically fused onto the surface of a ferritin cage, forming peptide bunches on nanocages (PBNCs) that offer synergistic increases in binding avidity. Here, we utilized two sites of the ferritin monomer, the N-terminus and the loop between the fourth and fifth helices, which are exposed on the surface of the assembled 24-subunit ferritin cage, to ligate one or two types of peptides to achieve "super affinity" and bispecificity, respectively. PBNCs formed by ligation of the IL-4 receptor-targeting peptide, AP1, to both sites (48AP1-PBNCs) tethered IL-4R, expressing tumor cells with greater affinity than did PBNCs with AP1 ligated to a single site (24AP1-PBNCs). Moreover, bispecific PBNCs containing 24 RGD peptides and 24 AP1 peptides (24RGD/24AP1-PBNCs) were capable of independently targeting cells expressing the corresponding receptors. Bispecific and superaffinity PBNCs could be useful for efficient targeting of ferritin-based therapeutic/diagnostic agents in a clinical setting.

Apoptosis imaging studies in various animal models using radio-iodinated peptide.

Apoptosis has a role in many medical disorders and treatments; hence, its non-invasive evaluation is one of the most riveting research topics. Currently annexin V is used as gold standard for imaging apoptosis. However, several drawbacks, including high background, slow body clearance, make it a suboptimum marker for apoptosis imaging. In this study, we radiolabeled the recently identified histone H1 targeting peptide (ApoPep-1) and evaluated its potential as a new apoptosis imaging agent in various animal models. ApoPep-1 (CQRPPR) was synthesized, and an extra tyrosine residue was added to its N-terminal end for radiolabeling. This peptide was radiolabeled with (124)I and (131)I and was tested for its serum stability. Surgery- and drug-induced apoptotic rat models were prepared for apoptosis evaluation, and PET imaging was performed. Doxorubicin was used for xenograft tumor treatment in mice, and the induced apoptosis was studied. Tumor metabolism and proliferation were assessed by [(18)F]FDG and [(18)F]FLT PET imaging and compared with ApoPep-1 after doxorubicin treatment. The peptide was radiolabeled at high purity, and it showed reasonably good stability in serum. Cell death was easily imaged by radiolabeled ApoPep-1 in an ischemia surgery model. And, liver apoptosis was more clearly identified by ApoPep-1 rather than [(124)I]annexin V in cycloheximide-treated models. Three doxorubicin doses inhibited tumor growth, which was evaluated by 30-40% decreases of [(18)F]FDG and [(18)F]FLT PET uptake in the tumor area. However, ApoPep-1 demonstrated more than 200% increase in tumor uptake after chemotherapy, while annexin V did not show any meaningful uptake in the tumor compared with the background. Biodistribution data were also in good agreement with the microPET imaging results. All of the experimental data clearly demonstrated high potential of the radiolabeled ApoPep-1 for in vivo apoptosis imaging.

Conversion of low-affinity peptides to high-affinity peptide binders by using a ß-hairpin scaffold-assisted approach.

Affinity maturation of protein-targeting peptides is generally accomplished by homo- or heterodimerization of known peptides. However, applying a heterodimerization approach is difficult because it is not clear a priori what length or type of linker is required for cooperative binding to a target. Thus, an efficient and simple affinity maturation method for converting low-affinity peptides into high-affinity peptides would clearly be advantageous for advancing peptide-based therapeutics. Here, we describe the development of a novel affinity maturation method based on a robust ß-hairpin scaffold and combinatorial phage-display technology. With this strategy, we were able to increase the affinity of existing peptides by more than four orders of magnitude. Taken together, our data demonstrate that this scaffold-assisted approach is highly efficient and effective in generating high-affinity peptides from their low-affinity counterparts.

Immune Checkpoint-Blocking Nanocages Cross the Blood-Brain Barrier and Impede Brain Tumor Growth.

Glioblastoma (GBM) is the deadliest tumor of the central nervous system, with a median survival of less than 15 months. Despite many trials, immune checkpoint-blocking (ICB) therapies using monoclonal antibodies against the PD-1/PD-L1 axis have demonstrated only limited benefits for GBM patients. Currently, the main hurdles in brain tumor therapy include limited drug delivery across the blood-brain barrier (BBB) and the profoundly immune-suppressive microenvironment of GBM. Thus, there is an urgent need for new therapeutics that can cross the BBB and target brain tumors to modulate the immune microenvironment. To this end, we developed an ICB strategy based on the BBB-permeable, 24-subunit human ferritin heavy chain, modifying the ferritin surface with 24 copies of PD-L1-blocking peptides to create ferritin-based ICB nanocages. The PD-L1pep ferritin nanocages first demonstrated their tumor-targeting and antitumor activities in an allograft colon cancer model. Next, we found that these PD-L1pep ferritin nanocages efficiently penetrated the BBB and targeted brain tumors through specific interactions with PD-L1, significantly inhibiting tumor growth in an orthotopic intracranial tumor model. The addition of PD-L1pep ferritin nanocages to triple in vitro cocultures of T cells, GBM cells, and glial cells significantly inhibited PD-1/PD-L1 interactions and restored T-cell activity. Collectively, these findings indicate that ferritin nanocages displaying PD-L1-blocking peptides can overcome the primary hurdle of brain tumor therapy and are, therefore, promising candidates for treating GBM.

IL4 receptor targeting enables nab-paclitaxel to enhance reprogramming of M2-type macrophages into M1-like phenotype via ROS-HMGB1-TLR4 axis and inhibition of tumor growth and metastasis.

Rationale: Nab-paclitaxel (Abx) is widely employed in malignant tumor therapy. In tumor cells and pro-tumoral M2-type macrophages, the IL4 receptor (IL4R) is upregulated. This study aimed to elucidate the selective delivery of Abx to M2-type macrophages by targeting IL4R and reprogramming them into an anti-tumoral M1-type. Methods: Abx was conjugated with the IL4R-binding IL4RPep-1 peptide using click chemistry (IL4R-Abx). Cellular internalization, macrophage reprogramming and signal pathways, and tumor growth and metastasis by IL4R-Abx were examined. Results: IL4R-Abx was internalized into M2 macrophages more efficiently compared to the unmodified Abx and control peptide-conjugated Abx (Ctrl-Abx), which was primarily inhibited using an anti-IL4R antibody and a receptor-mediated endocytosis inhibitor compared with a macropinocytosis inhibitor. IL4R-Abx reprogrammed the M2-type macrophages into M1-like phenotype and increased reactive oxygen species (ROS) levels and extracellular release of high mobility group box 1 (HMGB1) in M2 macrophages at higher levels than Abx and Ctrl-Abx. The conditioned medium of IL4R-Abx-treated M2 macrophages skewed M2 macrophages into the M1-like phenotype, in which an anti-HMGB1 antibody and a toll-like receptor 4 (TLR4) inhibitor induced a blockade. IL4R-Abx accumulated at tumors, heightened immune-stimulatory cells while reducing immune-suppressing cells, and hampered tumor growth and metastasis in mice more efficiently than Abx and Ctrl-Abx. Conclusions: These results indicate that IL4R-targeting allows enhancement of M2-macrophage shaping into M1-like phenotype by Abx through the ROS-HMGB1-TLR4 axis, improvement of antitumor immunity, and thereby inhibition of tumor growth and metastasis, presenting a new approach to cancer immunotherapy.

IRF3 Activation in Mast Cells Promotes FcεRI-Mediated Allergic Inflammation.

(1) Background: This study aims to elucidate a novel non-transcriptional action of IRF3 in addition to its role as a transcription factor in mast cell activation and associated allergic inflammation; (2) Methods: For in vitro experiments, mouse bone-marrow-derived mast cells (mBMMCs) and a rat basophilic leukemia cell line (RBL-2H3) were used for investigating the underlying mechanism of IRF3 in mast-cell-mediated allergic inflammation. For in vivo experiments, wild-type and Irf3 knockout mice were used for evaluating IgE-mediated local and systemic anaphylaxis; (3) Results: Passive cutaneous anaphylaxis (PCA)-induced tissues showed highly increased IRF3 activity. In addition, the activation of IRF3 was observed in DNP-HSA-treated mast cells. Phosphorylated IRF3 by DNP-HSA was spatially co-localized with tryptase according to the mast cell activation process, and FcεRI-mediated signaling pathways directly regulated that activity. The alteration of IRF3 affected the production of granule contents in the mast cells and the anaphylaxis responses, including PCA- and ovalbumin-induced active systemic anaphylaxis. Furthermore, IRF3 influenced the post-translational processing of histidine decarboxylase (HDC), which is required for granule maturation; and (4) Conclusion: Through this study, we demonstrated the novel function of IRF3 as an important factor inducing mast cell activation and as an upstream molecule for HDC activity.

Peptides as multifunctional players in cancer therapy.

Peptides exhibit lower affinity and a shorter half-life in the body than antibodies. Conversely, peptides demonstrate higher efficiency in tissue penetration and cell internalization than antibodies. Regardless of the pros and cons of peptides, they have been used as tumor-homing ligands for delivering carriers (such as nanoparticles, extracellular vesicles, and cells) and cargoes (such as cytotoxic peptides and radioisotopes) to tumors. Additionally, tumor-homing peptides have been conjugated with cargoes such as small-molecule or chemotherapeutic drugs via linkers to synthesize peptide-drug conjugates. In addition, peptides selectively bind to cell surface receptors and proteins, such as immune checkpoints, receptor kinases, and hormone receptors, subsequently blocking their biological activity or serving as hormone analogs. Furthermore, peptides internalized into cells bind to intracellular proteins and interfere with protein-protein interactions. Thus, peptides demonstrate great application potential as multifunctional players in cancer therapy.

Lupeol alleviates atopic dermatitis-like skin inflammation in 2,4-dinitrochlorobenzene/Dermatophagoides farinae extract-induced mice.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease that affects from children to adults widely, presenting symptoms such as pruritus, erythema, scaling, and dryness. Lupeol, a pentacyclic triterpenoid, has anti-inflammatory and antimicrobial activities. Based on these properties, the therapeutic effects of lupeol on skin disorders have been actively studied. In the present study, we aimed to determine the effectiveness of lupeol on AD. METHODS: We utilized tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated keratinocytes and 2, 4-dinitrochlorobenzene/Dermatophagoides farinae extract (DFE)-induced AD mice to confirm the action. RESULTS: Lupeol inhibited TNF-α/IFN-γ-stimulated keratinocytes activation by reducing the expressions of pro-inflammatory cytokines and chemokines which are mediated by the activation of signaling molecules such as signal transducer and activator of transcription 1, mitogen-activated protein kinases (p38 and ERK), and nuclear factor-κB. Oral administration of lupeol suppressed epidermal and dermal thickening and immune cell infiltration in ear tissue. Immunoglobulin (Ig) E (total and DFE-specific) and IgG2a levels in serum were also reduced by lupeol. The gene expression and protein secretion of T helper (Th) 2 cytokines, Th1 cytokines, and pro-inflammatory cytokine in ear tissue were decreased by lupeol. CONCLUSIONS: These results suggest that lupeol has inhibitory effects on AD-related responses. Therefore, lupeol could be a promising therapeutic agent for AD.

Endothelial argininosuccinate synthetase 1 regulates nitric oxide production and monocyte adhesion under static and laminar shear stress conditions.

Laminar shear stress (LSS) is known to increase endothelial nitric oxide (NO) production, which is essential for vascular health, through expression and activation of nitric oxide synthase 3 (NOS3). Recent studies demonstrated that LSS also increases the expression of argininosuccinate synthetase 1 (ASS1) that regulates the provision of L-arginine, the substrate of NOS3. It was thus hypothesized that ASS1 might contribute to vascular health by enhancing NO production in response to LSS. This hypothesis was pursued in the present study by modulating NOS3 and ASS1 levels in cultured endothelial cells. Exogenous expression of either NOS3 or ASS1 in human umbilical vein endothelial cells increased NO production and decreased monocyte adhesion stimulated by tumor necrosis factor-α (TNF-α). The latter effect of overexpressed ASS1 was reduced when human umbilical vein endothelial cells were co-treated with small interfering RNAs (siRNAs) for ASS1 or NOS3. SiRNAs of NOS3 and ASS1 attenuated the increase of NO production in human aortic endothelial cells stimulated by LSS (12 dynes·cm(-2)) for 24 h. LSS inhibited monocyte adhesion to human aortic endothelial cells stimulated by TNF-α, but this effect of LSS was abrogated by siRNAs of NOS3 and ASS1 that recovered the expression of vascular cell adhesion molecule-1. The current study suggests that the expression of ASS1 harmonized with that of NOS3 may be important for the optimized endothelial NO production and the prevention of the inflammatory monocyte adhesion to endothelial cells.

Synthesis and evaluation of a radioiodinated bladder cancer specific peptide.

Bladder cancer is the second most common cancer of the urinary tract, however the invasive cystoscopy is still the standard technique for diagnosis and surveillance of bladder cancer. Herein, we radiolabel bladder cancer specific peptide with radioactive iodine ((131/124)I) and evaluate its potential as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer. A 9-mer bladder cancer specific peptide (BP) was conjugated with tyrosine and cyclized by disulfide bond formation to give Y-BP, which was further radioiodinated to give [(131/124)I]Y-BP in good radiochemical yield. The biodistribution data showed the high selectivity of [(124)I]Y-BP in HT1376 human bladder cancer xenograft models with a tumor-to-muscle ratio of 6.2. This tumor targeting was not observed in control B16F10 melanoma tumor models. In microPET studies, while the control scrambled peptide, [(124)I]Y-sBP, did not accumulate in either the bladder cancer or melanoma, [(124)I]Y-BP showed high tumor uptake only in animals with HT1376 bladder cancer cells. Furthermore, [(124)I]Y-BP showed superior bladder cancer uptake even compared to most commonly used cancer imaging tracer, [(18)F]FDG. The experimental results suggest the potential of [(124)I]Y-BP as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer with high binding affinity and selectivity.