Topical drug delivery to retinal pigment epithelium with microfluidizer produced small liposomes.

Drug delivery from topically instilled eye drops to the posterior segment of the eye has long been one of the greatest challenges of ocular drug development. We developed methods of liposome preparation utilizing a microfluidizer to achieve adjustable nanoparticle size (even less than 80 nm) and high loading capacity of plasmid DNA. The microfluidizing process parameters were shown to affect the size of the liposomes. Higher operating pressures and passage for at least 10 times through the microfluidizer produced small liposomes with narrow size distribution. The liposomes were physically stable for several months at +4°C. In vivo distribution of the optimized liposome formulations in the rat eyes was investigated with confocal microscopy of the histological specimens. Transferrin was used as a targeting ligand directed to retinal pigment epithelium. Size dependent distribution of liposomes to different posterior segment tissues was seen. Liposomes with the diameter less than 80 nm permeated to the retinal pigment epithelium whereas liposomes with the diameter of 100 nm or more were distributed to the choroidal endothelium. Active targeting was shown to be necessary for liposome retention to the target tissue. In conclusion, these microfluidizer produced small liposomes in eye drops are an attractive option for drug delivery to the posterior segment tissues of the eye.

Delivery of siRNA to the brain using a combination of nose-to-brain delivery and cell-penetrating peptide-modified nano-micelles.

The potential for RNA-based agents to serve as effective therapeutics for central nerve systems (CNS) disorders has been successfully demonstrated in vitro. However, the blood-brain barrier limits the distribution of systemically administered therapeutics to the CNS, posing a major challenge for drug development aimed at combatting CNS disorders. Therefore, the development of effective strategies to enhance siRNA delivery to the brain is of great interest in clinical and pharmaceutical fields. To improve the efficiency of small interfering RNA (siRNA) delivery to the brain, we developed a nose-to-brain delivery system combined with cell-penetrating peptide (CPP) modified nano-micelles comprising polyethylene glycol-polycaprolactone (PEG-PCL) copolymers conjugated with the CPP, Tat (MPEG-PCL-Tat). In this study, we describe intranasal brain delivery of siRNA or dextran (Mw: 10,000 Da) as a model siRNA, by using MPEG-PCL-Tat. Intranasal delivery of dextran with MPEG-PCL-Tat improved brain delivery compared to intravenous delivery of dextran either with or without MPEG-PCL-Tat. We also studied the intranasal transfer of MPEG-PCL-Tat to the brain via the olfactory and trigeminal nerves, the putative pathways to the brain from the nasal cavity. We found that MPEG-PCL-Tat accelerated transport along the olfactory and trigeminal nerve pathway because of its high permeation across the nasal mucosa.

Variety and complexity of fluorine-18-labelled fluoro-2-deoxy-D-glucose accumulations in the oral cavity of patients with oral cancers.

OBJECTIVES: To elucidate the points that require attention when interpreting fluorine-18-labelled fluoro-2-deoxy-d-glucose ((18)F-FDG)/positron emission tomography (PET) images by demonstration of (18)F-FDG accumulation in various areas of the oral cavity other than primary lesions in patients with oral cancers. METHODS: (18)F-FDG accumulations with a maximal standardized uptake value of over 2.5 in various areas of the oral cavity other than primary lesions were identified in 82 patients with oral cancers. RESULTS: (18)F-FDG/PET-positive areas, excluding primary tumours, included the front intrinsic muscles of the tongue (89.0%), upper and lower marginal parts of the orbicularis oris muscle (64.6%), sublingual glands, palatine tonsil, pharyngeal tonsil, and lingual tonsil. In addition, some areas in the jaws also showed accumulation. CONCLUSIONS: In patients with oral cancers, areas of (18)F-FDG accumulation in the oral cavity should be precisely identified and appropriately diagnosed, because accumulations can be seen in areas other than the primary tumour.

Expression of P2Y1 receptors in rat taste buds.

Extracellular nucleotides such as ATP are the signaling molecules which bind to membrane receptors (P2X ligand-gated ion channels and G-protein-coupled P2Y families). In the gustatory system, it is known that P2X receptors are expressed exclusively in nerve fibers innervating the taste buds. Also, P2Y receptors are suggested to play some important roles in taste signal transductions on the basis of the physiological studies. In the present study, we examined the expression patterns of P2Y1 receptor subtype by using reverse transcript polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR analyses showed that P2Y1 receptor mRNAs appeared in circumvallate papillae. P2Y1 receptor mRNA was detected in a subset of taste bud cells by in situ hybridization. By immunohistochemical analyses, P2Y1 receptor was detected in a subset of taste bud cells of fungiform, foliate, and circumvallate papillae. We showed that ATP induced a biphasic intracellular Ca2+ increase in taste cells by a Ca2+ imaging method. Furthermore, we showed by double-immunolabeling methods that P2Y1-expressing cells coexpressed both IP3R3 and SNAP-25. These results suggest that ATP may activate P2Y receptors resulting in Ca2+ release from internal stores via IP3R3. Since many SNAP-25-immunoreactive taste bud cells coexpressed P2Y1 immunoreactivity, it is suggested that P2Y1-expressing cells may possess synapses with afferent nerve fibers. The results of the present study suggest that P2Y1 receptor may play some roles in ATP-mediated signal transductions between taste bud cells and afferent taste fibers.

Characterization of dental epithelial progenitor cells derived from cervical-loop epithelium in a rat lower incisor.

Dental epithelial progenitor cells differentiate into various cell types during development of tooth germs. To study this mechanism, we produced immortalized dental epithelial progenitor cells derived from the cervical-loop epithelium of a rat lower incisor. The expression patterns of cytokeratin 14, nerve growth factor receptor p75, amelogenin, Notch2, and alkaline phosphatase were examined by immunohistochemistry in both lower and higher cell densities. The patterns of each were compared in the dental epithelium of rat lower incisors. The results demonstrated that these cells could produce ameloblast lineage cells, stratum intermedium cells, stellate reticulum, and outer enamel epithelium. Furthermore, fibroblast growth factor 10 stimulated proliferation of dental progenitor cells and subsequently increased the number of cells expressing alkaline phosphatase. These results suggest that fibroblast growth factor 10 plays a role in coupling mitogenesis of the cervical-loop cells and the production of stratum intermedium cells in rat incisors.

[Strategy for post coronary artery bypass grafting in patients with bypass graft stenosis: comparison of percutaneous transluminal coronary angioplasty for the native coronary artery, internal mammary artery and saphenous vein graft].

OBJECTIVES: The strategy for post coronary artery bypass grafting (CABG) was investigated in patients with graft stenosis. METHODS: The study included 123 post-CABG patients with graft stenosis. The patients were divided into three groups according to target vessels; saphenous vein graft (SVG; n = 72), internal mammary artery (IMA; n = 21) and native coronary artery (n = 30). Furthermore, SVG lesions were divided into proximal anastomosis (n = 23), body (n = 40) and distal anastomosis (n = 9). The procedural success rate and late patency rate were compared between the three groups. Furthermore, the relationships between pre percutaneous transluminal coronary angioplasty (PTCA) percentage diameter stenosis, procedural success rate and late patency rate were evaluated. RESULTS: Procedural success rate was similar in the three groups, but late patency rate was higher in the IMA group. Procedural success rate and late patency rate were significantly lower in proximal anastomoses compared to other sites of SVG stenoses, IMA group and native coronary artery group (p < 0.05). Totally occluded native coronary artery lesions had a high procedural success rate compared with occluded IMA and SVG lesions, but the late patency rate was not higher. Procedural success rate showed no significant difference for 75-99% stenotic lesions, but the late patency rate was significantly higher in the IMA group (p < 0.05). Patients in the stenting group had a greater late patency rate compared with the balloon angioplasty group. There was no significant difference in late patency rate between the IMA group and SVG group. CONCLUSIONS: Late patency rate of the IMA is higher than that of the native coronary artery. SVG with proximal anastomosis and severe stenosis shows a significantly lower late patency rate than the native coronary artery. Therefore, PTCA should be considered for the native coronary artery in the absence of chronic total occlusion.

Evaluation of the mechanical destructive force in the stomach of dog.

Orally administered dosage forms receive a destructive force in the gastrointestinal (GI) tract due to peristalsis. In this study, the destructive force was measured with a 'destructive force-dependent release system' (DDRS). DDRS is a press-coated tablet with an extremely brittle outer layer composed of highly hydrophobic Teflon(R) powder, which is molded with a weak compression force. Teflon(R) powder forms a porous but water-impermeable layer around the core tablet. A marker drug contained in the core tablet is released only when the tablet receives a force larger than its pre-determined crushing strength. A comparison of the physiological conditions in the GI tract of dogs with those of humans, including the destructive force against tablets in the stomach, helps us to understand their difference in bioavailability of oral dosage forms. With DDRS, it is possible to evaluate the destructive force of both human and dog stomach using the same method. Therefore, the destructive force data from human and dog can be directly compared. The destructive force in the dog stomach was evaluated to be 3.2 N, which was considerably stronger than that of humans.

Functional significance of hemodynamic overload-induced expression of leukemia-inhibitory factor in the adult mammalian heart.

BACKGROUND: Leukemia-inhibitory factor (LIF) is a member of the interleukin-6 family of cytokines that utilize gp130 as a common signaling component. In the present study, we examined the mechanisms that govern LIF expression and functional effects in the adult heart. METHODS AND RESULTS: LIF mRNA and protein biosynthesis were examined in the adult feline heart after hemodynamic overloading ex vivo. Both LIF mRNA and protein expression were detected within 60 to 90 minutes after hemodynamic overloading. Studies in isolated adult cardiac myocytes showed that these cells synthesized both LIF mRNA and protein. The functional effects of LIF in the heart were demonstrated by studies that showed that LIF stimulation led to a significant increase in general protein synthesis and an increase in sarcomeric protein synthesis. Pretreatment with LIF also protected the cells against hypoxia/reoxygenation-induced cardiac myocyte apoptosis and cellular injury. Finally, LIF had no effect on isolated cardiac myocyte cell motion. CONCLUSIONS: Hemodynamic overload is a sufficient stimulus for LIF expression in the adult mammalian heart. Given that LIF confers both hypertrophic and cytoprotective responses in adult cardiac myocytes, this study suggests that the expression of LIF within the heart may play an important role in mediating homeostatic responses within the myocardium.

Synthesis and in vitro antifungal activities of novel triazole antifungal agent CS-758.

Synthesis and in vitro antifungal activities of a novel triazole antifungal agent CS-758 (former name, R-120758) are described. The minimum inhibitory concentrations (MICs) of a series of dioxane-triazole compounds related to R-102557 were examined. Variation of the length of the chain between the dioxane ring and the phenyl ring revealed that the linkage with two double bonds is the most preferable. When a cyano group was introduced to the C4 position on the benzene ring, MICs improved further. A fluorine atom was introduced to obtain CS-758. The MICs of CS-758 surpassed those of fluconazole and itraconazole against Candida, Aspergillus and Cryptococcus species. The precursor (E,E)-aldehyde was synthesized stereoselectively from 3-fluoro-4-methylbenzonitrile using the Horner-Wadsworth-Emmons reaction.

Effect of interleukin-18 on viral myocarditis: enhancement of interferon- gamma and natural killer cell activity.

Encephalomyocarditis virus causes viral myocarditis with myocyte necrosis and inflammatory cell infiltration in mice. Because previous studies have shown that some cytokines prevent the sequelae of myocarditis, we assessed the effect of a newly identified cytokine, interleukin-18 (IL-18), in preventing the sequelae of myocarditis. Murine IL-18 (10 microg/day/mouse) was given peritoneally for 10 days in C3H mice infected with EMC virus. Mice were divided into group IL-18 (infected-treated), saline group (infected-untreated), group IL-18-2 (treatment started on day 2), group IL-18-5 (treatment started on day 5). Although the 14-day survival rate in saline group was 20%, that in the group IL-18 increased to 80% (P<0.05). Either mice in group IL-18-2 or in group IL-18-5 did not survive longer than saline group. The viral titer of the heart on day 3 was lower in group IL-18 compared to the saline group (1.00+/-0.20 log(10)tissue culture infected dose (TCID)(50)/mg wet weight v 1.42+/-0.12 log(10)TCID(50)/mg, n=3 of each). Mice in group IL-18 had less myocardial necrosis and cellular infiltration than the saline group. The myocardial expression of interferon- gamma (IFN- gamma) mRNA in group IL-18 was significantly (P<0.05) greater than the saline group on days 1 and 3 after viral inoculation. Circulating IFN- gamma was significantly elevated on days 1, 5, and 7, but significantly reduced on day 3. The natural killer cell activities in the spleen on days 1, 3, and 5 were significantly (P<0.05) greater in group IL-18 than in the saline group (41+/-9%v 10+/-6% on day 3, 4 of each). We conclude that IL-18 reduces the severity of EMC viral myocarditis by inducing cardiac expression of IFN- gamma mRNA and increasing splenic natural killer cell activity.

A unique dosage form to evaluate the mechanical destructive force in the gastrointestinal tract.

The purpose of this study was to prepare tablets that could evaluate the destructive force in the gastrointestinal (GI) tract. Many factors are known to affect in vivo drug release from oral dosage forms. There is still relatively little information on the mechanical destructive force in the GI tract. Press-coated tablets with an extremely brittle outer layer were developed using a unique, highly hydrophobic Teflon powder that could be shaped with weak compression force. A marker drug contained in the tablets was released only when the tablets received a force larger than its predetermined crushing strength. We referred to this type of tablet as a 'destructive force dependent release system' (DDRS). A total of nine healthy, male subjects were orally administered the tablets under fed and/or fasting conditions. Tablets with a predetermined crushing strength of 1.50 N were crushed by all of the four subjects who took them under fed conditions and two of the five subjects under fasting conditions. Tablets with a crushing strength of 1.89 N were crushed by two of the six subjects who took them under fed conditions and none of the five subjects under fasting conditions. The range of mechanical destructive force in the human stomach was obtained.

Nitric oxide provokes tumor necrosis factor-alpha expression in adult feline myocardium through a cGMP-dependent pathway.

BACKGROUND: The mechanism(s) responsible for the persistent coexpression of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in the failing heart is unknown. METHODS AND RESULTS: To determine whether NO was sufficient to provoke TNF-alpha biosynthesis, we examined the effects of an NO donor, S-nitroso-N-acetyl penicillamine (SNAP), in buffer-perfused Langendorff hearts. SNAP (1 micromol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF-alpha mRNA and protein biosynthesis in adult cat hearts. The effects of SNAP were completely abrogated by a NO quenching agent, 2-(4-carboxyphenyl)-4, 4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO), and mimicked by sodium nitroprusside. Electrophoretic mobility shift assays demonstrated that SNAP treatment led to the rapid induction of nuclear factor kappa-beta (NF-kappaB) but not AP-1. The importance of the cGMP pathway in terms of mediating NO-induced TNF-alpha biosynthesis was shown by studies that demonstrated that 8-bromo-cGMP mimicked the effects of SNAP and that the effects of SNAP could be completely abrogated using a cGMP antagonist, 1H-(1,2, 4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or protein kinase G antagonist (Rp-8-Br-cGMPS). SNAP and 8-Br-cGMP were both sufficient to lead to the site-specific phosphorylation (serine 32) and degradation of IkappaBalpha in isolated cardiac myocytes. Finally, protein kinase G was sufficient to directly phosphorylate IkappaBalpha on serine 32, a critical step in the activation of NF-kappaB. CONCLUSIONS: These studies show that NO provokes TNF-alpha biosynthesis through a cGMP-dependent pathway, which suggests that the coincident expression of TNF-alpha and NO may foster self-sustaining positive autocrine/paracrine feedback inflammatory circuits within the failing heart.

Therapy with the nonpeptide endothelin receptor antagonist 97-139 in a murine model of congestive heart failure: reduction of cardiac mass and myofiber hypertrophy.

Endothelin-1 (ET-1) is a potent vasoconstrictor. This peptide exerts numerous effects on the heart, including regulation of cardiomyocyte growth during hypertrophy. The effects of the structurally novel, nonpeptide, ET-1-selective, competitive antagonist (ETA) 97-139 were investigated in mice with congestive heart failure (CHF) and myocardial hypertrophy. Morphological and microscopical analyses were conducted on day 56 after viral inoculation following 28 day treatment with 99-139. Eight week-old DBA2 mice were intraperitoneally inoculated with encephalomyocarditis virus at a dose of 500 pfu/mouse. The 30 mice were divided into two groups--an ETA treated group and an untreated group. Heart weight (HW) in the infected group was significantly (p < 0.05) increased compared to that in the uninfected group. HW and the HW/body weight (BW) ratio were significantly (p < 0.05) reduced in the ETA treated group compared with the untreated group (HW; 127.7 +/- 6.2 mg vs 144.3 +/- 4.2 mg, HW/BW; 4.9 +/- 0.9 x 10(-3) vs 5.4 +/- 0.5 x 10(-3)). Myofiber diameter in the ETA treated group was significantly reduced compared with the untreated group (12.1 +/- 1.5 microm vs 14.3 +/- 1.9 microm). These results suggest the ET-1 receptor antagonist 97-139 has an effect on the reduction of cardiac mass and myofiber hypertrophy, and that 97-139 may be a useful agent for CHF due to viral myocarditis.

Interleukin-18 in patients with congestive heart failure: induction of atrial natriuretic peptide gene expression.

Activated inflammatory responses appear to play a role in the development of congestive heart failure (CHF). We investigated interleukin-18 (IL-18), which is a cytokine synthesized by activated macrophage, changes in patients with CHF. We evaluated 11 Japanese patients with angina pectoris (n=4) or CHF (n=7). Blood was sampled immediately after admission and at 1, 2, 3, 6, and 9 hours after admission and then every 12 hours until 5 days after admission. Plasma IL-18 concentrations were measured by an enzyme-linked immunosorbent assay. Expression of atrial natriuretic peptide (ANP) mRNA and protein synthesis was examined in cardiac myocyte by stimulation of IL-18. Plasma IL-18 concentration was significantly higher in patients with CHF than in 15 healthy volunteers (51+/-21 pg/mL, and 28+11 pg/mL, respectively, P<0.05). Increased expression of ANP mRNA was demonstrated in IL-18 treated myocytes. Protein synthesis in myocytes was increased by IL-18 in a dose-dependent manner. Increased secretion of IL-18 is induced in patients with CHF and correlates with the severity of myocardial damage and dysfunction.

Cytokines in heart failure: pathogenetic mechanisms and potential treatment.

Recent studies have shown that patients with heart failure overexpress a class of biologically active molecules, generically referred to as pro-inflammatory cytokines. This article will review recent clinical and experimental material that suggests that pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and interleukin-6 (IL-6) may play a role in the pathogenesis of congestive heart failure. In addition, we will review recent studies that suggest that antagonizing cytokines may represent a novel target for heart failure therapy.

Safety and efficacy of a soluble P75 tumor necrosis factor receptor (Enbrel, etanercept) in patients with advanced heart failure.

BACKGROUND: Although previous studies suggested that TNF may contribute to heart failure progression, it is unclear whether antagonizing TNF is beneficial in heart failure patients. METHODS AND RESULTS: Eighteen NYHA class III heart failure patients were randomized into a double-blind dose-escalation study to examine the safety and potential efficacy of etanercept, a specific TNF antagonist (Enbrel). Patients received placebo (6 patients) or an escalating dose (1, 4, or 10 mg/m2) of etanercept (12 patients) given as a single intravenous infusion. Safety parameters and patient functional status were assessed at baseline and at days 1, 2, 7, and 14. There were no significant side effects or clinically significant changes in laboratory indices. There was, however, a decrease in TNF bioactivity and a significant overall increase in quality-of-life scores, 6-minute walk distance, and ejection fraction in the cohort that received 4 or 10 mg/m2 of etanercept; there was no significant change in these parameters in the placebo group. CONCLUSIONS: A single intravenous infusion of etanercept was safe and well tolerated in patients with NYHA class III heart failure. These studies provide provisional evidence that suggests that etanercept is sufficient to lower levels of biologically active TNF and may lead to improvement in the functional status of patients with heart failure.

Merkel cells are responsible for the initiation of taste organ morphogenesis in the frog.

Taste organs in the frog have a distinctive cell type located exclusively in the basal portion. In the same fashion as type III cells in mammalian taste buds, these basal cells show immunoreactivity for serotonin antibody. Further, these cells are morphologically similar to epidermal Merkel cells. To determine the significance of these serotonergic basal cells, we examined the early development of taste organs during metamorphosis of the frog by focusing on the origin and possible roles of serotonergic basal cells. For convenience of description, five stages of development (metamorphic stage to climax stages A-D) are defined. In the metamorphic stage, a few noninnervated Merkel cells appear at the upper layer of the lingual epithelium. No neuronal elements are seen in the epithelium at this stage. At climax stages A-B, immature fungiform papillae become discernible in the dorsal surface of the tongue, where the Merkel cells are located. Merkel cells then move downward and extend their cytoplasmic processes toward the basal lamina. These cells are identified by their intense immunoreactivity for serotonin. During the later stages, many nerve fibers in the subepithelial connective tissue approach the epithelium containing Merkel cells. At climax stages C-D, Merkel cells extend cytoplasmic processes along the basal lamina toward the center of the newly forming fungiform papillae. The morphology of these Merkel cells exactly coincides with that of serotonergic basal cells in adult taste organs. Profuse exocytotic release of dense-cored granules of Merkel cells toward the nerve fibers through the basal lamina is frequently seen in these stages. The present study indicates that serotonergic basal cells are derived from intraepithelial Merkel cells, which act as target sites for growing nerves and may be responsible for the initiation of taste organ morphogenesis.

Expression of Mash1 in basal cells of rat circumvallate taste buds is dependent upon gustatory innervation.

Mash1, a mammalian homologue of the Drosophila achaete-scute proneural gene complex, plays an essential role in differentiation of subsets of peripheral neurons. In this study, using RT-PCR and in situ RT-PCR, we investigated if Mash1 gene expression occurs in rat taste buds. Further, we examined dynamics of Mash1 expression in the process of degeneration and regeneration in denervated rat taste buds. In rat tongue epithelium, Mash1 gene expression is confined to circumvallate, foliate, and fungiform papilla epithelia that include taste buds. In taste buds, Mash1-expressing cells are round cells in the basal compartment. In contrast, the mature taste bud cells do not express the Mash1 gene. Denervation and regeneration experiments show that the expression of Mash1 requires gustatory innervation. We conclude that Mash1 is expressed in cells of the taste bud lineage, and that the expression of Mash1 in rat taste buds is dependent upon gustatory innervation.

Cardiac remodeling as a consequence and cause of progressive heart failure.

Natural history studies in heart failure have shown that increases in left ventricular (LV) volume and LV mass are directly related to future deterioration in LV performance and a less favorable clinical course. Despite the recognized importance of remodeling in heart failure, very little is known about the basic mechanisms that lead to cardiac remodeling. In this review, we will summarize recent clinical and experimental studies that highlight the importance of the remodeling process during the progression of heart failure. The intent of this review is to provide an integrated view of the mechanisms that contribute to LV remodeling at the cellular level, the myocardial level, and the level of the chamber.