Megumu Higaki

An important role of tumor necrosis factor-α in the induction of adhesion molecules in psoriasis

Abstract Recent studies have suggested that cell adhesion plays an important role in the development and regulation of inflammation. To elucidate the mechanisms of regulation of adhesion molecule expression by cytokines in psoriatic lesions, we compared the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, and P-selectin immunohistochemically in involved and uninvolved psoriatic skin with the expression of these molecules in normal skin, and measured the amounts of tumor necrosis factor-α, interferon-γ, interleukin-1α, and interleukin-1β in the supernatant of freeze-thawed skin specimens using an enzyme-linked immunosorbent assay. There was strong staining for P-selectin on endothelial cells from involved skin. There was also strong staining for intercellular adhesion molecule-1 on keratinocytes, dermal infiltrates, and endothelial cells from involved skin and on endothelial cells from uninvolved skin, and strong staining for vascular cell adhesion molecule-1 on dermal dendritic cells and fibroblasts and for E-selectin on endothelial cells from involved skin. Large amounts of tumor necrosis factor-α were detected in six out of ten specimens of involved skin, but not in uninvolved or normal skin, although interferon-γ was detected in both involved and uninvolved skin to the same extent. Neither interleukin-1α nor interleukin-1β was detected in involved skin. There was strong staining for tumor necrosis factor-α on keratinocytes and endothelial cells from involved skin. These findings suggest that tumor necrosis factor-αmight play an important role in the induction of vascular adhesion molecules in psoriatic lesions.

Drug delivery to the cochlea using PLGA nanoparticles.

Objectives: This study aimed to investigate the efficacy of encapsulating therapeutic molecules in poly lactic/glycolic acid (PLGA) nanoparticles for drug delivery to the cochlea.

Treatment of experimental arthritis with poly(d, l-lactic/glycolic acid) nanoparticles encapsulating betamethasone sodium phosphate

Objective: To examine the therapeutic activity of hydrophilic glucocorticoid encapsulated in PLGA nanoparticles, which have shown slow release and are targeted to inflamed joints after intravenous administration, in experimental arthritis models. Methods: Betamethasone sodium phosphate (BSP) encapsulated in PLGA nanoparticles with a size of 100–200 nm (PLGA-nanosteroid) was prepared using a modified oil in water emulsion solvent diffusion method with Zn ions and coated with lecithin. Rats with adjuvant arthritis (AA rats) and mice with anti-type II collagen antibody induced arthritis (AbIA mice) were treated intravenously with PLGA-nanosteroid after the initial sign of arthritis. Results: In AA rats, a 30% decrease in paw inflammation was obtained in 1 day and maintained for 1 week with a single injection of 100 μg of PLGA-nanosteroid. Soft x ray examination 7 days after this treatment showed decreased soft tissue swelling. Moreover, the PLGA-nanosteroid was also highly effective in AbIA mice. A single injection of 30 μg of the PLGA-nanosteroid resulted in almost complete remission of the inflammatory response after 1 week. In contrast, the same dose of free BSP after three administrations only moderately reduced the severity of inflammation. In addition, a histological examination 7 days after the treatment showed a significant decrease of the inflammatory cells in the joints. Conclusion: The observed strong therapeutic benefit obtained with PLGA-nanosteroid may be due to the targeting of the inflamed joint and its prolonged release in situ. Targeted drug delivery using a sustained release PLGA-nanosteroid is a successful intervention in experimental arthritis.

Ear involvement in patients with rheumatoid arthritis.

Objective: This study evaluates the degree of hearing impairment in patients with rheumatoid arthritis (RA) and examines the correlation between hearing impairment and the clinical data or chemical mediators. Background: Both sensorineural hearing loss (SNHL) and conductive hearing loss (CHL) have been reported in patients with RA, but the results of most studies are not in agreement, and the pathophysiology of hearing impairment in RA is not well known. Methods: Hearing in patients with RA and controls was examined using pure-tone audiometry and tympanometry. Also, the amounts of pro-inflammatory cytokines and matrix metalloproteinases in addition to antibodies against type II collagen in plasma of the patients with RA were determined using enzyme-linked immunosorbent assay. Results: The frequency of SNHL in the patients with RA was higher than in normal controls (36.1% versus 13.9%), and bone conduction at 2,000 Hz differed significantly between the patients with RA and the controls (p < 0.01). Moreover, the presence of SNHL was related to ESR (p < 0.05), plasma interleukin-6 (p < 0.05), and plasma matrix metalloproteinase-3 (p < 0.001). On the other hand, CHL was not observed, whereas As-type tympanograms increased in the patients with RA (p < 0.01). Abnormal tympanograms were not related to any clinical findings or any chemical mediators tested. Conclusion: We demonstrated that there is increased SNHL in patients with RA, which may result from systemic inflammation and tissue injury, and increased latent-type CHL caused by stiffness of the middle ear system whose mechanisms are not yet clear.

Treatment of Experimental Arthritis with Stealth-Type Polymeric Nanoparticles Encapsulating Betamethasone Phosphate

We examined the therapeutic activity of betamethasone disodium 21-phosphate (BP) encapsulated in biocompatible and biodegradable blended nanoparticles of poly (d,l-lactic/glycolic acid) (PLGA)/poly(d,l-lactic acid) (PLA) homopolymers and polyethylene glycol (PEG)-block-PLGA/PLA copolymers (stealth nanosteroid) in experimental arthritis models. Various stealth nanosteroids with a size of 45 to 115 nm were prepared and then intravenously administered to rats with adjuvant arthritis (AA) rats and mice with anti-type II collagen antibody-induced arthritis (AbIA). The accumulation of stealth nanoparticles with Cy7 in inflamed joints was determined using an in vivo imaging system. The type A stealth nanosteroid, composed of PLA (2.6 kDa) and PEG (5 kDa)-PLA (3 kDa), with a PEG content of 10% and a diameter of 115 nm, exhibited the highest anti-inflammatory activity. In AA rats, a 35% decrease in paw inflammation was obtained in 1 day and maintained for 9 days with a single injection of the type A stealth nanosteroid (40 μg of BP), whereas the same does of nonstealth nanosteroid and 3 times higher free BP showed a significantly weaker response. In AbIA mice, a single injection of the type A stealth nanosteroid (3 μg of BP) resulted in complete remission of the inflammatory response after 1 week. Furthermore, in AbAI mice, the accumulation of type A stealth nanoparticles in inflamed joints was shown to parallel the severity of inflammation. The observed strong therapeutic benefit obtained with the type A stealth nanosteroid in experimental arthritis may have been due to prolonged blood circulation and targeting to the inflamed joint in addition to its sustained release in situ.

Stereospecific analysis of omeprazole in human plasma as a probe for CYP2C19 phenotype.

Omeprazole is a class referred to as proton pump inhibitor; it acts to regulate acid production in the stomach and is used to treat various acid-related gastrointestinal disorders. In the liver, it is metabolized to varying degrees by several cytochrome P-450 (CYP) isoenzymes which are further categorized into subfamilies of related polymorphic gene products. The metabolism of omeprazole is to a large extent dependent on CYP3A4 and CYP2C19. Omeprazole is metabolized to two major metabolites, 5-hydroxyomeprazole (CYP2C19) and omeprazole sulfone (CYP3A4). Minor mutations in CYP2C19 affect its activity in the liver and, in turn, the metabolic and pharmacokinetic profiles of omeprazole. The frequency of CYP2C19 poor metabolizers in population of Asian descent has been reported to range from 10 to 20%. Accordingly, results from population studies indicate that omeprazole can be used as a probe drug for phenotyping CYP2C19. The optical isomers of omeprazole show a clear difference in their metabolism by human liver microsomes. This study demonstrates the stereospecific analysis of omeprazole in human plasma as a probe drug of CYP2C19 phenotyping. The chiral separation of omeprazole was achieved on a chiral column with circular dichroism (CD) detection and LC/MS. A good resolution of enantiomers was obtained. The column used for chiral separation was CHIRALPAK AD-RH column (4.6 x 150 mm) using phosphate buffer and (or ammonium acetate) acetonitrile as an eluent. After a single oral dose of omeprazole (20 mg), the plasma concentrations of the separate enantiomers of omeprazole were determined for 3.5 h after drug intake. The present study is useful because of the part polymorphism plays in the therapeutic effectiveness of proton pump inhibitors during the treatment of acid-related diseases.

The role of granzyme B-expressing CD8-positive T cells in apoptosis of keratinocytes in lichen planus.

Abstract Epidermal basal cell injury with colloid body formation is a characteristic feature of lichen planus. Infiltrated cells are thought to be responsible for the epidermal injury. Ultrastructural findings of colloid bodies are typical of apoptosis. Granzymes in cytotoxic T lymphocytes are involved in apoptosis probably together with perforin. Based on this background, we analyzed the role of granzyme B in the mechanisms of epidermal injury in lichen planus. On electron microscopy, basal and suprabasal cells showed condensed chromatin and fragmented nuclei which are typical morphological features of apoptosis. Nuclei of colloid bodies were positively stained by the in situ nick end labeling technique indicating that colloid bodies are subsequently formed in the process of apoptosis. Immunohistochemical staining showed CD8-positive infiltrating cells to contain granzyme B. Cells undergoing exocytosis also contained granzyme B. By immunoelectron microscopy, granzyme B molecules were observed to be secreted from a lymphocyte to an apoptotic keratinocyte. These findings suggest that granzyme B-positive CD8 cells seem to induce apoptosis of keratinocytes in lichen planus.

Effect of betamethasone phosphate loaded polymeric nanoparticles on a murine asthma model

Although inhaled steroids are the treatment of first choice to control asthma, administration of systemic steroids is required for treatment of asthmatic exacerbation and intractable asthma. To improve efficacy and reduce side effects, we examine the effects of betamethasone disodium phosphate (BP) encapsulated in biocompatible, biodegradable blended nanoparticles (stealth nanosteroids) on a murine model of asthma. These stealth nanosteroids were found to accumulate at the site of airway inflammation and exhibit anti-inflammatory activity. Significant decreases in BALF eosinophil number were maintained for 7 days with a single injection of nanosteroids containing 40 microg BP. Airway responsiveness was also attenuated by the injection of stealth nanosteroids. A single injection of 40 microg of free BP and 8 microg of free BP once daily for 5 days did not show any significant effects. We conclude that stealth nanosteroids achieve prolonged and higher benefits at the site of airway inflammation compared to free steroids.

Efficient encapsulation of a water-soluble corticosteroid in biodegradable nanoparticles

Solid nanoparticles consisting of biodegradable polymers have emerged as a promising carrier for various drugs, but unfortunately the encapsulation of drugs remains challenging. In this study, a technique for encapsulation of water-soluble drugs in solid nanoparticles was developed. Nanoparticles were prepared from a blend of biodegradable polymers, including poly(lactic acid) (PLA) and poly(lactic/glycolic acid) (PLGA), and monomethoxypolyethyleneglycol-polylactide block copolymer by an oil-in-water solvent diffusion method. Betamethasone sodium phosphate (BP) was not encapsulated by the nanoparticles due to its hydrophilicity, but it was effectively encapsulated in the presence of appropriate amounts of zinc and diethanolamine. It was found that BP formed an ionic complex with zinc at a certain pH range obtained by addition of diethanolamine. Furthermore, a carboxyl group located at the end of PLA/PLGA was shown to be essential for encapsulation of BP in nanoparticles, and the molar ratio among BP, zinc, and carboxyl groups in various nanoparticles was almost constant. These results strongly suggested that the encapsulation was promoted by zinc creating an ionic bridge between a carboxyl group on PLA/PLGA and a phosphate group on BP. This technique for entrapment of water-soluble drugs in solid biodegradable nanoparticles may expand the use of nanoparticles for various therapeutic applications.

Rheumatoid papules: A report on four patients with histopathologic analysis

BACKGROUND The rheumatoid papule is a recently described skin manifestation of rheumatoid arthritis. OBJECTIVE Rheumatoid papules from four patients with classic rheumatoid arthritis were examined to determine the origin of this palisading granulomatous reaction. METHODS Immunofluorescence and electron microscopic studies were performed on biopsy specimens of rheumatoid papules. RESULTS Leukocytoclastic vasculitis with collagen alteration and lymphohistiocytic infiltration were observed. The immunofluorescence study revealed deposits of immunoglobulins and complement in the vessel walls and in the area of collagen alteration. Electron microscopy revealed epithelioid cell-like histiocytes among altered collagen fibers. These cells contained abundant lysosomes and were connected to neighboring cells by well-developed intricate processes. CONCLUSION Vasculitis is important in the pathogenesis of rheumatoid papules. In patients with rheumatoid papules, systemic evaluation should be performed because these are a manifestation of rheumatoid vasculitis.