Pathogenic Mechanism of Der p 38 as a Novel Allergen Homologous to RipA and RipB Proteins in Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic relapsing pruritic disease encompassing skin inflammation and barrier dysfunction. House dust mites are key allergens that augment the development of atopic dermatitis. We aimed to investigate the pathogenic mechanism of AD due to Der p 38, recently identified by us. The frequency of IgE reactivity to Der p 38 in AD subjects was 52.6% (10/19) in the skin prick test and 57.9% (11/19) in the dot blot assay. In human keratinocyte HaCaT cells, Der p 38 triggered the impairment of filaggrin expression and induced pro-inflammatory cytokines such as IL-6, IL-8 and MCP-1 through TLR4, PI3K, AKT, c-Jun N-terminal kinase (JNK) and NF-κB pathway. Supernatants from Der p 38-treated cells blocked filaggrin expression and neutrophil apoptosis. The anti-apoptotic effect of the Der p 38-released molecules on neutrophils was accomplished by inhibition of the caspase 9/3 pathway, and by increased MCL-1 expression and BCL-2/BAX expression ratio. In C57BL/6 wild type (WT) mice, Der p 38 induced a dose-dependent increase of AD-like skin lesions, with enhanced expressions of total and Der p 38-specific IgE. Der p 38 also diminished the expressions of skin barrier proteins and induced JNK activation. However, the AD-like features following cutaneous Der p 38 exposure were observed to be reduced in the TLR4 knockout (KO) group, as compared to the WT group. Skin infiltration of neutrophils, eosinophils and mast cells was increased in the WT mice, but was not portrayed in the TLR4 KO mice. These findings indicate that Der p 38 is a novel mite allergen that triggers AD by lowering skin barrier proteins and increasing inflammatory cells. Results of this study have thereby paved the way to unveil the pathogenic mechanisms of AD.

Dual Stimuli-Triggered Nanogels in Response to Temperature and pH Changes for Controlled Drug Release.

Poly-N-isopropyl acrylamide (PNIPAM) nanogels have been modified with different acrylic acid (AAc) contents for the efficient control of lower critical solution temperature (LCST). In this study, PNIPAM-co-AAc nanogels nanogels showed two volume phase transitions in comparison with PNIPAM. The transition temperature of PNIPAM nanogels was increased with AAc contents. The controlled drug release performance of PNIPAM-co-AAc nanogels loaded with ß-lapachone was attributed to the AAc content ratio and was efficiently triggered in response to temperature and pH. Moreover, a colorimetric cell proliferation assay and direct fluorescence-based live/dead staining were used to confirm the concurrence on drug release profiles. Finally, PNIPAM-co-AAc20 showed a relatively low level of drug release in the range of acidic to neutral pH at body temperature, while maximizing drug release at basic pH. Therefore, we demonstrated that the PNIPAM-based nanogel with the temperature- and pH-responsive features could be a promising nanocarrier for potential intestine-specific drug delivery.

Analytical procedure to simultaneously measure trace amounts of trenbolone acetate and beta-trenbolone residues in porcine muscle using HPLC-UVD and MS.

The current study was undertaken to validate the performance for the determination of both TBA and beta-trenbolone (beta-TB) residues in porcine muscle at concentrations required to monitor compliance with the maximum residue limit (MRL). The method involves a one phase liquid-liquid extraction, cleanup with low-temperature fat precipitation, separation of the respective compounds by HPLC on a Capcell pak C(18) column, use of a methanol-water isocratic system as an eluent, and measurement by UV absorbance detection at 340 nm. Both compounds were confirmed using LC-MS/MS with electrospray interface (ESI) and a triple quadrupole (QqQ) analyzer. The method was found to be precise and accurate, with a linearity range of 1-10 microg/kg (r(2) >0.973). The intra- and interday precision showed good reproducibility with RSDs < or =13.25%. The LODs were 0.12 and 0.22 microg/kg, and the LOQs were 0.37 and 0.66 microg/kg, for TBA and beta-TB, respectively. The applicability of the method was demonstrated by analyzing real samples collected from major cities in the Republic of Korea. No residues of the selected compounds were detected in any of the samples. The advantages of our method are that it is: selective, sensitive, requires a short time for analysis (13 min), and performs simple sample extraction and clean-up procedure with low-temperature fat precipitation as compared to the previously published methods.

Comparison of different extraction methods for the simultaneous determination of pesticide residues in kiwi fruit using gas chromatography-mass spectrometry.

The methods of simultaneous extraction of iprodione, chlorpyrifos-methyl, EPN and endosulfan (with its metabolites) from kiwi fruit using accelerated solvent extraction (ASE), supercritical fluid extraction (SFE), and liquid-liquid extraction (LLE) were tested and compared in terms of their of limits of detection and quantification, as well as the highest pesticide recoveries with the lowest residues in the final extracts. The analysis was performed using gas chromatography-mass spectrometry in the selected ion monitoring mode. The proposed methods featured good sensitivity, pesticide quantification limits were low enough, and the precision (expressed as relative standard deviations) ranged from 0.56 to 7.17%. The recoveries obtained from ASE, SFE and LLE were 77.5-120, 71.9-109.1 and 75.6-127.1%, respectively. The proposed methods were successfully applied for the monitoring of the selected pesticide residues in kiwi fruit samples collected from Jollanamdo area, Republic of Korea. Iprodione was detected at a level lower than the maximum residue limit (MRL) established by the Korea Food and Drug Administration (5 ppm), while EPN was detected at a level higher than the Korea Food and Drug Administration MRL (0.1 ppm) in the real samples. The proposed sample preparations led to a higher preconcentration of the pesticide fraction, and allowed the sensitive and selective determination of pesticides with varied physicochemical properties in kiwi fruit.

Multiresidue analysis of four pesticide residues in water dropwort (Oenanthe javanica) via pressurized liquid extraction, supercritical fluid extraction, and liquid-liquid extraction and gas chromatographic determination.

The primary objective of this study was to simultaneously analyze the residues of the most commonly used pesticides, chlorpyrifos-methyl, endosulfan, EPN, and iprodione in the water dropwort, via accelerated solvent extraction (ASE), supercritical fluid extraction (SFE), and conventional solvent extraction (LLE) techniques. Residue levels were determined using GC with electron-capture detection (GC-ECD). The confirmation of pesticide identity was performed by GC-MS in a selected ion-monitoring (SIM) mode. In none of the ASE and SFE techniques were the extraction conditions optimized. Rather, the experimental variables were predicated on the author's experience. The ECD response for all pesticides was linear in the studied range of concentrations of 0.005-5.0 ppm, with correlation coefficients in excess of 0.9991. At each of the two studied fortification levels, the pesticides yielded recoveries in excess of 72% with RSDs between 1 and 19%. The LODs were achieved at a range of levels from 0.001 to 0.063 ppm, depending on the pesticide utilized. The LOQs, which ranged from 0.003 to 0.188 ppm, were lower than the maximum residue limits (MRLs) authorized by the Korean Food and Drug Administration (KFDA). All of the methods were applied successfully to the determination of pesticide residues in the real samples. It could, therefore, be concluded that any of the techniques utilized in this investigation might prove successful, given that the applied extraction conditions are wisely chosen.

Activation of selective transcription factors and cytokines by water-soluble extract from Lentinus lepideus.

We isolated a water-soluble extract, PG101, from cultured mycelia of Lentinus lepideus. Treatment of human peripheral blood mononuclear cells (PBMCs) with PG101 increased levels of TNF-alpha, IL-1beta, IL-10, and IL-12 by 100- to 1000-fold, whereas GM-CSF and IL-18 were activated by an order of magnitude. On the contrary, IFN-gamma and IL-4 were not affected. The response to PG101 occurred in a dose- and time-dependent manner. From the human PBMCs treated with PG101, TNF-alpha was a first cytokine to be activated, detectable at 2 hr post-treatment followed by IL-1beta at 6 hr post-treatment. IL-12 and IL-10 were the next to follow. GM-CSF and IL-18 both showed significant increases 24 hr after treatment. When PBMCs were sorted into various cell types, monocyte/macrophages, but not T and B cells, were the major target cell type responsive to PG101. Consistent with this result, the profile of cytokine expression upon PG101 treatment was comparable between PBMCs and a human promonocytic cell line (U937), whereas cell lines of T cell and myeloid origins did not respond to PG101. Data from a transient transfection assay involving specific reporter plasmids indicated that cellular transcription factor such as NF-kappaB, but not AP-1, was highly activated by PG101. Results from a gel retardation assay and the experiment involving a specific NF-kappaB inhibitor confirmed the involvement of NF-kappaB. Despite its significant biological effect on various cytokines, PG101 remained nontoxic in both rats and PBMCs even at a biological concentration approximately 20 times greater. PG101 demonstrates great potential as a therapeutic immune modulator.

Enhancement of repopulation and hematopoiesis of bone marrow cells in irradiated mice by oral administration of PG101, a water-soluble extract from Lentinus lepideus.

PG101 is a water-soluble extract from Lentinus lepideus. It is a potential biological response modifier that activates selective cytokines in vitro, mainly by controlling cellular transcription factor NF-kappaB. Effects of PG101 were tested on bone marrow cells in irradiated mice. Mice were irradiated with a dose of 6 Gy and were given PG101 by gavages daily for 24 days. In PG101-treated mice, the number of colony-forming cells, including colony-forming units (CFU)-granulocytes/macrophages (GM) and erythroid burst-forming units (BFU-E), were increased to almost the levels seen in nonirradiated control as early as 8 days after irradiation. Two-color flow cytometric analysis using antibodies to ER-MP12 and ER-MP20 suggested that in the bone marrow cell population, PG101 increased the number of granulocytes (ER-MP12(-)20(med)) and myeloid progenitors (ER-MP12(+)20(+)). Analysis of surface c-Kit and Gr-1 proteins in bone marrow cells indicated that PG101 might induce differentiation of progenitor cells to granulocytes and/or proliferation of the committed cells. Lastly, oral administration of PG101 highly increased serum levels of GM-CSF, IL-6, and IL-1beta. Interestingly, the level of TNF-alpha was elevated by irradiation in control mice, but was maintained at the background level in PG101-treated mice, suggesting that PG101 might effectively suppress TNF-alpha-related pathologic conditions. Our results strongly suggest the great potential of PG101 as an immune enhancer during radiotherapy and/or chemotherapy.