Structural evidence for intermediates during O2 formation in photosystem II.

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn4CaO5 cluster first stores four oxidizing equivalents, the S0 to S4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O-O bond formation chemistry1-3. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok's photosynthetic water oxidation cycle, the S3→[S4]→S0 transition where O2 is formed and Kok's water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn4CaO5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom Ox, which was introduced as a bridging ligand between Ca and Mn1 during the S2→S3 transition4-6, disappears or relocates in parallel with Yz reduction starting at approximately 700 µs after the third flash. The onset of O2 evolution, as indicated by the shortening of the Mn1-Mn4 distance, occurs at around 1,200 µs, signifying the presence of a reduced intermediate, possibly a bound peroxide.

Perturbative diffraction methods resolve a conformational switch that facilitates a two-step enzymatic mechanism.

Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to poor experimental access. This shortcoming is evident with Escherichia coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we describe ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments to map the conformational dynamics of the Michaelis complex of DHFR. We resolve coupled global and local motions and find that these motions are engaged by the protonated substrate to promote efficient catalysis. This result suggests a fundamental design principle for multistep enzymes in which pre-existing dynamics enable intermediates to drive rapid electrostatic reorganization to facilitate subsequent chemical steps.

Genetic parameter changes and age-age correlations in Pinus koraiensis growth over 40-year progeny testing.

BACKGROUND: Early selection in tree breeding could be achieved by addressing the longevity of tree improvement activities. Genetic parameter changes and age-age correlations are essential for determining the optimal timing of early selection. Practical tracking of genetic parameters of Pinus koraiensis, a major timber species with economic and ecological value, has become feasible as its progeny testing has entered the mid-term age in Korea. However, research on the age-age correlation of P. koraiensis as progeny trials approach rotation age is limited. This study aimed to investigate genetic parameter trends and age-age correlations in P. koraiensis progeny. P. koraiensis progeny were assessed at two sites using a linear mixed-effects model with two-dimensional spatial autoregressive structure. Height, diameter, and volume growth were measured in 11 assessments over 40 years. RESULTS: Genetic parameters, such as height and diameter, showed different patterns of change. The heritability ranged for the three growth traits in 0.083-0.710, 0.288-0.781, and 0.299-0.755 across the sites and age. Height heritability and its coefficient of variance decreased, whereas the diameter and volume estimates remained relatively constant. Correlations with Age 40 for phenotypic, genetic, and rank of breeding values ranged between 0.16 and 0.92, 0.594 and 0.988, and 0.412 and 0.965, respectively. These correlations generally increased as the age approached Age 40, with particularly high levels observed at Age 26 and Age 30. CONCLUSION: The observed genetic trends in P. koraiensis progeny testing offer valuable insights for early and precise selection. Notably, selecting superior genotypes at Ages 26-30 is supported by discernible genetic gains and robust correlations. Future research should integrate unbalanced data for selecting mother trees or families and conduct a comprehensive economic analysis of early selection to validate its practical benefits.

Anti-inflammatory effects of neuregulin-1 in HaCaT keratinocytes and atopic dermatitis-like mice stimulated with Der p 38.

Neuregulin (NRG)-1 plays fundamental roles in several organ systems after binding to its receptors, ErbB2 and ErbB4. This study examines the role of NRG-1 in atopic dermatitis (AD), a chronic skin disease that causes dryness, pruritus, and inflammation. In mice administered Der p 38, the skin presents AD-like symptoms including filaggrin downregulation and infiltration of neutrophils and eosinophils. Noticeably, there is an increased expression of NRG-1, ErbB2, and ErbB4 in the skin. Upregulation of these proteins is significantly correlated to the clinical skin severity score. In human keratinocyte HaCaT cells, exposure to Der p 38 decreased filaggrin expression, and NRG-1 alone had no effect on the expression. However, co-treatment of Der p 38 with NRG-1 enhanced the filaggrin expression decreased by Der p 38. Pre-treatment with AG879 (an ErbB2 inhibitor) or ErbB4 siRNA blocked the recovery of filaggrin expression in the cells after co-treatment with Der p 38 and NRG-1. Der p 38 treatment enhanced the secretion of interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein-1 (MCP-1). Co-treatment of Der p 38 with NRG-1 lowered the cytokine secretion increased by Der p 38, although NRG-1 alone was not effective on cytokine alteration. Neutrophil apoptosis was not altered by NRG-1 or supernatants of cells treated with NRG-1, but the cell supernatants co-treated with Der p 38 and NRG-1 blocked the anti-apoptotic effects of Der p 38-treated supernatants on neutrophils, which was involved in the activation of caspase 9 and caspase 3. Taken together, we determined that NRG-1 has anti-inflammatory effects in AD triggered by Der p 38. These results will pave the way to understanding the functions of NRG-1 and in the future development of AD treatment.

In Situ Structural Observation of a Substrate- and Peroxide-Bound High-Spin Ferric-Hydroperoxo Intermediate in the P450 Enzyme CYP121.

The P450 enzyme CYP121 from Mycobacterium tuberculosis catalyzes a carbon-carbon (C-C) bond coupling cyclization of the dityrosine substrate containing a diketopiperazine ring, cyclo(l-tyrosine-l-tyrosine) (cYY). An unusual high-spin (S = 5/2) ferric intermediate maximizes its population in less than 5 ms in the rapid freeze-quenching study of CYP121 during the shunt reaction with peracetic acid or hydrogen peroxide in acetic acid solution. We show that this intermediate can also be observed in the crystalline state by EPR spectroscopy. By developing an on-demand-rapid-mixing method for time-resolved serial femtosecond crystallography with X-ray free-electron laser (tr-SFX-XFEL) technology covering the millisecond time domain and without freezing, we structurally monitored the reaction in situ at room temperature. After a 200 ms peracetic acid reaction with the cocrystallized enzyme-substrate microcrystal slurry, a ferric-hydroperoxo intermediate is observed, and its structure is determined at 1.85 Å resolution. The structure shows a hydroperoxyl ligand between the heme and the native substrate, cYY. The oxygen atoms of the hydroperoxo are 2.5 and 3.2 Å from the iron ion. The end-on binding ligand adopts a near-side-on geometry and is weakly associated with the iron ion, causing the unusual high-spin state. This compound 0 intermediate, spectroscopically and structurally observed during the catalytic shunt pathway, reveals a unique binding mode that deviates from the end-on compound 0 intermediates in other heme enzymes. The hydroperoxyl ligand is only 2.9 Å from the bound cYY, suggesting an active oxidant role of the intermediate for direct substrate oxidation in the nonhydroxylation C-C bond coupling chemistry.

Neuregulin-1 reverses anxiety-like behavior and social behavior deficits induced by unilateral micro-injection of CoCl2 into the ventral hippocampus (vHPC).

Neuregulin-1 (NRG1) is an epidermal growth factor family member with essential roles in the developing and adult nervous systems. In recent years, establishing evidence has collectively suggested that NRG1 is a new modulator of central nervous system (CNS) injury and disease, with multifaceted roles in neuroprotection, remyelination, neuroinflammation, and other repair mechanisms. NRG1 signaling exerts its effects via the tyrosine kinase receptors ErbB2-ErbB4. The NRG1/ErbB network in CNS pathology and repair has evolved, primarily in recent years. In the present study, we demonstrated that a unilateral microinjection of CoCl2 into the ventral hippocampus (vHPC) induced hypoxic insult and led to anxiety-related behaviors and deficit sociability in mice. NRG1 treatment significantly alleviated the CoCl2-induced increase of hypoxic-related molecules and behavioral abnormalities. Furthermore, NRG1 reduced the CoCl2-induced neuroinflammation and neuronal deficits in the vHPC or primary hippocampal neurons in mice. Collectively, these results suggest that NRG1 ameliorates hypoxia by alleviating synaptic deficits and behavioral abnormalities of the CoCl2-induced vHPC hypoxic model.

Der p 38 Is a Bidirectional Regulator of Eosinophils and Neutrophils in Allergy.

The house dust mite is the most common cause of allergic diseases, and TLR4 acts as an overarching receptor for allergic responses. This study aimed to identify novel allergen binding to TLR4 in house dust mites and unveil its unique role in allergic responses. Der p 38 was purified and characterized by liquid chromatography tandem mass spectrometry-based peptide mapping. Biolayer interferometry and structure modeling unveiled TLR4-binding activity and the structure of recombinant Der p 38. The allergenicity of Der p 38 was confirmed by a skin prick test, and basophil activation and dot blot assays. The skin prick test identified 24 out of 45 allergic subjects (53.3%) as Der p 38+ subjects. Der p 38-augmented CD203c expression was noted in the basophils of Der p 38+ allergic subjects. In animal experiments with wild-type and TLR4 knockout BALB/c mice, Der p 38 administration induced the infiltration of neutrophils as well as eosinophils and exhibited clinical features similar to asthma via TLR4 activation. Persistent Der p 38 administration induced severe neutrophil inflammation. Der p 38 directly suppressed the apoptosis of allergic neutrophils and eosinophils, and enhanced cytokine production in human bronchial epithelial cells, inhibiting neutrophil apoptosis. The mechanisms involved TLR4, LYN, PI3K, AKT, ERK, and NF-κB. These findings may contribute to a deep understanding of Der p 38 as a bridge allergen between eosinophilic and neutrophilic inflammation in the pathogenic mechanisms of allergy.

Untangling the sequence of events during the S2 → S3 transition in photosystem II and implications for the water oxidation mechanism.

In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

Photoreversible interconversion of a phytochrome photosensory module in the crystalline state.

A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs - Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.

Pedigree reconstruction and spatial analysis for genetic testing and selection in a Larix kaempferi (Lamb.) Carrière plantation.

BACKGROUND: Larix kaempferi is one of the major timber species in Northeast Asia. Demand for the reforestation of the species is rising in South Korea due to an increase in large timber production and utilization. However, progeny trials for the species have not been explored, making it challenging to foster advanced generations of tree improvement. In the present study, genetic testing and selection for diameter growth were conducted using pedigree reconstruction and phenotypic spatial distribution analysis in a plantation of L. kaempferi. The aim of the present study was to select the superior larch individuals using the pedigree reconstruction and phenotypic spatial distribution to substitute progeny trials. The plantation of seed orchard crops was established in 1990 and one-hundred and eighty-eight trees were selected as the study material. Genetic variation was investigated first to validate its adequacy as breeding material. Genetic testing was carried out using a model considering pedigree information and spatial autoregression of the phenotypes. RESULTS: The expected heterozygosity of the mother trees and offspring were 0.672 and 0.681 presenting the corresponding level of genetic variation between two groups. The pedigree reconstruction using maternity analysis assigned one to six progenies to ninety-two candidate mothers. The accuracy of genetic testing was exceedingly increased with the animal model considering AR1 ⊗ AR1 structure compared to the animal model only. The estimated genetic variance of the former was 9.086 whereas that of the latter was 4.9E-5 for DBH. The predicted breeding values of the offspring for DBH were ranged from -5.937 cm to 5.655 cm and the estimated heritability of diameter growth was 0.344. CONCLUSIONS: The genetic testing approach based on pedigree reconstruction and phenotypic spatial distribution analysis was considered a useful analytical scheme that could replace or supplement progeny trials.

Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis.

S100 calcium-binding protein A8 (S100A8), a danger-associated molecular pattern, has emerged as an important mediator of the pro-inflammatory response. Some S100 proteins play a prominent role in neuroinflammatory disorders and increase the secretion of pro-inflammatory cytokines in microglial cells. The aim of this study was to determine whether S100A8 induced neuronal apoptosis during cerebral hypoxia and elucidate its mechanism of action. In this study, we reported that the S100A8 protein expression was increased in mouse neuronal and microglial cells when exposed to hypoxia, and induced neuroinflammation and neuronal apoptosis. S100A8, secreted from neurons under hypoxia, activated the secretion of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) through phosphorylation of extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in microglia. Also, phosphorylation of ERK via the TLR4 receptor induced the priming of the NLRP3 inflammasome. The changes in Cyclooxygenase-2 (COX-2) expression, a well-known inflammatory activator, were regulated by the S100A8 expression in microglial cells. Knockdown of S100A8 levels by using shRNA revealed that microglial S100A8 expression activated COX-2 expression, leading to neuronal apoptosis under hypoxia. These results suggested that S100A8 may be an important molecule for bidirectional microglia-neuron communication and a new therapeutic target for neurological disorders caused by microglial inflammation during hypoxia.

Der f 38 Is a Novel TLR4-Binding Allergen Related to Allergy Pathogenesis from Dermatophagoides farinae.

It is difficult to treat allergic diseases including asthma completely because its pathogenesis remains unclear. House dust mite (HDM) is a critical allergen and Toll-like receptor (TLR) 4 is a member of the toll-like receptor family, which plays an important role in allergic diseases. The purpose of this study was to characterize a novel allergen, Der f 38 binding to TLR4, and unveil its role as an inducer of allergy. Der f 38 expression was detected in the body and feces of Dermatophagoides farinae (DF). Electron microscopy revealed that it was located in the granule layer, the epithelium layer, and microvilli of the posterior midgut. The skin prick test showed that 60% of allergic subjects were Der f 38-positive. Der f 38 enhanced surface 203c expression in basophils of Der f 38-positive allergic subjects. By analysis of the model structure of Der p 38, the expected epitope sites are exposed on the exterior side. In animal experiments, Der f 38 triggered an infiltration of inflammatory cells. Intranasal (IN) administration of Der f 38 increased neutrophils in the lung. Intraperitoneal (IP) and IN injections of Der f 38 induced both eosinophils and neutrophils. Increased total IgE level and histopathological features were found in BALB/c mice treated with Der f 38 by IP and IN injections. TLR4 knockout (KO) BALB/c mice exhibited less inflammation and IgE level in the sera compared to wild type (WT) mice. Der f 38 directly binds to TLR4 using biolayer interferometry. Der f 38 suppressed the apoptosis of neutrophils and eosinophils by downregulating proteins in the proapoptotic pathway including caspase 9, caspase 3, and BAX and upregulating proteins in the anti-apoptotic pathway including BCL-2 and MCL-1. These findings might shed light on the pathogenic mechanisms of allergy to HDM.

High-Resolution XFEL Structure of the Soluble Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O2 activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here, microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≤35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage-free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.

Artificial Iron Proteins: Modeling the Active Sites in Non-Heme Dioxygenases.

An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. We have utilized biotin-streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the S112E mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic FeII complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from S112E. Two aqua ligands complete the primary coordination sphere of the FeII center with both involved in hydrogen bond networks within Sav. The corresponding FeIII protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The FeIII protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD.

Suppressive effects of S100A8 and S100A9 on neutrophil apoptosis by cytokine release of human bronchial epithelial cells in asthma.

S100A8 and S100A9 are important proteins in the pathogenesis of allergy. Asthma is an allergic lung disease, characterized by bronchial inflammation due to leukocytes, bronchoconstriction, and allergen-specific IgE. In this study, we examined the role of S100A8 and S100A9 in the interaction of cytokine release from bronchial epithelial cells, with constitutive apoptosis of neutrophils. S100A8 and S100A9 induce increased secretion of neutrophil survival cytokines such as MCP-1, IL-6 and IL-8. This secretion is suppressed by TLR4 inhibitor), LY294002, AKT inhibitor, PD98059, SB202190, SP600125, and BAY-11-7085. S100A8 and S100A9 also induce the phosphorylation of AKT, ERK, p38 MAPK and JNK, and activation of NF-κB, which were blocked after exposure to TLR4i, LY294002, AKTi, PD98059, SB202190 or SP600125. Furthermore, supernatants collected from bronchial epithelial cells after S100A8 and S100A9 stimulation suppressed the apoptosis of normal and asthmatic neutrophils. These inhibitory mechanisms are involved in suppression of caspase 9 and caspase 3 activation, and BAX expression. The degradation of MCL-1 and BCL-2 was also blocked by S100A8 and S100A9 stimulation. Essentially, neutrophil apoptosis was blocked by co-culture of normal and asthmatic neutrophils with BEAS-2B cells in the presence of S100A8 and S100A9. These findings will enable elucidation of asthma pathogenesis.

Efficacy of prophylactic central neck dissection in hemithyroidectomy for papillary thyroid carcinoma.

PURPOSE: To evaluate the efficacy of prophylactic central neck dissection (pCND) in hemithyroidectomy for clinically node-negative papillary thyroid carcinoma (PTC). METHODS: We retrospectively analyzed 299 patients who underwent thyroid lobectomy with or without pCND for unilateral PTC. Of the 299 patients, 245 (81.9%) underwent unilateral pCND along with lobectomy, and 54 (18.1%) patients underwent lobectomy without pCND. Propensity score matching was performed for five covariates to reduce selection bias. RESULTS: In the baseline cohort of 299 patients, mean age, extrathyroidal extension, T classification and stage were higher in the cases undergoing pCND than in those not undergoing pCND. After propensity score matching, the significant differences between the two groups seen in the baseline cohort disappeared. Recurrence rates and recurrence-free survival curves did not differ between the 2 matched groups each of 54 patients. CONCLUSION: The value of pCND in hemithyroidectomy for PTC is limited.

Chaenomeles sinensis Koehne extract suppresses the development of atopic dermatitis-like lesions by regulating cytokine and filaggrin expression in NC/Nga mice.

Chaenomeles sinensis Koehne (CS) has been used in a traditional oriental medicine for treating throat diseases, anaphylaxis, viral infection, and inflammation. This study investigated the underlying mechanism of anti-allergic effect of CS. Leaves of CS plants were dried, powdered, and then underwent extraction with DMSO. Both ELISA and western blotting were performed to evaluate cytokine concentration and the expression and activation of filaggrin and JNK. Five-week-old female NC/Nga mice were used as an AD-like mouse model by treating them with 2,4-dinitrochlorobenzene (DNCB). The secretion of TARC, MCP-1, and IL-8 is increased by TNF-α and IFN-γ in HaCaT cells, and CS extract inhibited the increased production of TARC, MCP-1, and IL-8. TNF-α and IFN-γ suppressed filaggrin expression by activating JNK. CS extract recovered the expression of filaggrin decreased by TNF-α and IFN-γ by blocking the activation of JNK. In vivo experiment, CS administration reduced thickening of the epidermis and infiltration of inflammatory cells into the dermis as compared to DNCB treatment. Moreover, the decrease of filaggrin expression due to DNCB treatment was recovered by CS administration. The serum IgE level was decreased by CS treatment. The levels of IL-4, IL-5, IL-13 and eotaxin in mouse splenocytes increased after treatment with concanavalin A, and the secretions of IL-4, IL-5, IL-13 and eotaxin were lower in the CS-treated group than in the DNCB group. These results may contribute to the development of a CS-based drug for the treatment of atopic dermatitis.

Poncirus Trifoliata (L.) Raf. Extract Inhibits the Development of Atopic Dermatitis-like Lesions in Human Keratinocytes and NC/Nga mice.

This study investigated the anti-allergic effect of Poncirus trifoliata (L.) Raf. (PT) on human keratinocytic HaCaT cells in vitro and on 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis-like lesions in vivo. The release of TARC, MCP-1, IL-6 and IL-8 is increased by IFN-γ and TNF-α in HaCaT cells, and PT extract suppressed the increased production of TARC, MCP-1, IL-6, and IL-8. PT extract recovered the expression of filaggrin decreased by IFN-γ and TNF-α. in vivo experiment, PT administration decreased the skin severity score, thickening of the epidermis, movement of inflammatory cells into the dermis, and serum IgE level as compared to DNCB treatment. Moreover, the decrease of filaggrin and loricrin induced by DNCB treatment was recovered by PT administration. The levels of IL-4, IL-5, IL-13 and eotaxin in mouse splenocytes increased after treatment with concanavalin A, and the secretions of IL-4, IL-5, IL-13 and eotaxin were lower in the PT-treated group than in the DNCB group. These findings may indicate that PT is useful in drug development for the treatment of AD.

Thymus and activation-regulated chemokine (TARC)/CCL17 and IgE are associated with elderly asthmatics.

BACKGROUND: The pathogenesis of asthma, which is an allergic lung disease, is associated with a variety of allergens such as house dust mite, pollen, and mould, IgE containing serum IgE and allergen-specific-IgE, and inflammatory cytokines including thymus and activation-regulated chemokine (TARC)/CCL17. Because aging is an essential factor in the pathogenesis of asthma, we examined biomarkers related to asthmatic subjects depending on age. RESULTS: Physiological indices such as FEV1(forced expiratory capacity in 1 s), FEV1 (% predicted), and FEV1/FVC(forced vital capacity) (%) in asthmatic subjects were lower than those in normal subjects. Total IgE, Der p1 specific IgE, and Der f1 specific IgE were elevated in serum of asthmatics relative to normal individuals. Regulated on activation, normal T cell expressed and secreted (RANTES)/CCL5 in serum and interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein (MCP)-1/CCL2, RANTES, and macrophage inflammatory protein (MIP)-1α/CCL3 in bronchoalveolar lavage fluid (BALF) of asthmatic subjects were higher than in normal individuals. Upon classification of experimental groups depending on age, physiological indices and Der p1-specific IgE (class) were decreased in middle aged adult and elderly adult groups relative to the young adult group. TARC levels in serum were strongly elevated in the elderly adult group relative to the young adult and the middle aged adult groups. TARC in serum was related to total IgE in serum in the elderly adult group. CONCLUSIONS: Taken together, although TARC in serum and BALF is not different between normal and asthmatic individuals, TARC increases in serum of elderly asthmatic subjects. The level of TARC has a positive effect on the level of IgE in the elderly adult group. These findings may help us better understand the relationship of pathogenesis of allergic diseases and aging.