Search

This study investigated youth’s modeling of and de-identification from parents in romantic relationships, using two phases of data from adolescent siblings, mothers, and fathers in 246 Mexican-origin families. Each parent reported his/her marital satisfaction and conflict, and youth reported on parent-adolescent warmth and conflict at Time 1. Youth’s reports of modeling of and de-identification from their mothers and fathers and three romantic relationship outcomes were assessed at Time 2. Findings revealed that higher parental marital satisfaction, lower marital conflict, and higher warmth and lower conflict in parent-adolescent relationships were associated with more modeling and less de-identification from parents. Moreover, higher de-identification was linked to a greater likelihood of youth being involved in a romantic relationship and cohabitation, whereas more modeling was linked to a lower likelihood of cohabitation and older age of first sex. Discussion underscores the importance of assessing parental modeling and de-identification and understanding correlates of these processes.

This paper concerned with the adaptive tracking control problem of an adaptive model-based fuzzy-neural networks (FNNs) sliding mode control (AFSMC) scheme for a class of nonlinear systems, which are represented by Takagi-Sugeno (T-S) fuzzy model to express a nonlinear systems model. Then, an adaptive parameter estimator is proposed to estimate the unknown nonlinear system parameters. Considering the online estimating error from the estimation model and nonlinear system model, a state estimation based feedback controller is derived by the proposed adaptive FNNs sliding mode control scheme and free parameters can be updated online by adaptive laws based on Lyapunov stability theorem. The proposed control scheme can guarantee that the unknown nonlinear system output can track to the states of reference model for any desired input signals when the stability condition is satisfied. Finally, simulation examples are provided to demonstrate the effectiveness of the proposed approaches.

Parental alcohol use disorders (AUDs) and parental separation are associated with increased risk for early use of alcohol in offspring, but whether they increase risks for early use of other substances and for early sexual debut is under-studied. We focused on associations of parental AUDs and parental separation with substance initiation and sexual debut to (1) test the strength of the associations of parental AUDs and parental separation with time to initiation (age in years) of alcohol, tobacco and cannabis use and sexual debut and (2) compare the strength of association of parental AUD and parental separation with initiation.Prospective adolescent and young adult cohort of a high-risk family study, the Collaborative Study on the Genetics of Alcoholism (COGA).Six sites in the United States.A total of 3257 offspring (aged 14-33 years) first assessed in 2004 and sought for interview approximately every 2 years thereafter; 1945 (59.7%) offspring had a parent with an AUD.Diagnostic interview data on offspring substance use and sexual debut were based on first report of these experiences. Parental life-time AUD was based on their own self-report when parents were interviewed (1991-2005) for most parents, or on offspring and other family member reports for parents who were not interviewed. Parental separation was based on offspring reports of not living with both biological parents most of the time between ages 12 and 17 years.Parental AUDs were associated with increased hazards for all outcomes, with cumulative hazards ranging from 1.19 to 2.71. Parental separation was also an independent and consistent predictor of early substance use and sexual debut, with hazards ranging from 1.19 to 2.34. The strength of association of parental separation with substance initiation was equal to that of having two AUD-affected parents, and its association with sexual debut was stronger than the association of parental AUD in one or both parents.Parental alcohol use disorders (AUDs) and parental separation are independent and consistent predictors of increased risk for early alcohol, tobacco and cannabis use and sexual debut in offspring from families with a high risk of parental AUDs.

Recently, a number of modern premium cars have been equipped with advanced driver assistance systems (ADAS), especially an adaptive cruise control (ACC) system, which was found to be one of the most widely and successful ADAS systems to date. However, the road topography on highways affects fuel consumption of vehicles. Rapid acceleration, rapid deceleration, and unreasonable gear use can all result in increasing fuel consumption. Therefore, an eco-cruising control (ECC) systems on roads with up-down slopes is a very critical significance for improving vehicle fuel economy based on the global positioning system (GPS) and geographic information system (GIS). This proposed approach combine the road elevation profile by digital road map and a nonlinear predictive control (NPC) strategy to deal with the vehicle velocity control; it is aimed at providing the most economical speed on road grade. The developed algorithms not only to provide the ecological driving guidance for driver, but also to achieve the driving safety. Extensive simulations illustrate that the proposed controller can significantly reducing fuel consumption of a vehicle and ensuring driving comfort and safety.

Background Few studies included information on components and sources when exploring the cardiovascular health effects from personal exposure to particulate matters (PM). We previously reported that exposure to PM between 1.0 and 2.5 μm (PM 2.5–1 ) was associated with increased cardio-ankle vascular index (CAVI, an arterial stiffness index), while exposure to PM smaller than 0.25 μm (PM 0.25 ) decreased the heart rate variability (HRV) indices. The purpose of this study was to investigate the association between PM elements and cardiovascular health effects and identify responsible sources. Methods In a panel study of seventeen mail carriers, the subjects were followed for 5–6 days while delivering mail outdoors. Personal filter samples of PM 2.5–1 and PM 0.25 were analyzed for their elemental concentrations. The source-specific exposures were further estimated by using absolute principal factor analysis. We analyzed the component- and source-specific health effects on HRV indices and CAVI using mixed models. Results Several elements in PM 2.5–1 (e.g., cadmium and strontium) were associated with the CAVI. Subsequent analyses showed that an interquartile range increase in exposure to PM from regional sources was significantly associated with a 3.28% increase in CAVI (95% confidence interval (CI), 1.47%–5.13%). This significant effect remained (3.35%, CI: 1.62%–5.11%) after controlling for the ozone exposures. For exposures to PM 0.25 , manganese, calcium, nickel, and chromium were associated with the CAVI and/or the HRV indices. Conclusions Our study suggests that PM 2.5–1 and PM 0.25 components may be associated with different cardiovascular effects. Health risks from exposure to PM from sources other than vehicle exhaust should not be underappreciated.

CD4+ memory/effector T cells play a central role in orchestrating the rapid and robust immune responses upon re-encounter with specific Ags. However, the immunologic mechanism(s) underlying these responses are still not fully understood. To investigate this, we generated an allergen (major house dust mite allergen, Blo t 5)-specific murine Th2 cell line that secreted IL-4, IL-5, IL-10, and IL-13, but not IL-9 or TNF-α, upon activation by the cognate Ag. These cells also exhibited CD44highCD62L− and CD127+ (IL-7Rα+) phenotypes, which are characteristics of memory/effector T cells. Experiments involving adoptive transfer of this Th2 cell line in mice, followed by three intranasal challenges with Blo t 5, induced a dexamethasone-sensitive eosinophilic airway inflammation. This was accompanied by elevation of Th2 cytokines and CC- and CXC-motif chemokines, as well as recruitment of lymphocytes and polymorphic mononuclear cells into the lungs. Moreover, Blo t 5-specific IgE was detected 4 d after the last intranasal challenge, whereas elevation of Blo t 5-specific IgG1 was found at week two. Finally, pulmonary delivery of the pVAX–IL-35 DNA construct effectively downregulated Blo t 5-specific allergic airway inflammation, and i.m. injection of pVAX–IL-35 led to long-lasting suppression of circulating Blo t 5-specific and total IgE. This model provides a robust research tool to elucidate the immunopathogenic role of memory/effector Th2 cells in allergic airway inflammation. Our results suggested that IL-35 could be a potential therapeutic target for allergic asthma through its attenuating effects on allergen-specific CD4+ memory/effector Th2 cell-mediated airway inflammation.

Background The prevalence of allergic diseases, such as asthma, allergic rhinitis, eczema and food allergy, has been increasing worldwide, as shown in a large number of studies, including the International Study of Asthma and Allergies in Childhood (ISAAC). However, there is significant variation in the prevalence of these diseases in different regions, suggesting that there may be location-specific factors such as environment and microbial exposure affecting allergic disease prevalence. Hence, in this study we determine if there is a difference in microbiota composition and allergen concentration of household dust collected from the homes of non-allergic and allergic subjects from the Growing Up in Singapore Towards Healthy Outcomes (GUSTO) cohort. Methods From the Growing Up in Singapore Towards Healthy Outcomes (GUSTO) cohort, 25 allergic subjects and 25 non-allergic subjects were selected at the year 5.5 follow up. Definitions of allergic outcomes were standardized in the questionnaires administered at 3, 6, 9, 12, 15, 18, 24, 36, 48 and 60 months to ensure consistency during interviews and home visits. Allergen sensitization was determined by skin prick testing (SPT) at 18, 36 and 60 months. Dust samples were collected from the subject’s bed, sofa, and play area. DNA extraction was carried out and V3-V4 hypervariable regions of bacterial 16S rRNA gene were sequenced. Protein extraction was performed and allergens assayed by using multiplex assay and ELISA. Results The most abundant phyla in house dust were Actinobacteria (29.8%), Firmicutes (27.7%), and Proteobacteria (22.4%). Although there were no differences in bacteria abundance and diversity between house dust samples of allergic and non-allergic subjects, the relative abundance of Anaplasmataceae, Bacteroidaceae, and Leptospiraceae were significantly higher in dust samples of allergic subjects as compared to non-allergic subjects in 2 or more locations. The concentration of Der p 1 was significantly lower in bed dust samples of allergic subjects (Median [Interquartile range], 174 ng/g [115–299 ng/g]) as compared to non-allergic subjects (309 ng/g [201–400 ng/g]; P

Fve is a fungal protein isolated from the golden needle mushroom Flammulina velutipes and has previously been reported to trigger immunological responses in both mouse and human lymphocytes. In this study, we evaluated the potential application of Fve as an adjuvant for tumour immunotherapy and examined the underlying mechanism(s). When the human papillomavirus (HPV)-16 E7 oncoprotein was used as a model antigen, mice coimmunized with HPV-16 E7 and Fve showed enhanced production of HPV-16 E7-specific antibodies as well as expansion of HPV-16 E7-specific interferon (IFN)-gamma-producing CD4(+) and CD8(+) T cells as compared with mice immunized with HPV-16 E7 alone. Tumour protection assays showed that 60% of mice coimmunized with HPV-16 E7 plus Fve, as compared with 20% of those immunized only with HPV-16 E7, remained tumour-free for up to 167 days after challenge with the tumour cells. Tumour therapeutic assays showed that HPV-16 E7 plus Fve treatment significantly prolonged the survival of tumour-bearing mice as compared with those treated only with HPV-16 E7. In vivo cell depletion and adoptive T-cell transfer assays showed that CD4(+) and CD8(+) T cells and IFN-gamma played critical roles in conferring the antitumour effects. Interestingly, Fve could stimulate the maturation of splenic dendritic cells in vivo and induce antigen-specific CD8(+) T-cell immune responses. In summary, Fve has potent adjuvant properties that enhance T helper type 1 antigen-specific humoral and cellular immune responses which confer strong antitumour effects. The use of Fve as an adjuvant could be an attractive alternative to the current vaccination strategy for cancer immunotherapy.

Lipopolysaccharide (LPS) is a major component of environmental microbial products. Studies have defined the LPS dose as a critical determining factor in driving differential T-cell polarization but the direct effects of LPS on individual antigen-presenting cells is unknown. Here, we investigated the effects of LPS doses on naive B cells and the subsequent modulatory effects of these LPS-activated B cells on T-cell polarization. The LPS was able to induce a proliferative response starting at a dose of 100 ng/ml and was capable of enhancing antigen internalization at a dose of 1 microg/ml in naive B cells. Following LPS stimulation, up-regulation of the surface markers CD40, CD86, I-Ad, immunoglobulin M, CD54 and interleukin-10 production, accompanied by down-regulation of CD5 and CD184 (CXCR4) were observed in a LPS dose-dependent manner. Low doses (10 ng/ml) of LPS-activated B cells drove T helper type 2 polarization whereas high doses (0.1 microg/ml) of LPS-activated B cells resulted in T regulatory type 1 cell polarization. In conclusion, LPS-activated B cells acquire differential modulatory effects on T-cell polarization. Such modulatory effects of B cells are dependent on the stimulation with LPS in a dose-dependent manner. These observations may provide one of the mechanistic explanations for the influence of environmental microbes on the development of allergic diseases.

Bifunctional apoptosis regulator (BAR) is an endoplasmic reticulum protein that interacts with both the extrinsic and intrinsic apoptosis pathways. We hypothesize that over-expression of BAR Delta RING prevents apoptosis and injury following ischaemia/reperfusion (I/R) and attenuates doxorubicin (DOX)-induced cardiotoxicity.We generated a line of transgenic mice that carried a human BAR Delta RING transgene under the control of the mouse alpha-myosin heavy chain promoter. The RING domain, which binds ubiquitin conjugating enzymes, was deleted to prevent auto-ubiquitination of BAR and allow accumulation of the BAR protein, which binds apoptosis-regulating proteins. High levels of human BAR Delta RING transcripts and 42 KDa BAR Delta RING protein were expressed in the hearts of transgenic mice. When excised hearts were reperfused ex vivo for 45 min as Langendorff preparations after 45 min of global ischaemia, the functional recovery of the hearts, expressed as left ventricular developed pressure x heart rate, was 23 +/- 1.7% in the non-transgenic hearts compared with 51.5 +/- 4.3% in the transgenic hearts (P0.05). For in vivo studies, mice were subjected to 50 min of ligation of the left descending anterior coronary artery followed by 4 h of reperfusion. The infarct sizes following I/R injury, expressed as the percentage of the area at risk, were significantly smaller in the transgenic mice than in the non-transgenic mice (29 +/- 4 vs. 55 +/- 4%, P0.05). In hearts of mice subjected to cardiac I/R injury, BAR transgenic hearts had significantly fewer in situ oligo-ligation-positive cardiac cells (5.0 +/- 0.4 vs. 13.4 +/- 0.5%, P0.05). Over-expression of BAR Delta RING also significantly attenuated DOX-induced cardiac dysfunction and apoptosis.Our results demonstrate that over-expression of BAR Delta RING renders the heart more resistant to I/R injury and DOX-induced cardiotoxicity, and this protection correlates with reduced cardiomyocyte apoptosis.

Previous studies have highlighted the importance of lung-draining lymph nodes in the respiratory allergic immune response, whereas the lung parenchymal immune system has been largely neglected. We describe a new in vivo model of respiratory sensitization to Blomia tropicalis, the principal asthma allergen in the tropics, in which the immune response is focused on the lung parenchyma by transfer of Th2 cells from a novel TCR transgenic mouse, specific for the major B. tropicalis allergen Blo t 5, that targets the lung rather than the draining lymph nodes. Transfer of highly polarized transgenic CD4 effector Th2 cells, termed BT-II, followed by repeated inhalation of Blo t 5 expands these cells in the lung >100-fold, and subsequent Blo t 5 challenge induced decreased body temperature, reduction in movement, and a fall in specific lung compliance unseen in conventional mouse asthma models following a physiological allergen challenge. These mice exhibit lung eosinophilia; smooth muscle cell, collagen, and goblet cell hyperplasia; hyper IgE syndrome; mucus plugging; and extensive inducible BALT. In addition, there is a fall in total lung volume and forced expiratory volume at 100 ms. These pathophysiological changes were substantially reduced and, in some cases, completely abolished by administration of neutralizing mAbs specific for IL-4 and IL-13 on weeks 1, 2, and 3. This IL-4/IL-13–dependent inducible BALT model will be useful for investigating the pathophysiological mechanisms that underlie asthma and the development of more effective drugs for treating severe asthma.

Understanding the determinants of brand value is a key theoretical and empirical issue in the fields of international marketing and international business. Researchers investigating the sources of brand value have most often focused on three primary levels of analysis: (1) brand image; (2) product category; and (3) country image. Despite an extensive literature on brand image, product category and country image effects, an examination based on a theoretical variance decomposition framework for investigating the relative influences of each effect on brand value is lacking. Therefore, acknowledging the increasing important role of brands, this study applies the variance components approach on the BusinessWeek/ Interbrand Best Global Brands database to explore the question of what matters most to superior brand value. In addition, this study also extends recent variance decomposition research by investigating three levels of effects using both the multilevel approach of hierarchical linear modeling and the conventional variance components approach. A variance components model is fitted to a new data set, and findings indicate that brand image effects dominate brand value while product category and country image effects have smaller but important impact on brand value. These findings support the suggestions of resource-based view of the firm.

Single-stranded telomeric DNA tends to form a four-base-paired planar structure termed G-quadruplex. This structure was easily formed in vitro in the presence of monovalent cations. However, the existence of this structure in native human telomeres is unclear. Here we address this important question through the distinctive properties of 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) upon binding to various DNA structures. Although the fluorescence of BMVC increases significantly in the presence of DNA, BMVC has high sensitivity and binding preference to quadruplex d(T(2)AG(3))(4) over duplex DNA. In addition, the fluorescent emissions were characterized around 575 nm for quadruplex d(T(2)AG(3))(4) and 545 nm for most of duplex DNA. The 575-nm fluorescence emissions were detected in the mixtures of 2 nM BMVC with the chromosomal DNA that were extracted from human cells, suggesting the presence of quadruplex structure in human nucleus. Further analyzing the BMVC fluorescence at the ends of metaphase chromosomes and other regions of chromosomes, we detected the quadruplex-binding BMVC fluorescence at telomere-proximal regions. Together these results provide the first evidence for the presence of quadruplex structures in human telomeres.

Pathogenesis of atopic dermatitis involved the interactions of immune and neuroendocrine systems. Here we describe a mouse model for atopic dermatitis with concomitant neurogenic inflammation, by epicutaneous sensitization with a dust mite allergen. Allergen patching resulted in localized dermatitis characterized by pronounced epidermal hyperplasia and spongiosis, which was associated with infiltration of eosinophils and neutrophils, degranulated mast cells, CD4+ and CD8+ T cells, and dendritic cells. There was increased innervation of calcium gene related peptides and substance P in inflamed skins, interactions between nerve fibers and mast cells were seen, indicating the coexistence of neurogenic inflammation. Splenic T cells produced T helper 2-polarized cytokines in response to allergen stimulation in vitro, indicating systemic allergen sensitization. This is the first report of a mouse model of eczema, accompanied by neurogenic inflammation, which shows close resemblance to human allergic diseases. This work supports the notion that the skin is an important site for the initiation of primary allergen sensitization. Besides, this model may also be useful for study of other stress-associated neuroinflammatory skin disorders such as neurogenic pruritus and psoriasis.

Summary Background House dust mite allergy is closely associated with allergic diseases. Blomia tropicalis mite species is an important clinical species in the tropics. The cDNA clone encoding Blo t 3, a group 3 allergen from B. tropicalis, has been isolated in our laboratory. Objective This study was designed to generate Blo t 3-specific monoclonal antibodies (mAbs) for the detection, characterization and purification of this allergen. Methods Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 3 gene with in vivo electroporation. Hybridomas were generated by the fusion of the splenocytes to X63-Ag8.653 myeloma cells. Purified native Blo t 3 was obtained by mAb immuno-affinity purification and the allergenicity of native Blo t 3 was determined by human IgE enzyme-linked immunosorbent assay (ELISA). Results A panel of class-switched and high-affinity mAb recognizing a wide spectrum of Blo t 3 epitopes have been generated. These mAbs are useful for western immunoblot assay, sandwich ELISA and affinity purification of native Blo t 3. Allergenicity of native Blo t 3 protein was examined with 44 mite-allergic sera and approximately 57% of the tested sera had positive serum IgE reactivity to the native Blo t 3. Conclusions These results demonstrated that intramuscular injection of naked DNA encoding Blo t 3 gene combined with in vivo electroporation is an effective and simple method to raise monoclonal antibodies that can be used for characterization and purification of Blo t 3.

This work demonstrates the feasibility of gallium nitride (GaN)-based light-emitting diodes (LEDs) with a metal-via-hole structure embedded in a reflector on the backside of sapphire substrate. Luminescence intensity for the surface-emitting LEDs is enhanced by mirroring efficaciously the downward light emitted from the InGaN/GaN multiquantum wells (MQWs) owing to the deep ladder-shaped inclined reflector on the backside of substrate. A metal-via-hole structure with a deep ladder shape was also processed with a wet-etching method at a high temperature to pattern the sapphire substrate deeply. The electroplating method was used to fill the area of patterned sapphire substrate with copper subsequently to produce a heat spreader path through the metal-via-hole and to strengthen the sapphire substrate with the void structure. Experimental results indicate that the GaN-based surface-emitting LEDs with a reflector for the planar, deep ladder-shaped sapphire, and metal-via-hole structures exhibit a luminescence intensity of 37%, 178%, and 226%, respectively, which are higher than the conventional ones under an injection current of 20 mA. A more stable peak wavelength shift is also observed for the metal-via-hole LED structure.

The gene of Blomia tropicalis group 5 allergen (Blo t 5) was cloned and expressed in Pichia pastoris KM71. Selected KM71 clones were cultivated in a fed-batch bioreactor feeding first glycerol then followed by methanol. Recombinant Blo t 5 constituted about 30% of the total broth protein after 60 h cultivation. The harvested broth was purified to >95% purity by a two-step anion exchange chromatography. The overall yield was 37 mg Blo t 5 per litre broth.

Crystals of the 14-kDa group 5 allergen from Dermatophagoides pteronyssinus (Der p 5) have been obtained at low pH and diffract to 3-A resolution using a conventional x-ray source. The crystals belong to tetragonal space group P41212 or P43212, with unit cell parameters a = b = 114 A and c = 234 A. A self-rotation search revealed a 432 point symmetry and thus suggested 96 molecules in one unit cell, hence 12 monomers in each asymmetric unit.

Background: Eczema is a known risk factor for the development of wheeze in childhood. Cord blood T-cell cytokine responses have been shown to be associated with the development of both early childhood eczema and wheeze. Our objective is to study and compare the influence of intrinsic T-cell cytokine responses on the development of wheezing and eczema in the first 2 years of life in a birth cohort of at risk (first degree family with atopic disease) infants. Methods: Cord blood samples were collected from 195 eligible subjects of a birth cohort of 253 subjects. The subjects studied were those who developed either wheezing (n = 34) or eczema (n = 29) in the first 2 years of life, and 65 healthy infants served as control. Cytokines from phytohaemagglutinin stimulated mononuclear cells were analyzed using multiplex cytokine assays and the cytokine profiles in the 3 groups were compared. Results: Most of the subjects were non-atopic with only 3/34 (9%) wheeze and 9/29 (31%) eczema subjects sensitized to the common dietary or inhalant allergens. After adjustment for potential risk factors, wheeze, but not eczema subjects, presented with hyper-responsive cytokine profiles with increased production of T-cell cytokines IL-2 and IL-5. IL-5 was the strongest risk factor associated to the development of wheeze at 2 years of age (OR, 35; 95% CI, 5.0 – 246.7). Conclusion: Cord blood cytokine responses in early onset wheeze and eczema are distinctly different. This suggests that the tendency to develop early onset wheeze may be influenced by preexisting immune factors independent to those for eczema.

The fungal immunomodulatory protein from the edible golden needle mushroom (Flammulina velutipes), designated Fve, is a single polypeptide consisting of 114 amino-acid residues. It is believed to trigger the mitogenic proliferation of T lymphocytes and Th1 cytokine production. Here, it is demonstrated that Fve forms a homodimer in nature. In order to understand the relationship between its structure and function, Fve was crystallized using the hanging-drop method; the protein formed well diffracting crystals within 3-5 d in 2.5% PEG 400, 2.0 M ammonium sulfate and 0.1 M Tris base buffer pH 8.5. The space group of the Fve crystals is either P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 96.92, c = 61.42 A. The crystal contains two molecules per asymmetric unit and diffracts to 1.4 A resolution when exposed to synchrotron radiation.

Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.

The effects on heart rate variability (HRV) and arterial stiffness from exposure to ambient particulate matter and ozone have not been studied simultaneously. The aim of this study was to analyze these effects with refined exposure estimates from personal measurements of ozone and size-resolved particulate matter mass concentrations. The authors recruited 17 mail carriers in a panel study in Taipei County, Taiwan, during February-March, 2007, and each subject was followed for 5-6 days. Personal ozone and size-fractionated particulate matter exposures were monitored during working hours while carriers delivered mail outdoors. Cardiovascular effects were evaluated with heart rate variability (HRV) indices and an arterial stiffness index, the cardio-ankle vascular index (CAVI). The authors used linear mixed models to examine the association between personal exposure data and the HRV index and CAVI. They found that an interquartile range increase in personal exposure to ozone and particulate matter of between 1.0 and 2.5 microm was associated with a 4.8% and 2.5% increase in CAVI, respectively, in the single-pollutant models. In contrast, the personal exposure data showed no significant effects on HRV. In 2-pollutant models, personal ozone exposure remained significantly associated with the CAVI measurements. The study results indicate that vascular function may be more sensitive to air pollutants than the autonomic balance.

Purpose of review Antiallergy DNA vaccine is an attractive alternative for the prevention and treatment of allergic diseases. This review covers recent studies to enhance potency and safety of antiallergy DNA vaccines. Recent findings Dendritic cell-targeted allergen gene vaccination using fascin gene promoter inhibited IgE production and allergic inflammation but not airway hyperresponsiveness. Targeting allergen expression at immature dendritic cells or induction of tolerogenic dendritic cells could induce antiallergic T regulatory cells. Vaccination with DNA-encoded Ag85B and AIMP1 as adjuvants could downregulate established Th2-mediated allergic responses. Forced ubiquitation or targeting allergens to lysosomal/endosomal compartments could avoid risk of allergen sensitization. Gene gun delivery of conventional antiallergy DNA vaccine is a risk factor. Replicase-based allergy DNA vaccines showed enhanced immunogenecity and safety as compared to conventional DNA vaccines. TANK-binding kinase-1 (TBK1) is a novel key molecule in DNA vaccine-induced immunogenicity. Summary Dendritic cell-based approach has been actively explored to enhance immunogenicity of antiallergy DNA vaccines. Codelivery of hypoallergenic DNA vaccines with potent adjuvants via a desirable delivery mode will help to fulfill the requirements for clinical application of antiallergy DNA vaccines. Activation of TBK1 signaling pathway could be a novel strategy to enhance immunogenicity of DNA vaccines.

Carbazole derivatives that stabilized G-quadruplex DNA structure formed by human telomeric sequence have been designed and synthesized. Among them, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) showed an increase in G-quadruplex melting temperature by 13°C and has a potent inhibitory effect on telomerase activity. Treatment of H1299 cancer cells with 0.5 μmol/L BMVC did not cause acute toxicity and affect DNA replication; however, the BMVC-treated cells ceased to divide after a lag period. Hallmarks of senescence, including morphologic changes, detection of senescence-associated β-galactosidase activity, and decreased bromodeoxyuridine incorporation, were detected in BMVC-treated cancer cells. The BMVC-induced senescence phenotype is accompanied by progressive telomere shortening and detection of the DNA damage foci, indicating that BMVC caused telomere uncapping after long-term treatments. Unlike other telomerase inhibitors, the BMVC-treated cancer cells showed a fast telomere shortening rate and a lag period of growth before entering senescence. Interestingly, BMVC also suppressed the tumor-related properties of cancer cells, including cell migration, colony-forming ability, and anchorage-independent growth, indicating that the cellular effects of BMVC were not limited to telomeres. Consistent with the observations from cellular experiments, the tumorigenic potential of cancer cells was also reduced in mouse xenografts after BMVC treatments. Thus, BMVC repressed tumor progression through both telomere-dependent and telomere-independent pathways. (Mol Cancer Res 2008;6(6):955–64)

Blomia tropicalis allergens are the most important mite allergens in tropical regions. Most of them only have 30-40% sequence identity with their Dermatophagoides counterparts and they share low IgE cross reactivity and exhibit different immunobiology. Unlike the pyroglyphid counterparts, Blo t 5 is the major allergen whereas Blo t 1 only has modest allergenicity.

Introduction From a birth cohort of at-risk Asian infants, we prospectively investigated the role of early onset allergen sensitization and clinical phenotypes as risk factors for atopic disorders at the age of 5 years.Methods and materials The study recruited 253 families with a history of allergic disease in a first degree relative from an antenatal clinic in Singapore. The children were followed prospectively to assess clinical outcomes and skin prick test was performed at 2 and 5 years of age.Results Allergen sensitization (food and/or house dust mites) alone at 2 years of age was not associated with increased risk of wheeze and eczema at 5 years. However, the clinical phenotype (eczema and wheeze) with or without the presence of concomitant allergen sensitization at 2 years increased this risk. For eczema, eczema alone at year 2 increased the risk of eczema at year 5 (adjOR = 7.1; 95 % CI: 1.8–27.8) and this was further increased by the presence of allergen sensitization (adjOR = 25.4; 95 % CI: 4.7–138.5) and the concomitant presence of both wheeze and allergen sensitization (adjOR = 64.9; 95 % CI: 4.7–900.0). For wheeze, wheeze alone at 2 years (adjOR = 4.5; 95 % CI: 1.4 -14.8), and wheeze with concomitant allergen sensitization and eczema (adjOR = 13.9; 95 % CI: 1.2–168.5) increased the risk of wheeze at 5 years. The exception was rhinitis, where allergen sensitization alone at 2 years (adjOR = 5.6; 95 % CI: 1.1–29.2) increased the risk of rhinitis at 5 years. Early onset of eczema at 2 years also increased the risk of rhinitis (adjOR = 6.8; 95 % CI: 2.0–23.1).Conclusion In this Asian birth cohort, the clinical phenotype (eczema and wheeze) with or without concomitant allergen sensitization in the first 2 years of life were strong predictors of atopic disorders at 5 years. Keywords: Eczema, Wheeze, Rhinitis, Allergen sensitization

We have investigated a novel compound, 3,6-bis[2-(1-methylpyridinium)vinyl]carbazole diiodide (BMVC), for inhibiting telomerase activity and distinguishing human lung H1299 and oral Ca9-22 cancer cells from lung IMR90 and skin Detroit-551 normal fibroblast cells. The telomeric repeat amplification protocol (TRAP) assay shows that the concentration of BMVC that inhibits 50% of the telomerase activity (IC50) is ca. 0.05 microM. On the other hand, the cell-viability assay indicates that the cytotoxicity was less than 15% to the H1299 and Ca9-22 cancer cells, and almost negligible to the MRC-5 and Detroit-551 normal cells after incubation with 0.5 microM BMVC for 72 h. The low concentration of 0.05 microM of BMVC can inhibit telomerase activity but does not have general toxic effects to normal cells, implying that BMVC is a promising telomerase inhibitor. Moreover, wide-field fluorescence images of 0.1 microM BMVC-treated cells show bright fluorescence spots in the nuclei of the most H1299 and Ca9-22 cancer cells. Interestingly, similar fluorescence spots are hardly observed in the nuclei of the IMR90 and Detroit-551 normal cells, implying that BMVC might be a useful marker to distinguish tumor cells and normal cells.

Summary Background Sensitization to mite and cockroach allergens is common, and diagnosis and therapy of allergy can be further complicated by the presence of allergen isoforms and panallergens. Purified recombinant and native allergens are useful for studies to resolve such problems. Objective To assess the allergenicity of native and recombinant mite glutathione S-transferase (GST) (Der p 8) and study the IgE cross-reactivity between Der p 8 and cockroach GST. Methods Der p 8 cDNA encoding a new isoform was isolated and expressed in yeast. Native Der p 8 was affinity purified from mite extract. IgE reactivity to native and recombinant Der p 8 was assessed by ELISA using sera from allergic subjects from Taiwan, Singapore and Malaysia. IgE cross-reactivity between Der p 8 and cockroach GST was examined by IgE inhibition assays. Results Our Der p 8 cDNA encoded a basic isoform (pI=8.5) containing six polymorphic residues located at positions 46, 106, 149, 160, 167 and 184. At least 8 isoforms of native Der p 8 were detected by two-dimensionalgel and immunoblot analyses. Sera from Taiwanese asthmatics showed 96% and 84% IgE reactivity to native Der p 8 and recombinant Der p 8, respectively. Native Der p 8 showed 75% and 65% IgE reactivity with sera from Malaysia and Singapore, respectively. Conclusions A high frequency of sensitization to mite GST among allergic subjects was observed but the titres of IgE reactivity were low. The IgE cross-reactivity between mite and cockroach GST suggests that GST is a panallergen.

Background Previous studies demonstrated that allergen gene vaccination induced TH1-skewed responses and inhibited IgE production. This study evaluated and characterized the immune responses induced by three DNA constructs encoding different forms of Der p 2 for safe and efficacious vaccination against mite allergy. Methods Mice were immunized intramuscularly with DNA constructs encoding a major mite allergen, Der p 2, without a signal peptide (p2), with a signal peptide (p52), and with a signal peptide plus lysosomal-targeting sequence (p52-LA), respectively, followed by TH2-skewed protein challenge. Antibody and T-cell cytokine responses were assessed by ELISA. Primed dendritic cells (DCs) were adoptively transferred to naive mice and humoral responses were examined after protein challenge. The circulating Der p 2 protein was detected by sandwich ELISA. Results Mice immunized with p52-LA showed strong and clear-cut TH1-type response, as evident by high IFN-γ production and elevated levels of Der p 2-specific IgG2a production whereas construct p2 induced only moderate levels of TH1 response. In contrast, mice immunized with construct p52 showed a mixed TH1/TH2 phenotype and produced substantial circulating Der p 2 protein. Mice adoptively transferred with DCs primed by p52 construct, but not by the p2 or p52-LA constructs, were sensitized to produce high levels of Der p 2-specific IgE. Conclusions Immunization with DNA construct encoding a signal peptide could potentially prime TH2-skewed responses and IgE production. The additional inclusion of lysosomal-targeting sequences to such construct could improve the safety and efficacy of DNA vaccination against allergy.

Control of colon cell fate in adenocarcinomas is disrupted, in part, due to aberrant Wnt/β-catenin signaling. The nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) has been implicated in the development of colon cancers. In the adenomatous polyposis coli multiple intestinal neoplasia (APC Min ) mouse cancer model, PPARγ expression in the colonic mucosa is markedly altered. In addition, PPARγ protein levels are elevated, possibly through sequestration by activated β-catenin in colon cancer cell lines. Induction of the Wnt/β-catenin pathway by LiCl also elevated PPARγ levels and induced PPARγ-dependent reporter and endogenous target genes. Mechanistically, PPARγ, through interactions with β-catenin and T cell transcription factor (Tcf)-4, may be a determinant of cell fate and is likely a target of the Wnt pathway in cancer cells.

Induced circular dichroism has been used to study the binding of thionin molecule to twelve guanine-rich quadruplexes. Substituting the base in the loops and varying the length of the tails could substantial change the induced circular dichroism spectra, which allow us to distinguish structural isomers and to determine the binding sites of quadruplexes. Our results show that external stacking on the end surface of the G-quartet associated with electrostatic interaction with the loops or tails is the major binding mode of thionin with these G-quadruplexes.

Protein tyrosine kinase signaling cascade plays a pivotal role in the activation of inflammatory cells. The purpose of this study was to investigate the effects of genistein, a broad-spectrum protein tyrosine kinase inhibitor, on airway inflammation in an in vivo guinea pig model of asthma. Guinea pigs were actively sensitized by intraperitoneal injections of ovalbumin. Aerosolized ovalbumin induced acute bronchoconstriction in conscious animals in a dose-dependent manner. Genistein (15 mg/kg given intraperitoneally) markedly inhibited ovalbumin-induced, but not histamine- and methacholine-induced, acute bronchoconstriction. In addition, genistein significantly reduced ovalbumin-induced increases in total cell counts and eosinophils recovered in bronchoalveolar lavage fluid, airway eosinophilia, and eosinophil peroxidase activity in cell-free bronchoalveolar lavage fluid and markedly attenuated ovalbumin-induced airway hyperresponsiveness to inhaled methacholine. Immunoblot analysis of lung lysates isolated from genistein-pretreated animals showed that epidermal growth factor-induced tyrosine phosphorylation in lung tissues was inhibited by genistein. These results implicate that inhibition of tyrosine kinase signaling cascade may have therapeutic potential for allergic airway inflammation.

It remains puzzling how immunodeficiency with hypo-responsive cells can be associated with increased immune cell activation to seemingly harmless substances such as allergens. Here, we have investigated skin pathology in the severe primary immunodeficiency Wiskott-Aldrich syndrome (WAS). Wildtype and WAS protein-deficient (WASp KO) mice were challenged with the allergen Der p 2 to induce eczema or infected with Leishmania major to induce robust skin inflammation. Der p 2 challenge of wildtype mice induced eczema with epidermal hyperplasia and egress of Langerhans cells from the skin. WASp KO mice had increased accumulation of mature DCs in the skin and, in contrast to wildtype cells, Der p 2 induced expansion of WASp KO CD8+ T cells that produced IFNγ. Similar to challenge with Der p 2 and unlike wildtype mice, WASp KO mice infected with L. major showed a skewed immune response with increased expansion of IFNγ-producing CD8+ T cells in the draining lymph node. Finally, we show that WASp KO DCs have increased capacity to cross-present exogenous antigen and activate CD8+ T cells. Our results imply that WASp deficiency in skin inflammation causes a breach in tolerance and skews the immune system to DC mediated CD8+ T cell responses at incorrect sites.

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.

Although a skin test is the primary option for detecting allergen-specific IgE in clinics, the serum IgE immunoassay is also important because it allows for the diagnosis of allergy without any accompanying adverse effect on the patient. However, the low detection limit of IgE levels by immunoassay may restrict the use of the method in some occasions, and improving its sensitivity would thus have a significant implication in allergy-immunology clinics. Methods: In this study, we attempted to detect specific serum IgE by using immuno-polymerase chain reaction (IPCR) which combines the antigen-antibody specificity of enzyme-linked immunosorbent assays (ELISAs) with the amplification power of PCR. Results: Our results demonstrated that Blo t5-specific serum IgE can be detected by IPCR with a 100-fold higher sensitivity than ELISA, and cross-reactivity of serum IgE to other mite allergens is able to be analyzed by using only 0.3μl of serum sample. Use of real-time IPCR seemed to permit more convenient determination of specific serum IgE as well. Conclusion: We believe that IPCR can serve as a valuable tool in determining specific serum IgE, especially when the amount of serum sample is limited.

House dust mites are main source of indoor allergens triggering the development of allergic diseases such as atopic dermatitis, perennial rhinitis and asthma. Blomia tropicalis (Family: Echimyopodidae) is a predominant and clinically important mite species in tropical and subtropical countries. B. tropicalis allergens are shown to be distinct and have relatively low to moderate IgE cross-reactivity with Dermatophagoides pteronyssinus mite allergens. Group 5 allergen, Blo t 5 is the major allergen from this mite and shows up to 90% IgE reactivity with asthmatic sera (Kuo et al. 2003). 13 C and/or 15 N isotopically enriched Blo t 5 samples were prepared as described previously (Arruda et al. 1997). Practically complete backbone (99.4%) and side-chain (95%) assignments were achieved by standard triple resonance experiments using Bruker Avance spectrometers equipped with cryoprobe. The chemical shifts are deposited in the BioMagResBank with accession number 7276. Chemical shifts index analysis of the data predicts Blo t 5 as an all-helical protein comprising of three long a-helices.

Summary Blo t 5 is the major allergen from Blomia tropicalis mites and shows strong IgE reactivity with up to 90% of asthmatic and rhinitis patients' sera. The NMR solution structure of Blo t 5 comprises three long α helices, forming a coiled-coil, triple-helical bundle with a left-handed twist. TROSY-NMR experiments were used to study Blo t 5 interaction with the Fab′ of a specific monoclonal antibody, mAb 4A7. The mAb epitope comprises two closely spaced surfaces, I and II, connected by a sharp turn and bearing critical residues Asn46 and Lys47 on one surface, and Lys54 and Arg57 on the other. This discontinuous epitope overlaps with the human IgE epitope(s) of Blo t 5. Epitope surface II is further predicted to undergo conformational exchange in the millisecond to microsecond range. The results presented are critical for the design of a hypoallergenic Blo t 5 for efficacious immunotherapy of allergic diseases.