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Objective The study evaluated the potential of apple aroma to alleviate physiological stress response induced by a short-term cognitive stressor. It investigated the effects of apple aroma on cardiac and peripheral autonomic nervous system activity under stressful conditions.Methods Within-subject experiments were conducted with 25 healthy male university students with a 30-minute calculation task under three conditions: Apple, Chamomile (CHA), and Di propylene glycol (DPG) (scentless air). The aroma administration was precisely controlled and counterbalanced. Cardiac activity on electrocardiograms and nose tip temperature were recorded throughout the experiment. A visual analog scale and a scent questionnaire were subjective measures.Results Apple aroma demonstrated a significantly smaller increase in heart rate (t [24] = 3.36, p = 0.008 vs. DPG, t [24] = 4.06, p = 0.001 vs. CHA), and a decrease in the high-frequency component of heart rate variability (t [24] = 2.81, p = 0.029 vs. DPG, t [24] = 3.48, p = 0.006 vs. CHA) compared to the other conditions, representing a smaller enhancement of cardiac sympathetic nervous system activity and smaller suppression of cardiac parasympathetic nervous system activity respectively.Conclusion Apple aroma showed efficacy in alleviating the physiological stress response in terms of cardiac activity. Apple was also considered significantly preferable and comfortable compared to the other conditions, which would be beneficial in the context of aromatherapy.

Introduction: Mature adipocyte-derived dedifferentiated fat cells (DFATs) are mesenchymal stem cell (MSC)-like cells with high proliferative ability and multilineage differentiation potential. In this study, we first examined whether DFATs can be prepared from infrapatellar fat pad (IFP) and then compared phenotypic and functional properties of IFP-derived DFATs (IFP-DFATs) with those of subcutaneous adipose tissue (SC)-derived DFATs (SC-DFATs). Methods: Mature adipocytes isolated from IFP and SC in osteoarthritis patients (n = 7) were cultured by ceiling culture method to generate DFATs. Obtained IFP-DFATs and SC-DFATs were subjected to flow cytometric and microarray analysis to compare their immunophenotypes and gene expression profiles. Cell proliferation assay and adipogenic, osteogenic, and chondrogenic differentiation assays were performed to evaluate their functional properties. Results: DFATs could be prepared from IFP and SC with similar efficiency. IFP-DFATs and SC-DFATs exhibited similar immunophenotypes (CD73+, CD90+, CD105+, CD31-, CD45-, HLA-DR-) and tri-lineage (adipogenic, osteogenic, and chondrogenic) differentiation potential, consistent with the minimal criteria for defining MSCs. Microarray analysis revealed that the gene expression profiles in IFP-DFATs were very similar to those in SC-DFATs, although there were certain number of genes that showed different levels of expression. The proliferative activity in IFP-DFATs was significantly (p

Abstract Background Prepancreatic portal vein (PPV) is a congenital anatomical variant of the portal vein (PV). PPVs are extremely rare and generally classified into two categories, prepancreatic preduodenal portal vein and prepancreatic postduodenal portal vein (PPPV). Prepancreatic preduodenal portal veins are rare, with approximately 100 reported cases globally; PPPVs are even more atypical, with less than 20 documented cases globally. Despite the extremely low occurrence, PPPV knowledge and recognition are important, especially for hepatobiliary-pancreatic (HBP) surgeries, such as pancreaticoduodenectomy (PD) for patients of a PPPV. Here, we report a case of PPPV and a literature review. Case presentation A 73-year-old-male with ampullary carcinoma underwent PD at our hospital. Preoperative enhanced CT revealed an abnormal L-shaped PV, identified as a PPPV. Both the PPPV and the postpancreatic “normal” superior mesenteric vein (SMV) divaricated from the SMV at the caudal side of the pancreas. A splenic vein and inferior mesenchymal vein flowed into the postpancreatic “normal” PV, which encircled the common bile duct and potentially flowed into the liver, forming a cavernous transformation at the hilar plate. During surgery, we attempted to isolate the PV from the pancreas and common bile duct. However, it was difficult to isolate from the pancreas. The PPPV was so fragile that bleeding from the PPPV became uncontrollable. To remove the tumor, we resected the PPPV and reconstructed a “normal” PV as an autogenous graft. To maintain intraoperative hepatic blood flow and avoid small bowel congestion, an antithrombogenic bypass catheter was placed between the SMV and umbilical vein during reconstruction. After surgery, several complications occurred, such as PV thrombosis and hyperammonemia. The patient was discharged on postoperative day 45. Conclusions PPPV is a rare vascular variant but is easily diagnosed preoperatively due to its distinct shape on CT imaging. However, isolating the PPPV from the pancreas and bile duct is incredibly difficult and potentially associated with increased operative risks and postoperative complications. PV resection rather than isolation is a potential solution to reduce the risk of hemorrhage, even in the absence of invasion.

Abstract Background Anorectal malignant melanoma (ARMM) has an extremely poor prognosis, and there is no report of resection of liver metastases so far. We report herein a rare case of postoperative laparoscopic partial hepatic S7 resection for rectal malignant melanoma. Case presentation A 51-year-old female patient with a diagnosis of an ARMM underwent a laparoscopic rectal amputation. Eleven months later, computed tomography (CT) revealed a 14-mm nodule in liver segment 7 (S7), which was diagnosed as a hepatic recurrence of the ARMM. Because no other recurrences were found, a laparoscopic partial resection of S7 was performed. Pathological analysis found intracellular melanin deposition, and immunostaining was S-100 (+), HMB-45 (+), and SOX-10 (+). Based on these findings, a liver metastasis of malignant melanoma was diagnosed. The patient is alive 7 months after the second surgery and has so far experienced no recurrences. Conclusion We reported an extremely rare case of a laparoscopic resection of a liver metastasis following surgery for ARMM.

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is a practical kind of bacterial polyhydroxyalkanoates (PHAs). A previous study has established an artificial pathway for the biosynthesis of P(3HB-co-3HHx) from structurally unrelated sugars in Ralstonia eutropha, in which crotonyl-CoA carboxylase/reductase (Ccr) and ethylmalonyl-CoA decarboxylase (Emd) are a key combination for generation of butyryl-CoA and the following chain elongation. This study focused on the installation of the artificial pathway into Escherichia coli. The recombinant strain of E. coli JM109 harboring 11 heterologous genes including Ccr and Emd produced P(3HB-co-3HHx) composed of 14 mol% 3HHx with 41 wt% of dry cellular weight from glucose. Further investigations revealed that the C6 monomer (R)-3HHx-CoA was not supplied by (R)-specific reduction of 3-oxohexanoyl-CoA but by (R)-specific hydration of 2-hexenoyl-CoA formed through reverse β-oxidation after the elongation from C4 to C6. While contribution of the reverse β-oxidation to the conversion of the C4 intermediates was very limited, crotonyl-CoA, a precursor of butyryl-CoA, was generated by dehydration of (R)-3HB-CoA. Several modifications previously reported for enhancement of bioproduction in E. coli were examined for the copolyester synthesis. Elimination of the global regulator Cra or PdhR as well as the block of acetate formation resulted in poor PHA synthesis. The strain lacking RNase G accumulated more PHA but with almost no 3HHx unit. Introduction of the phosphite oxidation system for regeneration of NADPH led to copolyester synthesis with the higher cellular content and higher 3HHx composition by two-stage cultivation with phosphite than those in the absence of phosphite.

Abstract Background In Malaysia, the domestic water buffaloes (Bubalus bubalis) are classified into the swamp and the murrah buffaloes. Identification of these buffaloes is usually made via their phenotypic appearances. This study characterizes the subspecies of water buffaloes using karyotype, molecular and phylogenetic analyses. Blood of 105 buffaloes, phenotypically identified as swamp, murrah and crossbred buffaloes were cultured, terminated and harvested using conventional karyotype protocol to determine the number of chromosomes. Then, the D-loop of mitochondrial DNA of 10 swamp, 6 crossbred and 4 murrah buffaloes which were identified earlier by karyotyping were used to construct a phylogenetic tree was constructed. Results Karyotypic analysis confirmed that all 93 animals phenotypically identified as swamp buffaloes with 48 chromosomes, all 7 as crossbreds with 49 chromosomes, and all 5 as murrah buffaloes with 50 chromosomes. The D-loop of mitochondrial DNA analysis showed that 10 haplotypes were observed with haplotype diversity of 0.8000 ± 0.089. Sequence characterization revealed 72 variables sites in which 67 were parsimony informative sites with sequence diversity of 0.01906. The swamp and murrah buffaloes clearly formed 2 different clades in the phylogenetic tree, indicating clear maternal divergence from each other. The crossbreds were grouped within the swamp buffalo clade, indicating the dominant maternal swamp buffalo gene in the crossbreds. Conclusion Thus, the karyotyping could be used to differentiate the water buffaloes while genotypic analysis could be used to characterize the water buffaloes and their crossbreds.

Purpose: Although olfactory decline and visual hallucinations are useful in distinguishing dementia with Lewy bodies (DLB) from Alzheimer's disease (AD) in a clinical setting, neither is easy to evaluate objectively. The pareidolia test is used to assess susceptibility to visual hallucinations, while in Japan, the Odor Stick Identification Test for the Japanese (OSIT-J) is used to objectively quantify olfactory decline. The present study investigated the efficacy of these olfactory and pareidolia tests in differentiating AD from DLB. Their usefulness was then compared with that of the indicative biomarkers in neuroimaging for a clinical diagnosis of DLB listed in the Fourth Consensus Report of the Dementia with Lewy Bodies Consortium.Methods: A total of 24 probable DLB and 22 probable AD patients were enrolled. All underwent 4 diagnostic procedures: uptake of dopamine transporter in single photon emission computed tomography (DaT-SPECT) and meta-iodobenzylguanidine (MIBG) in myocardial scintigraphy, the pareidolia test, and OSIT-J. The sensitivity, specificity, and accuracy of these methods in differentiating DLB from AD were compared.Results: Sensitivity and specificity in differentiating DLB from AD were 86 and 100% by the heart-to-mediastinum ratio of MIBG uptake; 82 and 96% by the specific binding ratio on DaT-SPECT; 77 and 67% by the combination of OSIT-J and pareidolia test scores; 73 and 62% by the pareidolia test scores; and 77 and 58% by the OSIT-J scores, respectively.Conclusions: The present results suggest that the pareidolia and OSIT-J tests may be considered before resorting to nuclear neuroimaging in the diagnosis of DLB.

When a nanoparticle is irradiated by an intense laser pulse, it turns into a nanoplasma, a transition that is accompanied by many interesting nonequilibrium dynamics. So far, most experiments on nanoplasmas use ion measurements, reflecting the outside dynamics in the nanoparticle. Recently, the direct observation of the ultrafast structural dynamics on the inside of the nanoparticle also became possible with the advent of x-ray free electron lasers (XFELs). Here, we report on combined measurements of structural dynamics and speeds of ions ejected from nanoplasmas produced by intense near-infrared laser irradiations, with the control of the initial plasma conditions accomplished by widely varying the laser intensity (9×10^{14} W/cm^{2} to 3×10^{16} W/cm^{2}). The structural change of nanoplasmas is examined by time-resolved x-ray diffraction using an XFEL, while the kinetic energies of ejected ions are measured by an ion time-of-fight method under the same experimental conditions. We find that the timescale of crystalline disordering in nanoplasmas strongly depends on the laser intensity and scales with the inverse of the average speed of ions ejected from the nanoplasma. The observations support a recently suggested scenario for nanoplasma dynamics in the wide intensity range, in which crystalline disorder in nanoplasmas is caused by a rarefaction wave propagating at a speed comparable with the average ion speed from the surface toward the inner crystalline core. We demonstrate that the scenario is also applicable to nanoplasma dynamics in the hard x-ray regime. Our results connect the outside nanoplasma dynamics to the loss of structure inside the sample on the femtosecond timescale.

In this study we develop a model that optimizes the price for new and remanufactured short life-cycle products where demands are time-dependent and price sensitive. While there has been very few published works that attempt to model remanufacturing decisions for products with short life cycle, we believe that there are many situations where remanufacturing short life cycle products is rewarding economically as well as environmentally. The system that we model consists of a retailer, a manufacturer, and a collector of used product from the end customers. Two different scenarios are evaluated for the system. The first is the independent situation where each party attempts to maximize his/her own total profit and the second is the joint profit model where we optimize the combined total profit for all three members of the supply chain. Manufacturer acts as the Stackelberg leader in the independently optimized scenario, while in the other the intermediate prices are determined by coordinated pricing policy. The results suggest that (i) reducing the price of new products during the decline phase does not give better profit for the whole system, (ii) the total profit obtained from optimizing each player is lower than the total profit of the integrated model, and (iii) speed of change in demand influences the robustness of the prices as well as the total profit gained.

An experimental study is performed on a two-dimensional offset jet with a dielectric barrier discharge plasma actuator (PA) as a flow control device that introduces a periodic disturbance to the system. The offset jet is produced by a flow of air emitted from the end of a long parallel channel, and the offset ratio H/h (H: step height, h: channel height) is 1.0. The plasma actuator is installed on the lower wall of the jet exit, and is operated using burst-modulated or continuous waveforms. The Reynolds number based on the mean velocity and the hydraulic diameter of the inlet channel is 2.0 × 103, and the flow at the channel exit is laminar. The wall static pressure and the heat transfer coefficient of the offset plate are measured, and the pressure loss and average Nusselt number are processed. The flow field is examined by flow visualization using a high-speed camera, and the velocity profiles are measured using a particle image velocimetry system. The results show that the reattachment length decreases and the Nusselt number in the recirculation region increases with the induced flow due to the PA. These effects are remarkable at a burst frequency of 100 Hz, which matches the acceptance frequency of the dividing shear layer. At a duty ratio of D = 50 %, due to the existence of a periodic large vortex structure, the reduction of the reattachment length is similar and the Nusselt number is higher compared to those obtained for continuous operation (D = 100 %).

In-situ X-ray computed tomography (CT) was used to observe microstructure formations during freeze-drying of a dextrin solution. A specially designed freeze-drying stage was equipped at the X-ray CT stage. Frozen and dried microstructures were successfully observed. The CT images of the frozen solution clarified the ice crystal size increase and obvious boundary formation between the ice and freeze-concentrated phases upon performing post-freezing annealing at −5°C. These structural modifications emerged owing to Ostwald ripening and glassy phase relaxation. During the freeze-drying, pore microstructures formed as a consequence of water removal. The pores were replicas of the original ice microstructures; some pore microstructures newly formed by the removal of water. The latter mechanism was more obvious in the non-annealed sample than in the annealed sample. The glassy phase in the non-annealed solution was not perfectly freeze-concentrated; water was rapidly removed from this phase, losing its original microstructure. At this moment, the freeze-concentrated region piled up to new pore walls, which consequently thickened the pore walls. An image analysis estimated that the mean pore wall thicknesses for the non-annealed and annealed samples were 13.5 and 8.6 μm, respectively. It was suggested that the advantages of annealing are not only to reduce drying time owing to the modification of ice crystal morphologies but also to avoid quality loss related to the structural deformation of the glassy matters.

Background: Neural pathways are thought to be directly involved in the pathogenesis of rheumatoid arthritis (RA). Although synovial mast cells (MCs) are activated by substance P (SP), the role of MCs in neural pathways in RA remains unknown. The aims of this study were to investigate 1) whether tachykinins are produced by synovial MCs and whether production differs in RA and osteoarthritis (OA) patients, and 2) what is the responsible receptor for SP in synovial MCs. Methods: Synovial tissues were obtained from patients with RA or OA undergoing joint replacement surgery. Cultured synovium-derived MCs were generated by culturing dispersed synovial cells with stem cell factor. SP expression was investigated using immunofluorescence and enzyme immunoassays. Mas-related gene X2 (MrgX2) expression was reduced in human MCs using a lentiviral shRNA silencing technique. Results: SP expression was localized around the cell membrane in 41% (median) of the MCs in synovium from RA but in only 7% of that from OA, suggesting the activation of MCs. Synovial MCs expressed tachykinin (TAC) 1 mRNA, the expression of which was upregulated by the aggregation of FcɛRI or the addition of aggregated IgG. However, the released SP appeared to be rapidly degraded by MC chymase. Synovial MCs were activated with SP through MrgX2 to release histamine without producing proinflammatory cytokines. Conclusions: Activated synovial MCs may rapidly degrade SP, which may downregulate the SP-mediated activation of synoviocytes in RA. On the other hand, SP activates MCs to induce inflammatory mediators, suggesting the dual regulation of SP-mediated inflammation by MCs in RA.

Background: Interleukin (IL)-17A plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). The expression of IL-17A in synovial mast cells (MCs) in RA and osteoarthritis (OA) has been reported, but the frequencies of IL-17A expression in synovial MCs have varied. The aim of this study was to investigate whether IL-17A expression is upregulated in human synovial MCs in RA and to elucidate the mechanism of IL-17A expression in synovial MCs. Methods: Synovial tissues were obtained from patients with RA or OA undergoing joint replacement surgery, and synovial MCs were enzymatically dispersed. Synovium-derived cultured MCs were generated by culturing synovial cells with stem cell factor. IL-17A expression was investigated using immunofluorescence in synovial tissues. IL-17A mRNA expression and its production from MCs were examined using RT-PCR and ELISA, respectively. Results: The number of IL-17A-positive (+) synovial MCs and the percentage of IL-17A+ MCs among all the IL-17A+ cells from RA patients were not significantly increased compared with those from OA subjects. The synovium-derived cultured MCs spontaneously released small amounts of IL-17A. Neither IgE- nor IgG-dependent stimulation increased IL-17A production from the MCs. IL-33, tumor necrosis factor-α, C5a, lipopolysaccharide or IL-23 plus IL-1β did not affect IL-17A production in MCs. Conclusions: The synovial MCs are not a main source of IL-17A in RA.

An asymmetric Claisen rearrangement using Oppolzer’s camphorsultam was developed. Under thermal conditions, a geraniol-derived substrate underwent the rearrangement with good stereoselectivity. The absolute configuration of the newly formed all-carbon quaternary stereocenter was confirmed by the total synthesis of (+)-bakuchiol from the rearrangement product.

Phaeohyphomycosis (PHM) is a rare, deep fungal infection of the skin and subcutaneous tissues caused by dematiaceous fungi. In this report, we describe a case of PHM caused by Phaeoacremoniumrubrigenum, which is generally known to infect woody plants. We detected the gray-blackish villi by biopsy culture material, and slide culture revealed the conidia arising from slightly tapering phialides. Furthermore, we differentiated these fungi as P.rubrigenum by Basic Local Alignment Search Tool (BLAST) algorithm. We performed surgical debridement of disseminated nodules and administered oral itraconazole for a duration of 4 weeks. One year after stopping itraconazole, there was no sign of relapsing subcutaneous nodules. To our knowledge, this is the third case report of PHM developing from skin infection by P. rubrigenum in human.

We extend the modularity lifting result of P. Kassaei (‘Modularity lifting in parallel weight one’,J. Amer. Math. Soc. 26 (1) (2013), 199–225) to allow Galois representations with some ramification at $\def \xmlpi #1{}\def \mathsfbi #1{\boldsymbol {\mathsf {#1}}}\let \le =\leqslant \let \leq =\leqslant \let \ge =\geqslant \let \geq =\geqslant \def \Pr {\mathit {Pr}}\def \Fr {\mathit {Fr}}\def \Rey {\mathit {Re}}p$ . We also prove modularity mod 5 of certain Galois representations. We use these results to prove new cases of the strong Artin conjecture over totally real fields in which 5 is unramified. As an ingredient of the proof, we provide a general result on the automatic analytic continuation of overconvergent $p$ -adic Hilbert modular forms of finite slope which substantially generalizes a similar result in P. Kassaei (‘Modularity lifting in parallel weight one’, J. Amer. Math. Soc. 26 (1) (2013), 199–225).

Emerging evidence suggests that the increasing prevalence of food allergies is associated with compositional and functional changes in our gut microbiota. Microbiota-host interactions play a key role in regulating the immune system. Development of a healthy gut microbiota and immune system occurs early in life and is largely shaped by exposure to maternal microbes through vaginal/natural delivery and breast milk, whereas use of antibiotics can disrupt gut homeostasis and significantly raise the risk of allergic diseases. Thus, changes in the quantity or diversity of gut microbes affect oral toleranace through interations of microbial molecules with pattern recognition receptors on immune cells and confer susceptibility to food allergies. On the other hand, short chain fatty acids which are fermentation end products of insoluble fibers by intestinal micoorganisms have been shown to confer protective effects on food allergy. As a preventive and therapeutic treatment for food allergies, probiotics have gained widespread attention in recent years. Reintroducing certain commensal microbes, such as Clostridia, both in animal models and clinical trials led to the prevention or resolution of allergic symptoms. This review highlights recent progress in our understanding of the gut microbiota's role in food allergy. However, mechanistic details underlying the anti-allergic effects of probiotics and the interaction between the gut microbiota and the immune system remain circumstantial and are not fully understood. Future studies should address possible factors and underlying mechanisms for microbiota-host interactions and gut immunity, as well as the efficacy, safety, and appropriate use of probiotics in establishing a standard treatment regimen for food allergies.

The one-bead-one-compound (OBOC) combinatorial peptide library is a powerful tool to identify ligand and receptor interactions. Here, we applied the OBOC library technology to identify mimotopes specific to the immunoglobulin E (IgE) epitopes of the major shellfish allergen tropomyosin. OBOC peptide libraries with 8-12 amino acid residues were screened with serum samples from patients with shellfish allergy for IgE mimotopes of tropomyosin. Twenty-five mimotopes were identified from the screening and their binding reactivity to tropomyosin-specific IgE was confirmed by peptide ELISA. These mimotopes could be divided into seven clusters based on sequence homology, and epitope mapping by EpiSearch of the clustered mimotopes was performed to characterize and confirm the validity of mimotopes. Five out of six of the predicted epitopes were found to overlap with previously identified epitopes of tropomyosin. To further confirm the mimicry potential of mimotopes, BALB/c mice were immunized with mimotopes conjugated to keyhole limpet hemocyanin and assayed for their capacity to induce tropomyosin-specific antibodies. BALB/c mice that received mimotope immunization were found to have an elevated level of tropomyosin-specific immunoglobulin G, but not mice that received an irrelevant mimotope. This study pioneers the successful application of the OBOC libraries using whole sera to screen and identify multiple shrimp allergen mimotopes and validates their mimicry potential using in vitro, in vivo, and in silico methods.Cellular & Molecular Immunology advance online publication, 14 september 2015; doi:10.1038/cmi.2015.83.

Background: The efficacy and safety of allergen-specific immunotherapy (AIT) are highly dose-dependent., Methods: We investigated the dosage effects of AIT and the underlying mechanisms in a murine model of shrimp hypersensitivity. BALB/c mice were sensitized with recombinant shrimp allergen rMet e 1 and challenged orally with a high dose of rMet e 1 to elicit an allergic response. These sensitized mice were then treated with a low (0.01 mg), medium (0.05 mg), or high dosage (0.1 mg) of rMet e 1 intraperitoneally before receiving a second oral challenge. The allergic responses and immunological changes in the gut were compared between animals receiving different dosages., Results: We found that all sensitized mice that received rMet e 1 immunotherapy were desensitized, regardless of the dosage, and protected at the second oral challenge. Nevertheless, the mice in the high-dosage group experienced severe systemic reactions during the treatment phase. In contrast, regulatory T (Treg) cell-associated genes were upregulated only in the low- and medium-dosage groups, and Foxp3+ cells were more abundant in the gut lymphoid tissues than in the high-dosage group., Conclusions: Our results demonstrate that low-dosage immunotherapy favors the induction of local Foxp3+ Treg cells and the upregulation of regulatory cytokines. The safety advantages and long-term efficacy of low-dosage immunotherapy should be taken into consideration when developing immunotherapy dose schedules., (© 2017 S. Karger AG, Basel.)

Advances in understanding the immunological and molecular basis of autoimmune diseases have made gene therapy a promising approach to treat the affected patients. Gene therapy for autoimmune diseases aims to regulate the levels of proinflammatory cytokines or molecules and the infiltration of lymphocytes to the effected sites through successful delivery and expression of therapeutic genes in appropriate cells. The ultimate goal of gene therapy is to restore and maintain the immune tolerance to the relevant autoantigens and improve clinical outcomes for patients. Here, we summarize the recent progress in identifying genes responsible for autoimmune diseases and present examples where gene therapy has been applied as treatments or prevention in autoimmune diseases both in animal models and the clinical trials. Discussion on the advantages and pitfalls of gene therapy strategies employed is provided. The intent of this review is to inspire further studies toward the development of new strategies for successful treatment of autoimmune diseases.

Background: Shellfish hypersensitivity is among the most common food allergies. A murine model of IgE-mediated shrimp allergy has been established in our laboratory. The aim of this study is to determine the intestinal histological changes and cytokine expression profile of this model sensitized with the major shellfish allergen tropomyosin., Methods: Female Balb/c mice orally sensitized and challenged with recombinant tropomyosin were sacrificed. Continuous sections of duodenum, jejunum and ileum were prepared using the Swiss roll technique for histological and immunological analysis. Duodenal epithelial cell apoptosis and migration were examined. mRNA expression of IL-4, IL-6, IL-10, IL-13, IL-18 and IFN-γ in intestinal tissue was measured via RT-PCR., Results: In tropomyosin-sensitized and challenged mice, an increased number of eosinophils, mast cells and goblet cells was found 24 h after challenge. There were also increased mast cell and goblet cell numbers at 72 h after challenge, but the level of eosinophils decreased. Differences compared with control mice are most prominent at the duodenum compared to the distal regions. In addition, TUNEL assay indicates a significantly higher apoptosis rate in sensitized mice sacrificed 72 h after challenge, and mRNA expression showed a biased Th2/Th1 cytokine profile and a higher level of murine mast cell protease 1., Conclusions: This study documented a multitude of histological and immunological changes in the gut in a murine model of shrimp allergy. Even without repetitive intragastric challenge, shrimp tropomyosin induces an increase in the number of inflammatory cells to varying degrees within the small intestine. This model provides an important tool for testing new therapeutic interventions., (© 2015 S. Karger AG, Basel.)

Designer proteins deprived of its IgE-binding reactivity are being sought as a regimen for allergen-specific immunotherapy. Although shrimp tropomyosin (Met e 1) has long been identified as the major shellfish allergen, no immunotherapy is currently available. In this study, we aim at identifying the Met e 1 IgE epitopes for construction of hypoallergens and to determine the IgE inhibitory capacity of the hypoallergens. IgE-binding epitopes were defined by three online computational models, ELISA and dot-blot using sera from shrimp allergy patients. Based on the epitope data, two hypoallergenic derivatives were constructed by site-directed mutagenesis (MEM49) and epitope deletion (MED171). Nine regions on Met e 1 were defined as the major IgE-binding epitopes. Both hypoallergens MEM49 and MED171 showed marked reduction in their in vitro reactivity towards IgE from shrimp allergy patients and Met e 1-sensitized mice, as well as considerable decrease in induction of mast cell degranulation as demonstrated in passive cutaneous anaphylaxis assay. Both hypoallergens were able to induce Met e 1-recognizing IgG antibodies in mice, specifically IgG2a antibodies, that strongly inhibited IgE from shrimp allergy subjects and Met e 1-sensitized mice from binding to Met e 1. These results indicate that the two designer hypoallergenic molecules MEM49 and MED171 exhibit desirable preclinical characteristics, including marked reduction in IgE reactivity and allergenicity, as well as ability to induce blocking IgG antibodies. This approach therefore offers promises for development of immunotherapeutic regimen for shrimp tropomyosin allergy.

Global and regional studies on the prevalence of food allergies are plagued by inconsistent methodologies, variations in interpretation of results, and non-standardized study design. Hence, it becomes difficult to compare the prevalence of food allergies in different communities. This information would be useful in providing critical data that will enhance research to elucidate the nature of food allergies, and the role of gene-environment interactions in the sensitization of children and adults to foods. Testing methodologies range from questionnaires to objective in vitro and in vivo testing, to the gold standard, double-blind placebo-controlled food challenge (DBPCFC). Although considered the most accurate and reliable method in detecting the prevalence of food allergy, DBPCFC is not always practical in epidemiological studies of food allergy. On the other hand, multiple logistic regression studies have been done to determine predictability of the outcome of food challenges, and it appears that skin prick testing and in vitro-specific serum IgE are the best predictors. Future studies directed towards confirming the validity of these methods as well as developing algorithms to predict the food challenge outcomes are required, as they may someday become accessory tools to complement DBPCFC.

The science of food allergy has been rapidly evolving before our eyes in the past half century. Like other allergic disorders, the prevalence of food allergies has dramatically increased, and coupled with the increased public awareness of anaphylaxis due to food allergy, this has driven an explosion in basic and clinical research in this extremely broad subject. Treatment of food allergies has evolved and practices such as food challenges have become an integral part of an allergy practice. The impact of the increase of food allergy has driven package labeling laws, legislation on emergency treatment availability in schools and other public places, and school policy. But to this day, our knowledge of the pathogenesis of food allergy is still incomplete. There are the most obvious IgE-mediated immediate hypersensitivity reactions, but then multiple previously unidentified conditions such as eosinophilic esophagitis, food protein-induced enterocolitis syndrome, milk protein allergy, food-induced atopic dermatitis, oral allergy syndrome, and others have complicated the diagnosis and management of many of our patients who are unable to tolerate certain foods. Many of these conditions are not IgE-mediated, but may be T cell-driven diseases. The role of T regulatory cells and immune tolerance and the newly discovered immunological role of vitamin D have shed light on the variable clinical presentation of food allergy and the development of new methods of immunotherapy in an example of bench-to-bedside research. Component-resolved diagnostic techniques have already begun to allow us to more precisely define the epitopes that are targeted in food allergic patients. The development of biological modulators, research on genomics and proteomics, and epigenetic techniques all offer promising avenues for new modes of therapy of food allergy in the twenty-first century.

Unlabelled: Antimitochondrial antibodies (AMAs) directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2) are detected in 95% of patients with primary biliary cirrhosis (PBC) and are present before the onset of clinical disease. The recent demonstration that AMAs recognize xenobiotic modified PDC-E2 with higher titers than native PDC-E2 raises the possibility that the earliest events involved in loss of tolerance are related to xenobiotic modification. We hypothesized that reactivity to such xenobiotics would be predominantly immunoglobulin M (IgM) and using sera from a large cohort of PBC patients and controls (n = 516), we examined in detail sera reactivity against either 6,8-bis(acetylthio) octanoic acid (SAc)-conjugated bovine serum albumin (BSA), recombinant PDC-E2 (rPDC-E2) or BSA alone. Further, we also defined the relative specificity to the SAc moiety using inhibition enzyme-linked immunosorbent assay (ELISA); SAc conjugate and rPDC-E2-specific affinity-purified antibodies were also examined for antigen specificity, isotype, and crossreactivity. Reactivity to SAc conjugates is predominantly IgM; such reactivity reflects a footprint of previous xenobiotic exposure. Indeed, this observation is supported by both direct binding, crossreactivity, and inhibition studies. In both early and late-stage PBC, the predominant Ig isotype to SAc is IgM, with titers higher with advanced stage disease. We also note that there was a higher level of IgM reactivity to SAc than to rPDC-E2 in early-stage versus late-stage PBC. Interestingly, this finding is particularly significant in light of the structural similarity between SAc and the reduced form of lipoic acid, a step which is similar to the normal physiological oxidation of lipoic acid., Conclusion: Specific modifications of the disulfide bond within the lipoic-acid-conjugated PDC-E2 moiety, i.e., by an electrophilic agent renders PDC-E2 immunogenic in a genetically susceptible host., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Recent advancement in immunology, molecular biology, and bioinformatics has yielded extensive information on the pathophysiological mechanisms of autoimmunity, which has greatly facilitated the identification of potential therapeutic targets and the development of gene therapy in the treatment of autoimmune disease. Preclinical studies were carried out in animal models. This phenomenon is well illustrated in two prototypic animal models of autoimmune disease: the autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) and collagen-induced arthritis (CIA) model in rheumatoid arthritis (RA). Here we discuss the current data on the development and validation of gene therapy in autoimmunity in these two models. The success in preclinical animal model studies provides the proof-of-concept of gene therapy for potential future applications in the treatment of autoimmune diseases. Furthermore, the identification of risk factors from epidemiological studies reveals further potential therapeutic targets to be examined in animal models., (2009 Elsevier B.V. All rights reserved.)

Flavanols and procyanidins isolated from cocoa have been reported to possess multiple activities potentially relevant to oxidant defenses, vascular function, and immune function. In a combination of in vivo and in vitro studies, we and others have observed that cocoa can be an anti-inflammatory modulator and that compounds in cocoa are capable of modulating eicosanoid production, platelet aggregation, and the pool size of nitric oxide. The present study extends these findings by examining the in vitro effects of cocoa procyanidins on polymorphonuclear cells (PMNs). PMNs, part of the innate arm of the immune system, represent 50-60% of the total peripheral white blood cells and are the first cells to be recruited to the sites of inflammation or injury secondary to bacterial infections. Herein, we demonstrate that certain flavanols and procyanidins isolated from cocoa can moderate a subset of signaling pathways derived from lipopolysaccharide (LPS) stimulation of PMNs, mainly, PMN oxidative bursts and activation markers, and they can influence select apoptosis mechanisms. We hypothesize that flavanols and procyanidins can decrease the impact of LPS on the N-formyl-Met-Leu-Phe-primed PMN ability to generate reactive oxygen species by partially interfering in activation of the mitogen-activated protein kinase pathway.

Unlabelled: Primary biliary cirrhosis (PBC) is an organ-specific autoimmune liver disease characterized by the presence of antimitochondrial antibodies and the destruction of small intrahepatic bile ducts with portal inflammation. In previous studies, we reported that both CD1d expression and the frequency of CD1d-restricted natural killer T (NKT) cells were increased in the livers of patients with PBC. To define a specific role of CD1d-restricted NKT cells in the pathogenesis of PBC, particularly early events, we investigated the function of hepatic CD1d-restricted NKT cells in our transforming growth factor beta (TGF-beta) receptor II dominant-negative (dnTGFbetaRII) mouse model of PBC. We generated CD1d(-/-) and CD1d(+/-) dnTGFbetaRII mice and performed a comparative study of liver immunopathology. We report herein that these dnTGFbetaRII mice demonstrate a massive increase of hyperactive CD1d-restricted NKT cells within the hepatic tissues. CD1d(-/-)dnTGFbetaRII mice, which lack CD1d-restricted CD1d-restricted NKT cells, exhibit significantly decreased hepatic lymphoid cell infiltrates and milder cholangitis compared with CD1d(+/-)dnTGFbetaRII mice. Interestingly, there was a significant increase in the production of interferon-gamma in hepatic CD1d-restricted NKT cells activated by alpha-galactosylceramide in young but not older dnTGFbetaRII mice, suggesting an age-dependent role of CD1d-restricted NKT cells., Conclusion: These data demonstrate that CD1d-restricted NKT cells in dnTGFbetaRII mice are a critical factor in liver injury.

Current models of adult haematopoiesis propose that haematopoietic stem cells (HSCs) differentiate into common lymphoid (CLP) and common myeloid (CMP) progenitors and establish an early separation between myeloid and lymphoid lineages. Nevertheless, the developmental potential of CMP-associated B cells suggests the existence of alternate pathways for B lymphopoesis. The aim of this study was to compare the developmental and functional properties of CMP- and CLP-derived B cells. While both populations matured through pro-B cell and transitional B cell intermediates in the bone marrow and spleen, respectively, following transfer into irradiated mice, mature CMP- and CLP-derived B cells exhibit distinct functional responses. Specifically, CMP-derived B cells did not respond to mitogenic stimulation to the same degree as their CLP-derived counterparts and secrete lower levels of IgM and the inflammatory cytokines such as interleukin (IL)-6 and IL-10. Together, these data suggest the existence of multiple pathways for generating functionally distinct B cells from bone marrow precursors.

While only a small percentage of the liver as dendritic cells, they play a major role in the regulation of liver immunity. Four major types of dendritic cell subsets include myeloid CD8alpha(-)B220(-), lymphoid CD8alpha(+)B220(-), plasmacytoid CD8alpha(-)B220(+), and natural killer dendritic cell with CD8alpha(-)B220(-)NK1.1(+) phenotype. Although these subsets have slightly different characteristics, they are all poor naïve T cell stimulators. In exchange for their reduced capacity for allostimulation, hepatic DCs are equipped with an enhanced ability to secrete cytokines in response to TLR stimulation. In addition, they have increased level of phagocytosis. Both of these traits suggest hepatic DC as part of the innate immune system. With such a high rate of exposure to the dietary and commensal antigens, it is important for the hepatic DCs to have an enhanced innate response while maintaining a tolerogenic state to avoid chronic inflammation. Only upon secondary infectivity does the hepatic DC activate memory T cells for rapid eradication of recurring pathogen. On the other hand, overly tolerogenic characteristics of hepatic DC may be responsible for the increase prevalence of autoimmunity or liver malignancies.

The role of the liver in the initiation and maintenance of tolerance is a critical immune function that involves multiple lineages of immune cells. Included within these populations are liver dendritic cells (DCs). Although there has been significant work on the phenotypic and functional roles of splenic and bone marrow dendritic cells, as well as their subsets, comparable studies in liver have often been difficult. To address this issue we have isolated, from C57BL/6 mice, relatively pure populations of DCs and compared phenotype and function to the data from spleen using flow cytometry, cell sorter assisted purification and culture, morphology by cytospin and May-Giemsa staining, cell cycle progression, antigen uptake, cytokine production and allo-activation potential. natural killer (NK)1.1(-)CD11c(+) liver DC subsets (conventional DCs, T cell receptor (TcR)beta(-)NK1.1(-)CD11c(+)B220(-) and plasmacytoid DCs, TcRbeta(-)NK1.1(-)CD11c(+)B220(+)) efficiently endocytose dextran and produce significant levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12 p40 in response to Toll-like receptor (TLR) ligands, with responses higher than splenic DCs. There is also a differential capability of hepatic DCs to respond to innate signals. Indeed, CD11c(+) hepatic DCs have a greater capacity to respond to innate stimulation but are less capable of inducing CpG activated-allogeneic T cells. These data suggest that hepatic dendritic cells function as a critical bridge between innate and adaptive immunity and are capable of inducing stronger innate responses with a lower capacity for allo-stimulation than splenic dendritic cells. These properties of liver dendritic cells contribute to their unique role in the induction of tolerance.

B and T lymphocyte attenuator (BTLA), a recently identified immune inhibitory receptor, has been demonstrated to have the ability to maintain self-tolerance and transplant-tolerance in mice. However, little is known about the effects of immunosuppressive drugs on the expression of BTLA. In the present study, we observed that the immunosuppressive drug cyclosporin A (CsA) could significantly reduce BTLA but not CD25 and CD69 expression on CD4+ T cells during activation in vitro, while rapamycin (RPM) had little effect on it. Exogenous interleukin-2 (IL-2) failed to reverse the inhibitory effect that CsA had on BTLA expression. Furthermore, phorbol 12-myristate 13-acetate (PMA) or ionomycin alone could efficiently induce BTLA protein expression on CD4+ and CD8+ T cells, while CsA significantly suppressed BTLA expression in this system. The present data indicate that the regulation of BTLA expression on CD4+ T cells does not depend on IL-2 and T cell activation but depends on calcineurin-dependent and calcineurin-independent pathways. The observation that CsA significantly inhibits BTLA expression on CD4+ T cells during activation, suggests that CsA might block the immune tolerance induced by BTLA and potentially increase the susceptibility to autoimmune diseases and graft rejection.

A method of antibody elution from red blood cells using xylene is described. It can be completed in 15 minutes after cell washing and requires no special equipment. For warm antibodies, the eluates prepared were more potent than those prepared by Rubin's ether method. It was as effective as Landsteiner and Miller's heat elution method in investigations of ABO hemolytic disease of the newborn. The clinical application of this method in the investigation of hemolytic disease of the newborn, delayed transfusion reaction and autoimmune hemolytic anemia was described.