Escherichia coli (E. coli) to develop an effective virus-like-particles (VLPs) vaccine and evaluated its immunogenicity. Methods: The VLPs produced in E. coli were used as an immunogen mixed with a water-in-mineral-oil adjuvant  or a white oil (7#) adjuvant. VLPs without an adjuvant, commercial subunit vaccine, inactivated vaccine, and attenuated vaccine were used as controls. These test vaccines were intramuscularly injected into 19-day-old SPF chickens, which were challenged with the IBDV virulent strain at 30 days after vaccination. Results: The adjuvants boosted antibody production, and the adjuvant groups (except white oil) produced higher antibody levels than the non-adjuvanted controls and the commercial vaccine groups. In terms of cellular immunity, the VLPs plus adjuvant combinations produced higher levels of cytokines, IL-2, IL-4, and IFN-γ than the controls. Conclusion: IBDV VLPs plus the ISA 71 RVG adjuvant can be used as an optimal vaccine combination for improving the immune efficacy of IBD subunit vaccines, which can protect against the virulent strain.Objectives: Infectious bursal disease virus (IBDV) is a highly contagious, acutely infectious agent that causes immunosuppression in chickens. We expressed IBDV VP2 proteins in  IBDV is responsible for the acute, contagious, and immunosuppressive infectious bursal disease (IBD) in chicks that causes huge economic losses in the chicken industry worldwide . IBDV maEscherichia coli [baculovirus [yeast [plants [baculovirus system is the most frequently used to produce IBDV VLPs, it is inefficient, low-yield, and high cost [E. coli expression systems are more convenient, economical, and fast for the production of recombinant proteins on an industrial scale. Studies have shown that recombinant VP2 proteins can be efficiently expressed in E. coli and self-assembled into VLPs [E. coli lacks the ability to produce modifications after protein expression. VLPs are a powerful tool for enhancing the immunogenicity of IBDV subunit vaccines because they are similar in structure to natural viral capsids and are effective at inducing a humoral response [E. coli expression system affords a potential strategy for the development of IBDV subunit vaccines.At present, VP2 for use in developing subunit vaccines has been produced in different expression systems, such as hia coli , baculovulovirus , yeast [s [yeast , and pla [plants . Althougigh cost ,13. In cnto VLPs ,13,14,15response . TherefoTM ISA series of water-in-oil emulsion adjuvants, which has been widely used in animal models, is very effective at enhancing the humoral and/or cellular immune response and producing a stronger protective effect than traditional adjuvants [Research has shown that when VP2 is mixed with various adjuvants, such as oil , Freund’djuvants . Previoudjuvants . Using tE. coli expression system. The VLPs were then mixed with different adjuvants and used as an immunogen. The test vaccines were intramuscularly injected into 19-day-old SPF chickens, after which they were challenged with the standard virulent strain BC6/85 of IBDV and their protective effects were evaluated. Comparison allowed us to optimize the vaccine/adjuvant combination, which offers better options for IBD subunit vaccines.In this study, we developed an economical and effective subunit vaccine against IBDV using the VP2 protein expressed and self-assembled into VLPs in an Escherichia coli containing the VP2 gene fragment of the IBDV B87 strain  were grown in 150 mL of fresh LB medium in the presence of kanamycin (100 μg/mL) in a shaker (200× g) to an OD600 of 0.5 at 37 °C, then induced with 0.2 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) for 21 h at 37 °C [g for 10 min at 4 °C, resuspended in phosphate-buffered saline , and then disrupted using sonication. The cell lysates containing VP2 proteins were centrifuged at 10,000× g for 20 min at 4 °C and supernatants were collected. Proteins were precipitated with the addition of saturated ammonium sulfate to the supernatants, dissolved in Tris buffer , and purified using anionic ion-exchange chromatography (IEC) and hydrophobic interaction chromatography (HIC)  according to the manufacturer’s instructions. The purity and bioactivity of the proteins were determined with 12% SDS-PAGE and Western blot, respectively. Western blot analysis was performed with anti-His tag mouse monoclonal antibody  and goat anti-mouse IgG antibody .Recombinant w/v) phosphotungstic acid (pH 6.8). The VP2 protein assemblies were observed under a transmission electron microscope  and imaged to indicate shape and size. The size and uniformity of the VLPs in suspension were measured with dynamic light scattering (DLS).The purified VP2 proteins in PBS were assembled into VLPs. Nickel grids (200-mesh) with a carbon-coated collodion film were placed upside down on a drop of VLP suspension for 2 min, after which the grids were washed with PBS and stained with 3%  at a 30:70 ratio (w:w) and with white oil adjuvant at 50:50, and emulsified by vortexing for 5 min. The commercial vaccines were used according to the manufacturer’s instructions.Purified protein solutions were filtered through a 0.22 μm membrane and quantified with a BCA protein assay kit . Aliquots of VLPs (0.05 mg/mL) were mixed with MontanideAll animal experiments were approved by the Animal Ethics Committee of the Animal Experiment Committee of the Henan Academy of Agricultural Sciences . To mitigate the potential impact of the immunogen on the physical and mental well-being of birds during the experiment, a total of 135 specific-pathogen-free (SPF) white Leghorn chickens (19 days old) were procured from Spencer  and housed in negative-pressure filtered-air isolators. They were sacrificed using the cervical dislocation method under xanthine anesthesia to reduce the pain of the birds.g.The immunization program is shown in The IBDV standard virulent strain BC6/85  was used for the challenge. On the fourth day after the challenge, we strangled the chicken to suffocate and remove it quickly. Under visual observation, the bursa of Fabricius appeared swollen or showed clear yellow exudation, jelly samples, or bleeding. We then determined the chicken’s infectious bursal disease by viewing typical lesions. The challenge virus was prepared by orally inoculating SPF chickens at five weeks of age and harvesting the bursae at three days post-infection. The bursae were homogenized in PBS (1:10 ratio) and stored at −80 °C.50 was conducted following Karber’s method of biological statistics, as described in Veterinary Microbiology by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences . Calculation formula: logBID50 = L − d (S − 0.5). In the formula: L = logarithm of the lowest dilution, d = the difference between the logarithms of dilution, and S = total rate of bursal lesions.The statistical analysis of the BC6/85 strain BID−5 BID50 of the standard virulent IBDV strain BC6/85, and the chickens were observed for clinical symptoms and survival status over 4 days. At 4 days post-infection, the chickens were euthanized and necropsied, and the leg muscles, bursae of Fabricius, and other major organs (liver and kidney) were examined for pathogenic gross lesions. The bursae to body weight (BF/BW) ratio was calculated as bursae of Fabricius weight/body weight ×1000. All groups except group I (negative control) were challenged at 30 days after vaccination via the oral route with 10Sera were obtained using low-speed centrifugation of clotted blood samples. The VP2-specific antibody titer was determined using a commercial antibody ELISA (CK113) test kit , following the manufacturer’s instructions. The absorbance was determined with a microplate reader at a wavelength of 405 nm. The IFN-γ, IL-2, and IL-4 levels in the serum before challenging the chickens were determined with ELISA using IFN-γ (SEA049Ga), IL-4 (SEA077Ga), and IL-2 (SEA073Ga) kits , following the manufacturer’s instructions. The absorbance at 450 nm was determined with a microplate reader. The bursae of Fabricius were removed and fixed in formalin for scoring of the histopathological lesion severity . The effp < 0.05 for all tests.Analysis of variance (ANOVA) and other statistical analyses (antibody titers and cytokines) were performed using GraphPad Prism version 5.00 . Data are shown as the mean ± SD, and statistical significance was set at E. coli culture was 0.054 mg/L.The expression of the IBDV VP2 protein was confirmed using SDS-PAGE and Western blot A,C. The E. coli were capable of forming VLPs after purification with IEC and HIC, the purified VP2 proteins were analyzed using TEM and DLS. TEM images of the purified VP2 revealed that it did self-assemble into VLPs (Previous studies have shown that VP2 proteins can self-assemble into VLPs with a diameter of 25 nm . To detento VLPs A. The DLnto VLPs B.p < 0.01). The groups receiving VLPs plus adjuvant  produced antibodies earlier than those given VLPs alone or inactivated vaccine, and they had higher antibody titers than the VLP-only group. The antibody titers in the VLPs with adjuvant groups continued to increase from 0 to 28 dpv, but the antibody levels in the VLP group only showed a downward trend on day 28 compared with day 21. On day 28 pv, the antibody titers in the groups receiving VLPs plus the three adjuvants were: ISA 71 RVG (log103.14) > ISA 71 VG (log102.96) > white oil (log102.51). Furthermore, the antibody titers recorded for the commercial vaccine group were as follows: inactivated vaccine (log103.10) > subunit vaccine (log102.87) > attenuated vaccine (log102.46). In addition, the antibody titers of the ISA 71 RVG group were similar to those of the inactivated vaccine group and were higher than those of the commercial subunit vaccine group and the attenuated vaccine group. The ISA 71 RVG group had higher antibody titers than the groups given commercial vaccines . The results demonstrate that the E. coli-synthesized VLPs had good immunogenicity, that ISA adjuvants seem to be more efficient than white oil, and that it is as efficient as commercial vaccines, which are really good results.The antibody titers against IBDV were determined with ELISA at 0, 7, 14, 21, and 28 days post-vaccination (dpv). The mean serum antibody titers in each group are shown in p < 0.001, ** p < 0.01, * p < 0.05). The results showed that there was no significant difference in IL-4 levels between the antigen groups and the PBS control. The IL-2 level in the VLP plus ISA 71 RVG group was significantly higher than that of the PBS group (* p < 0.05) only, but the difference was not significant with the other antigen groups (p > 0.05). For the IFN-γ levels, the antigen groups were significantly higher than the PBS group (** p < 0.01), and the VLP plus adjuvant groups were significantly higher than the commercial groups  (* p < 0.05). In addition, the VLP-immunized groups exhibited the same trend in cytokine production, IFN-γ > IL-2 > IL-4, and the levels of all three cytokines in the VLP plus ISA 71 RVG group were a bit higher than in the other groups.The IL-2, IL-4, and IFN-γ cytokine levels in serum samples from immunized chickens were evaluated with ELISA , and the−5 BID50 per bird), except for group I. No mortality was observed in any group of chickens in this experiment. The chickens were dissected at four days post-challenge. Necropsy of the diseased chickens showed varying degrees of hemorrhage in the thigh muscles, bursal atrophy, cyst formation, yellow gelatinous coatings, or hemorrhages , and the immune protection rate of the three adjuvants was: ISA 71 RVG (13/15) > ISA 71 VG (11/15) > white oil (10/15). The VLPs plus ISA 71 RVG produced a higher protection rate than the commercial subunit vaccine (10/15) and the attenuated vaccine (8/15). These results indicated that the combination of VLPs with ISA 71 RVG gave the best protection against IBDV. The BF/BW ratios, bursal lesion scores, and protection rates were also determined . The VLPThe pathological changes in the bursae of all groups were revealed with hematoxylin and eosin (H&E) staining . The burE. coli expression system is more convenient, more economical, and faster for manufacturing owing to the ease of growth and genetic manipulation. Chelating Ni2+ Sepharose columns [E. coli cells and purified with saturated ammonium sulfate precipitation, IEC, and HIC; this guarantees the vaccine’s safety and ease of biological process amplification. The VP2 protein was purified to a level of 3.5 mg/L, which is significantly higher than previously reported [E. coli expression system and the purification method could produce high yields of IBDV VLPs.The VP2 protein of IBDV is a major structural and host-protective protein and has been used as a target for the development of subunit vaccines. Compared with other expression systems, the  columns , sucrose columns , and ces columns  are oftereported . The purreported  and yeasreported . Our res3.14) produced the highest level of antibodies and had levels comparable to the three commercial vaccine groups  in the bursae of Fabricius, and > 70% of the follicles were atrophied and necrotic. Follicular atrophy has been shown to cause immunosuppression. In the results of challenge protection, the adjuvant groups were significantly higher than the adjuvant-free group . Both ISEscherichia coli was used to express IBDV VP2 proteins, which could be isolated using convenient and low-cost purification methods and demonstrated the ability to self-assemble into VLPs that possessed immunogenicity. The results of animal experiments showed that the VLPs assembled by prokaryotic expression had good immunogenicity in combination with different adjuvants , and ISA 71 RVG was the optimum adjuvant for the IBDV VLPs. The VLPs, in combination with the ISA 71 RVG adjuvant, induced high levels of immune response and enhanced protection against IBDV. Our alternative vaccine against IBDV offers a new option for improving the immune efficacy of IBD subunit vaccines.In conclusion, this report documents the efficacy of different adjuvants combined with VLPs for IBDV subunit vaccination. In this experiment,


The most common cause of sensorineural hearing loss is damage of auditory hair cells. Tumor necrosis factor-alpha (TNF-α) is closely associated with sensorineural hearing loss. The present study examined the preconditioning effect of dexamethasone (DEX) on TNF-α-induced ototoxicity in mouse auditory hair cells (HEI-OC1) and cochlear explants. Treatment of HEI-OC1 with 10 ng/ml TNF-α for 24 h decreased cell viability, increased the accumulation of reactive oxygen species (ROS), and induced caspase-mediated apoptotic signaling pathways. Pretreatment with 10 nM DEX for 6 h before TNF-α exposure restored cell viability, decreased ROS accumulation, and attenuated apoptotic signaling activation induced by TNF-α. Incubation of cochlear explants with 20 ng/ml TNF-α for 24 h resulted in significant loss of both inner hair cells (IHCs) and outer hair cells (OHCs) and an increase in apoptotic activation accessed by annexin V staining. The cochlear explants pre-incubated with 10 nM DEX attenuated TNF-α ototoxicity in both IHCs and OHCs and apoptotic cell death. These results indicated that DEX plays a protective role in ototoxicity induced by TNF-α through attenuation of caspase-dependent apoptosis signaling pathway and ROS accumulation. The most common cause of sensorineural hearing loss is damage of auditory hair cells. Many causes including aging process, noises, ototoxic drugs, trauma, vascular disorders, viral infections, and immune-related mechanisms act as insults to the auditory hair cell and result in sensorineural hearing loss –3. BecauTNF-α is a well-established proinflammatory cytokine that can induce apoptosis in various types of cells , 10. TNFCorticosteroids or synthetic glucocorticoids have been used to treat various inner ear diseases, including sudden sensorineural hearing loss, immune-related hearing loss, Ménière’s disease, and inner ear diseases –19. IntrThe present study examined the effects of TNF-α on cell viability, accumulation of reactive oxygen species (ROS), and changes in apoptosis-related protein expression in mouse auditory hair cells (HEI-OC1). Moreover, we investigated whether the beneficial effects of dexamethasone (DEX) treatment on TNF-α could apply to mouse cochlear explants.The components of the cell culture medium were procured from Thermo Fisher Scientific  unless indicated otherwise. TNF-α and DEX were bought from BioLegend  and Enzo Life Sciences , respectively. Polyclonal antibodies against Bcl-2 (3498), caspase-3 (9662), cleaved caspase-3 (9661), cleaved caspase-7 (9491), and poly (ADP-ribose) polymerase (PARP)/cleaved PARP (9542) were purchased from Cell Signaling Technology , Bax (556467) from BD Biosciences , caspase-7 (sc-81654) from Santa Cruz Biotechnology , and β-actin (A5441) from Sigma-Aldrich . Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Bethyl Laboratories .2 at 33°C. For experiments involving TNF-α or/and DEX treatment, cells were seeded into appropriate culture dishes and incubated for 24 hours under standard conditions. After 3 hours incubation in a serum-free medium, cells were treated to various concentrations of TNF-α or DEX. In DEX pretreatment studies, cells were pretreated with 10 nM DEX for 6 hours. DEX containing culture medium was discarded, and cells were treated with 10 ng/ml TNF-α for additional 24 hours.The immortalized HEI-OC1 mouse auditory cells were kindly provided by Dr. Federico Kalinec . The HEI-OC1 cells were previously characterized in a publication . The HEI3 cells/well and incubated under standard conditions for 24 hours. The serum-starved cells were treated to various concentrations of TNF-α (0–20 ng/ml), DEX (0–20 nM), or DEX (10 nM) plus TNF-α (10 ng/ml). The amount of formazan dye product was established by measuring absorbance at 450 nm with a microplate spectrophotometer . The absorbance values were converted into percentages relative to the untreated control.A colorimetric D-Plus CCK cell viability assay kit  was utilized to evaluate the viability of cells following to the manufacturer’s protocol. Cells were seeded in 96-well plates at a density of 4 × 10The quantification of intracellular ROS was conducted using 5-(and-6)-chloromethyl-2′-7′-dichlorodihydrofluorescein diacetate, acetyl ester . Cells were seeded in 96-well plates and then incubated with DEX, TNF-α, or both sequentially, as described previously. Subsequently, cells were washed with Hank’s balanced salt solution (HBSS) and incubated with 5 μM CM-H2DCFDA for 20 minutes at 33°C in the dark. For quantification of ROS levels, DCF fluorescence was determined by a spectrofluorometer  at 485 nm (excitation) / 535 nm (emission).Total protein was extracted by supplementing the RIPA lysis buffer (Sigma-Aldrich) with protease and phosphatase inhibitors. Protein concentrations were quantified with the Pierce BCA Protein Assay Kit (Thermo fisher). After separating equal amounts of protein via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), they were transferred to nitrocellulose membranes . Next, membranes were washed thrice with Tween 20-containing TBS (TBST), blocked with 5% skim milk, and incubated overnight with the appropriate primary antibodies. After three washes with TBST, the membranes were incubated for 1 hour with HRP-conjugated secondary antibodies. The immunoreactive bands were developed by an enhanced chemiluminescence assay kit  and detected by the ImageQuant LAS 500 biomolecular imager .Annexin V/PI staining was carried out in accordance with the manufactor’s instruction using an Annexin V-FITC Apoptosis staining/Detection kit . The cells cultured on coverslips in 6-well plates were treated with TNF-α or 10 nM DEX for 6 hours followed by TNF-α, and further incubated for 24 hours. After washing with binding buffer, cells were incubated for 10 minutes with Annexin V-FITC and PI solution in the dark. Cells were mounted on glass slides and visualized using the appropriate filter of an Olympus IX71 fluorescence microscope. Green fluorescence (ex/em ∼495/515 nm) was used to detect early apoptotic cells, while red fluorescence (ex/em ∼561/615 nm) was used to detect late apoptotic and necrotic cells.The protocol of animal experiments was approved by the Committee on Use and Care of Animals of the University of Ulsan . The animal experiments were performed according to the guidelines of the National Institutes of Health and the principles of the Declaration of Helsinki. All the animal care was conducted under the supervision of the Laboratory Animal Unit of the Asan Institute for Life Sciences . The pregnant C57BL/6J mice  were obtained from Central Laboratory Animal Inc. , and 3-days neonatal mice were used for explant culture studies. All the neonatal mice were euthanized by decapitation to collect their cochleae, and all efforts were made to minimize suffering.2 overnight. Culture medium was substituted with fresh medium (control) or medium containing different concentrations of TNF-a (10–40 ng/ml) and incubated for 24 hours. For DEX-mediated inhibitory experiment, explants were incubated with medium containing 10 nM DEX for 6 hours prior to treatment with 20 ng/ml TNF-a.To provoke the auditory hair cell damage under more physiologic condition, murine cochleae were isolated. Mice of postnatal day 3 (P3) were euthanized via decapitation, and ∼ 40 cochleae were carefully dissected in ice-cold HBSS solution. The stria vascularis, spiral ligament, and the Reissner’s membrane were meticulously eliminated to isolate cochlear explants. The middle-turn cochlear explants were plated onto 4-well culture dishes and incubated in culture media  at 37°C in 5% COAfter washing twice with PBS and the cochlear explants fixed with 4% paraformaldehyde for 20 minutes. The explants were permeabilized in 0.5% Triton X-100, blocked in 5% normal goat serum (NGS), and incubated overnight with the primary antibody against myosin 7a  at 4°C. Explants were then washed three times with NGS and incubated for 1.5 hours with Alexa Fluor 564 goat anti-mouse secondary antibody . After washing with NGS and distilled water, explants were counterstained with DAPI for 5 minutes and briefly washed with distilled water. Finally, explants were dehydrated in the air and mounted with fluorescence mounting medium . Images of the stained middle-turn cochlear explants were observed with a Zeiss LSM 880 confocal microscope .For Annexin V staining, permeabilized explants were washed with PBS and binding buffer and were incubated for 10 minutes with buffer containing Annexin V-FITC in the dark. The procedure of myosin 7a staining and the observation of fluorescent images were performed as described above. The numbers of myosin 7a and annexin V positive inner hair cells (IHCs) and outer hair cells (OHCs) were counted over a distance of 160 um in the middle turn of each cochlear explant.The data are presented as means ± standard error (SE) from three or four independent experiments. Statistical analyses were carried out by Student’s t-test or one-way analysis of variance (ANOVA) using SigmaPlot software . A p-value less than 0.05 was considered statistically significant.Reports show that various cells exposed to TNF-α induce apoptotic cell death , 25. To The expression of apoptosis-related proteins is shown in To investigate the protective effect of DEX on TNF-α-induced cytotoxicity, HEI-OC1 cells were pretreated with 10 nM DEX for 6 hours, followed by treatment with 10 ng/ml TNF-α for a further 24 hours. DEX pretreatment restored cell viability by ∼20%, which was reduced by TNF-α-treatment . ConcomiFinally, to determine the inhibitory effect of DEX on TNF-α-induced apoptosis, we performed the Annexin V/PI staining assay. As shown in ex vivo.The otoprotective effect of DEX against TNF-α-induced hair cell loss is shown in Immune-related hearing loss is characterized by frequent recurrence and a chronic course and mediated by autoimmune response . ReportsL were associated with production of ROS in reaction to TNF-α [TNF-α-mediated signaling involves two distinct pathways: the prosurvival/proinflammatory pathway and the pro-apoptotic pathway. The former activates NF-κB and MAPK through TNF-α-induced complex I. The latter stimulates production of ROS and the caspase cascade activation in mitochondria through TNF-α-induced complex II . This stto TNF-α . TherefoDEX, a synthetic corticosteroid agonist, functions as an inflammation and immune responses regulator. In general, glucocorticoid induction is mediated by a glucocorticoid receptor that binds to the glucocorticoid, then translocates to the nucleus, and binds to a glucocorticoid response element within the promoter regions . ReportsPrevious reports have shown an anti-apoptotic effect of DEX in various types of cells. For example, DEX pretreatment decreased NADPH oxidase-mediated generation of ROS in BV-2 microglial cells treated to lipopolysaccharides  and suppTreatment of auditory hair cells with ototoxic drugs, such as gentamicin and cisplatin, has shown increased expression of TNF-α mRNA , 41, andIn conclusion, we have shown that DEX pretreatment protects TNF-α-induced ototoxicity in both auditory hair cells and cochlear explants. The otoprotective effect of DEX is achieved through attenuation of caspase-dependent apoptosis signaling pathway and ROS accumulation induced by TNF-α. Our findings broaden the understanding of the beneficial effects of DEX pretreatment on TNF-α-induced ototoxicity and also provide basis for designing therapeutic approaches to treat or prevent inflammatory-related hearing loss.S1 Raw images(PDF)Click here for additional data file.


Body weight, white adipose tissue weight, and the levels of serum lipid and inflammatory markers were measured. The expression of genes related to lipid metabolism in the liver were determined. The results showed that PLs decreased body weight, fat storage, and circulating lipid levels, and EPA-PLs had the best efficiency. Serum TNF-α, MCP-1 levels were significantly reduced via treatment with DHA-PLs and PS groups. Mechanistic investigation revealed that PLs, especially EPA-PLs and PSs, reduced fat accumulation through enhancing the expression of genes involved in fatty acid β-oxidation  and downregulating lipogenesis gene  expression. These data suggest that EPA-PS exhibits the best effects among other PLs in terms of ameliorating obesity, which might be attributed to the fatty acid composition of phospholipids, as well as their headgroup.The weight loss effects of dietary phospholipids have been extensively studied. However, little attention has been paid to the influence of phospholipids (PLs) with different fatty acids and polar headgroups on the development of obesity. High-fat-diet-fed mice were administrated with different kinds of PLs (2%,  In recent decades, obesity has become a growing public health problem worldwide . ImportaA typical diet contains approximately 2–8 g of phospholipids (PLs), of which 10–40% (or 0.8 g) are egg-derived PLs . StructuAlthough PS is less abundant than PC is in dietary intake, there have been several studies regarding the neuroprotective effect of PS ,16. Recep < 0.01). This decline may be attributed to the excessive body fat of the mice in the model group. After the administration of different phospholipids, there was an increasing trend in the muscle index of mice. Specifically, the muscle index significantly increased in the Soy-PS group and the EPA-PS group. Moreover, the high-fat diet increased lipid accumulation in white adipose tissues, including visceral white adipose tissue (VAT) and subcutaneous white adipose tissue (SAT), which further confirmed the success of the obesity model   . There wp < 0.01). Dietary phospholipids reduced the serum level of TNF-α compared to that of the model group. Moreover, the mice in the Egg-PS group, Soy-PS group, DHA phospholipid group, and EPA-PS group had significantly lower levels of TNF-α than those in the model group. With the exception of Egg-PC, dietary phospholipids also significantly decreased the MCP-1 level compared to that in the model group.The inflammatory cytokines were measured in the serum. Pparα and Acox1 among the phospholipid groups and the model group. However, compared to the model group, EPA-PE and EPA-PS significantly upregulated the expression of Cd36 .The observations are summarized in In the current study, we compared the anti-obesity effects of 12 kinds of phospholipids with varying fatty acids and polar headgroups and linked the underlying mechanisms to chronic inflammation and lipid metabolism. Among all phospholipids, EPA-enriched phospholipids and PS were the most effective in reversing HFD-induced obesity and hyperlipidemia in mice. These improvements were accompanied by an increase in fatty acid β-oxidation and a decrease in lipid synthesis. Moreover, DHA-enriched phospholipid groups, along with PS groups, exhibited the strongest anti-inflammatory effects compared to other phospholipid groups.The present study revealed varying degrees of weight loss in mice across different phospholipid groups, indicating variations in the anti-obesity effects of these phospholipids. The health effects of the phospholipids are highly correlated with their fatty acid compositions. Shirouchi et al. compared the effects of egg-PC and phosphatidylcholine enriched with n-3 PUFAs (n3-PC) using OLETF rats . The finObesity is characterized by chronic low-grade inflammation, and adipose tissue is known to release many inflammatory factors . Obese iAcox1 and Cpt1 [Cpt1a, Cpt2, and Acaa1a) compared to other types of phospholipids. Previous studies have also found that EPA is a stronger activator of PPARα compared to alpha-linolenic acid (C18:3) [The liver plays a key role in lipid metabolism. When energy intake increases, there is a disruption in hepatic lipid metabolism, resulting in the inhibition of lipoprotein secretion and abnormal lipid accumulation in the liver . Our preand Cpt1 . Moreoveand Cpt1 . Differe (C18:3) . The str (C18:3) .Several studies have investigated the activation of PPARα by specific phospholipid species, including phosphatidylcholines PC 16:0/18:1) or PC (18:0/18:1) . Althoug:1 or PC Srebp1c, known for its role in regulating fatty acid synthesis, is a target of PUFAs control in the liver [Srebp1c and its downstream genes involved in fatty acid synthesis [Fas, Acc, and other lipogenic target genes through the post-transcriptional regulation of SREBP-1 [Fas, Scd1, and Acc), suggesting that PS has a larger effect on inhibiting the expression of genes involved in lipogenesis.The nuclear transcription factor he liver . Numerouynthesis ,35. Our ynthesis . The inf SREBP-1 ,36. AddiCucumaria frondosa) and squid (Illex argentinus) roe, respectively, and the preparation procedures were performed as described in the literature [EPA- and DHA-PC were obtained from sea cucumber . Experimental groupings were as follows: the control group was fed a standard diet, the model group was fed a high-fat diet (HFD), and the PL groups were fed a HFD plus 2% of the corresponding PL. The formulations of the experimental feeds are shown in g for 15 min at 4 °C. Livers, adipose tissues, muscle, and other tissues were harvested from these mice, and stored at −80 °C for further analysis.All aspects of the animal care and experimental protocols were approved by the Ethical Committee of the College of Food Science and Engineering of the Ocean University of China. Six-week-old male C57BL/6J mice were purchased from Vital River Laboratory Animal Technology Co. . After an acclimatization period of one-week, mice were randomly divided into 14 groups , triglyceride (TG), LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) levels were determined using commercial kits . Serum concentrations of tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP-1) were measured using commercial ELISA kits .Total hepatic RNA was extracted using Trizol Reagent  following the supplier’s instructions. RNA was reversed to cDNA using a random primer . The target genes were amplified using SYBR Green I Master Mix  in an iCycler iQ5 system  with specific primers. The thermal conditions were as follows: 1 cycle of 95 °C for 10 min, 45 cycles of 95 °C for 15 s, 55–60 °C for 20 s, and 72 °C for 30 s. The primer list is given in Hepatic samples were lysed in RIPA lysis buffer  following the manufacturers’ instructions. The protein concentrations were determined using a BCA Protein Assay kit . Proteins were separated on 8% acryl amide gels and transferred to a PVDF membrane . Membranes were blocked for 2 h at RT in a 5% nonfat milk solution in Tris-buffered saline containing 0.5% Tween 20, and incubated with antibodies for 2 h. HRP detection was carried out using ECL plus reagent  in accordance with the manufacturer’s instructions. Western blotting was used to determine the expression of FAS ) and ACC  in the liver. Protein loading was evaluated using β-actin antibody ). The protein bands were quantified via band intensity and band area.The pGMPPAR-Lu reporter plasmid was obtained from Genomeditech Co., Ltd. . Human embryonic kidney (HEK) 293 cells were seeded on a 24-well plate and transfected with 450 ng of pGMPPAR-Lu and 50 ng of pRL-TK per well. After 24 h of transfection, the cells were treated with EPA-PC/PE/PS and DHA-PC/PE/PS  for 24 h. Following cell lysis, luciferase activities were analyzed using Dual-Luciferase Reporter Assay System  in accordance with the manufacturer’s recommended procedure.p-value less than 0.05. Graphs were generated using the Prism 5 software .Data were presented as the mean ± SEM. A two-tailed t-test was used to compare two groups, and one-way ANOVA with a post hoc Tukey test was used to compare multiple groups (SPSS version 19.0). A statistically significant result was defined as a In conclusion, the present study demonstrates that dietary PLs were capable of ameliorating weight loss and fat accumulation in HFD-induced mice. Among the phospholipids with different fatty acid compositions, EPA-PLs showed more pronounced benefits in terms of weight loss. Furthermore, the anti-obesity and lipid-lowering effects of PS were superior to those of PC and PE. Mechanistic investigation revealed that EPA-PS reduced fat storage by activating PPARα and regulating genes related to lipid metabolism. The novelty in this work is that we investigated the anti-obesity effects of multiple phospholipids that vary in acyl-chain and polar headgroup composition in one study. Compared to soy phospholipids and egg phospholipids, marine phospholipids have a more unique fatty acid composition. These phospholipids, which contain specific fatty acids, such as EPA and DHA, could have a more significant impact on human health. Moreover, phospholipids products such as soy lecithin are a mixture of various phospholipids, and the production of specific phospholipid species  deserves further attention.


Potentilla chinensis on TNF-α-induced ROS and MMP-1 production were explored. Consequently, we verified the skin-protective effects of these flavonol and flavone glucuronides, including potentilloside A, from P. chinensis, and conducted a structure–activity relationship analysis as part of our ongoing research. We investigated the protective effects of the extract and its 11 isolates on TNF-α-stimulated normal human dermal fibroblasts (NHDFs). Ten flavonol and flavone glucuronides significantly inhibited ROS generation (except for 7) and suppressed MMP-1 secretion, except for 2. In contrast, six isolates  showed a significant reverse effect on COLIA1 secretion. Comparing the three experimental results of each isolate, potentilloside A (1) showed the most potent skin cell-protective effect among the isolates. Evaluation of the signaling pathway of potentilloside A in TNF-α-stimulated NHDF revealed that potentilloside A inhibits the phosphorylation of ERK, JNK, and c-Jun. Taken together, these results suggest that compounds isolated from P. chinensis, especially potentilloside A, can be used to inhibit skin damage, including aging.Skin aging is a complex biological process influenced by a variety of factors, including UV radiation. UV radiation accelerates collagen degradation via the production of reactive oxygen species (ROS) and cytokines, including TNF-α. In a prior investigation, the inhibitory properties of flavonol and flavone glucuronides derived from  Skin aging is a complex biological process that is influenced by several factors. Aging is divided into two types: intrinsic and extrinsic aging. Intrinsic aging is programmed aging caused by interior factors in the human body. Extrinsic aging is caused by environmental factors, such as smoking, pollution, and UV radiation . UV radiUV radiation exposure triggers the release of tumor necrosis factor-α (TNF-α), a cytokine involved in the inflammatory response of the skin. TNF-α exacerbates skin inflammation and plays a role in the aging process. UV radiation induces the production of reactive oxygen species (ROS) in skin cells. ROS are highly reactive molecules that contribute to oxidative stress and are significant factors in skin aging ,4. A posBoth ROS and TNF-α activate matrix metalloproteinases (MMPs). MMPs, particularly MMP-1, are enzymes responsible for collagen degradation within the ECM. Collagen breakdown leads to visible skin-aging effects. Consequently, MMP-1 and proinflammatory cytokines also play a role in age-related skin conditions, such as psoriasis, acne vulgaris, and atopic dermatitis ,8,9. ROSHence, the inhibition of oxidative stress and TNF-α activity holds significant potential as a promising approach for the creation of novel therapeutic agents aimed at enhancing skin aging or managing inflammatory skin conditions.Potentilla chinensis is widespread throughout East Asia, particularly in China and Korea [The genus Potentilla contains approximately 500 known species, some of which are associated with the Latin word “potens”, which means “strong”. nd Korea . Previound Korea .P. chinensis leaves and discovered their potential biological activity. All isolates were tested for the inhibition of TNF-α-induced ROS at each concentration, with no effect on cell proliferation or inhibition, and the isolates showed a significant inhibitory effect. Among the isolates, four flavonol-bis-glucuronides, which are rare in nature and whose pharmacological activity is not well known, were tested for their MMP-1 inhibitory effect, and potentilloside A and quercetin-bis-3,7-O-β-d-glucuronide showed significant inhibition [In a previous study, we isolated and identified 11 compounds, including the new compound potentilloside A, from hibition . Thus, tP. chinensis and to analyze the structure–activity relationship. In addition, we will identify the compounds with the strongest effects and propose them as representative active ingredients through mechanistic studies.In this study, as part of ongoing research, we aimed to determine the skin-protective effects of flavonol and flavone glucuronides from v/v)  and 100 U/mL of antibiotics  at 37 °C and incubated in a 5% CO2 atmosphere; the cells within passage numbers 6–10 were used for subsequent experiments.Normal human dermal fibroblasts (NHDFs) were acquired from PromoCell GmbH . NHDFs were cultured in DMEM  containing FBS . The indicated concentrations of the extract and all the isolates were treated for 24 h. A cell viability assay was conducted using the Ez-Cytox solution. Briefly, 10% Ez-Cytox solution was added into each well and reacted at 37 °C for 1 h. Viability was measured using a microplate reader at 450 nm.NHDFs were plated in 96-well flat-bottomed microplates (5 × 104 cells/well). NHDFs were pre-treated with the indicated concentrations of isolates for 1 h. Next, the cells were treated with 20 ng/mL TNF-α and 10 μM DCF-DA  for 15 min in 48-well plates. Next, the medium was removed, and the cells were washed with DPBS . Cells were stained with DCFDA and visualized under a fluorescence microscope . To quantify intracellular ROS generation levels, cells were seeded on a 96-well black plate (5 × 103 cells/well) and treated with the concentrations mentioned above. Relative fluorescence intensities were measured using a microplate reader  at excitation and emission wavelengths of 485/535 nm. The levels of intracellular ROS were expressed as fold changes.NHDFs were plated in 48-well flat-bottomed microplates (1 × 104 cells/well) for 24 h in DMEM. After 24 h, the cells were starved overnight in serum-free DMEM. Eleven isolates and extract were added at the indicated concentrations  and incubated for 1 h. TNF-α was then added for 24 h to measure MMP-1 and COLIA1 secretion. Commercial ELISA kits  were used to quantify MMP-1 and COLIA1 concentrations, according to the manufacturer’s protocol. The absorbance at 450 nm was measured using a microplate reader.NHDFs were cultured in 48-well plates (2 × 105 cells/well) in complete DMEM and incubated for 24 h . After incubation, cells were treated with 1 at concentrations of 10, 30, and 100 µM for 1 h. After 1 h, the cells were treated with 20 ng/mL of TNF- for 24 h. After 24 h, the cells were lysed by scraping with RIPA buffer to collect the proteins. The mixture was sonicated and centrifuged at 14,000 rpm for 10 min at 4 °C. The protein concentration was determined using BCA protein method. Proteins were separated using a 4–20% premade protein gel (SDS-PAGE) and transferred to PVDF membranes . Membranes were blocked with 5% nonfat milk in Tris-buffered Tween20 for 1 h and incubated with primary antibodies  overnight at 4 °C. Membranes were re-incubated with HRP-conjugated secondary antibody  for 1 h. Membranes were developed using an enhanced chemiluminescence (ECL) plus reagent .NHDFs were incubated in 6-well microplates (1.53 × 10p < 0.05.Data are expressed as means ± standard deviation (SD). Statistical significance was determined via one-way analysis of variance (ANOVA) using GraphPad Prism version 9. Tukey’s multiple comparison test was used to assess the differences between groups, with statistical significance set at P. chinensis were collected in August at Evada Botanical Garden (GPS coordinates: X: 126.926768 and Y: 37.2482236) and dried for extraction. Four flavonol-di-glucuronides (1 and 3–5), five flavonol-mono-glucuronides (2 and 8–11), and two flavone-mono-glucuronides (6 and 7) were isolated from the 30% ethanol extract of P. chinensis leaves , quercetin-3′-O-β-d-glucuronide (2), quercetin-di-3,7-O-β-d-glucuronide (3), isorhamnetin-di-3,7-O-β-d-glucuronide (4), kaempferol-di-3,7-O-β-d-glucuronide (5), luteolin-7-O-β-d-glucuronide (6), apigenin-7-O-β-d-glucuronide (7), quercetin-3-O-β-d-glucuronide (8), isorhamnetin-3-O-β-d-glucuronide (9), kaempferol-3-O-β-d-glucuronide (10), and quercetin-3-O-β-d-glucuronide-6″-methyl ester (11).The chemical structures were determined by comparing their spectral data with those reported in the literature as quercetin-di-3,3′-To evaluate the protective effects of the extract and isolates on TNF-α-induced NHDFs in the non-cytotoxic range, a viability assay was conducted. In the concentration range of 1–100 μM, the extract and isolates did not show remarkable cytotoxicity toward NHDFs .1 significantly inhibited the ROS generation at all tested concentrations (1–100 μM) to 1.32 ± 0.02-fold (p < 0.001), 1.27 ± 0.08-fold (p < 0.001), 1.25 ± 0.15-fold (p < 0.001), 1.06 ± 0.29-fold (p < 0.001), and 0.88 ± 0.16-fold (p < 0.001) compared to the TNF-α-treated group . Additionally, compound 8 significantly decreased ROS generation at all treatment dosages compared with that of the TNF-α-treated group. In addition, compound 2 diminished ROS generation to 0.96 ± 0.23-fold (p < 0.001) and 0.82 ± 0.08-fold (p < 0.001) at 30 and 100 μM, respectively. Compound 10 decreased ROS generation to 0.93 ± 0.04-fold (p < 0.001) and 0.80 ± 0.11-fold (p < 0.001) at 30 and 100 μM, respectively.Next, we determined the inhibitory effect of the extract and isolates on ROS generation in TNF-α-induced NHDFs at the same concentrations  in 2, significantly inhibited the MMP-1 secretion at 100 μM. The extract caused significant concentration-dependent suppression at all concentrations. Treatment with 30 and 100 μM of compound 1 dramatically (potentilloside A) decreased MMP-1 secretion by 1.96 ± 0.14-fold (p < 0.001) and 0.56 ± 0.05-fold (p < 0.001), respectively, compared to the TNF-α group . Additionally, compounds 5 and 9 significantly decreased MMP-1 secretion at all tested treatment concentrations. Furthermore, compounds 7 and 10 significantly inhibited MMP-1 secretion at 100 μM to 0.45 ± 0.08-fold (p < 0.001) and 1.09 ± 0.06-fold (p < 0.001), respectively.ELISA was conducted to determine the inhibitory effects of the extract and isolates on MMP-1 secretion in TNF-α-treated NHDFs. As shown in 1, 4, 5, 6, 9, 10, and 11) significantly increased COLIA1 secretion at different concentrations. However, the extract did not show an increase compared to that in the TNF-α-treated group. Compound 1 at 3, 10, and 30 µM potently increased COLIA1 secretion to 0.94 ± 0.02-fold (p < 0.001), 0.86 ± 0.09-fold (p < 0.001), and 0.77 ± 0.04-fold (p < 0.01), respectively, compared to the TNF-α-treated group . In particular, 100 µM of compound 5 almost recovered the COLIA1 secretion by 0.96 ± 0.05-fold (p < 0.001).Next, we evaluated COLIA1 secretion in the TNF-α-treated condition. As shown in A spider chart was used to evaluate the following three factors: inhibition of MMP-1 secretion, inhibition of ROS generation, and stimulation of COLIA1 expression. The rates of inhibition or increase at each concentration were calculated by converting the difference between the TNF-α-treated group and the untreated group to 100%. These were then assigned as follows: 0 (x < 20%), 1 (20 ≤ x < 40%), 2 (40 ≤ x < 60%), 3 (60 ≤ x < 80%), and 4 (80 ≤ x < 100%). The scores for the three experiments were then summed up. The total scores were divided into three ranges: 0 (y ≤ 4), 1 (5 ≤ y ≤ 9), 2 (10 ≤ y ≤ 14), and 3 (15 ≤ y).1 had the highest overall score, followed by compounds 5, 9, and 10, respectively. Notably, compounds 1, 5, 7, 9, 10, and 11 exhibited MMP-1 suppression. Among the flavonols with a glucuronide moiety at position 3, compounds 1 and 5 obtained the highest scores for stimulating COLIA1, followed by 9, 10, and 11. All compounds, except compound 7, demonstrated substantial ROS-inhibiting activity. Consequently, according to the spider chart evaluation, potentilloside A (1) was identified as the most effective compound.As shown in 1) on intracellular ROS generation in TNF-α-induced NHDFs, the cells were visualized using a DCFDA staining assay. As shown in 1) conspicuously inhibited fluorescence, indicating ROS generation, compared to that of the TNF-α-treated group.To clarify the inhibitory effect of potentilloside A (1) plays a critical role in TNF-α-induced MAPK phosphorylation in NHDFs to analyze the MAPK biomarkers. As shown in p < 0.001). In contrast, treatments of 10, 30, and 100 μM potentilloside A (1) significantly decreased the phosphorylation of ERK to 1.21 ± 0.08-fold (p < 0.01), 1.02 ± 0.05-fold (p < 0.001), and 0.62 ± 0.03-fold (p < 0.001), respectively. TNF-α treatment increased JNK phosphorylation by 1.46 ± 0.04-fold (p < 0.001), and 30 and 100 μM potentilloside A (1) markedly decreased it by 1.12 ± 0.03-fold (p < 0.001) and 0.37 ± 0.01-fold (p < 0.001), respectively. However, potentilloside A (1) failed to inhibit p38 phosphorylation.Western blotting was performed to ascertain whether potentilloside A (1) in TNF-α-treated NHDF, we examined the expression levels of proteins in TNFα-stimulated intracellular pathways, NF-κB and c-Jun. As shown in 1 did not decrease NF-κB phosphorylation compared with that of the TNF-α-treated group. TNF-α treatment increased c-Jun phosphorylation by 4.12 ± 0.13-fold (p < 0.001), and treatment with 100 μM potentilloside A (1) significantly decreased it by 3.28 ± 0.08-fold (p < 0.001).To explore the possible protective mechanism of potentilloside A  and 3-O-glucuronide structures (8 and 9). Furthermore, through a comparison of the di-glucuronide and mono-glucuronide structures, it was demonstrated that the inhibitory effect increased when the glucuronide was attached to the C-3 only, rather than when it was attached to both the C-3 and C-7. As a result, it was confirmed that the inhibitory effect increased when the functional group was not attached to position 3′  and quercetin (hydroxy at position 3′), quercetin was found to be more active than isorhamnetin in the 3,7-di-glucuronide , which exhibited a higher inhibitory effect on MMP-1 secretion than quercetin-3-O-glucuronide (8).However, there was an exception to this trend for quercetin-3,3′-1, 5, and 11 exhibited significant effects at concentrations of 1, 3, 10, and 30 μM, with compound 1 showing the highest efficacy among them. Additionally, compounds 5, 6, and 9 induced a notable increase in COLIA1 secretion at a concentration of 100 μM. Although no specific structural requirements were observed for the increased secretion of COLIA1, compound 1 was confirmed to be an effective agent. Therefore, potentilloside A (1) exhibited the most effective reduction in MMP-1 secretion and simultaneous increase in COLIA1 secretion. However, further investigations with a larger number of samples are needed to provide evidence of the structure–activity relationship.In the evaluation of increased COLIA1 secretion, compounds O-α-l-rhamnopyranosyl-(1″′ → 6″)-β-d-galactopyranoside, hyperin, afzelin, and cryptochlorogenic acid methyl ester were shown to inhibit MMP-1 expression in UVB-irradiated human dermal fibroblasts (WS-1 cells). These three compounds were found to increase the secretion of pro-collagen type 1. Among these, hyperin was shown to reduce ROS generation [P. chinensis isolates can prevent aging by inhibiting MMP-1, ROS generation, and collagen degradation.Potentilloside A was found to be the most effective isolate among the others. Previous studies were conducted to identify the signaling pathways involved. Similar to our results, flavonoid compounds were also shown to suppress ROS generation and MMP-1 expression. Additionally, flavonoid compounds have been shown to increase COLIA1 expression in human skin cells. Hesperidin and hesperetin were shown to inhibit MMP-1 production in physiologically aged NHDFs . In addineration . These p1 inhibited ERK and JNK phosphorylation in TNF-α-treated NHDFs. ROS upregulate NF-κB phosphorylation in human dermal fibroblasts and keratinocytes [1) did not inhibit NF-κB phosphorylation in the TNF-α-treated NHDFs. These results demonstrate that potentilloside A (1) inhibits MMP-1 expression and secretion by inhibiting c-Jun phosphorylation via decreasing ERK and JNK phosphorylation.TNF-α-induced ROS generation downregulates several signaling pathways, including MAPKs and NF-κB . MAPKs rinocytes ,36. In tinocytes . Figure 8) was reported to have skin-protective properties through the NF-κB and AP-1 pathways in human keratinocytes and melanoma cells [Similar to our results, flavonoids were shown to inhibit ROS generation, MMP-1, and increase COLIA1 via MAPK/AP-1 or NF-κB in human skin cells. Quercetin-3-glucuronide  exhibited stronger inhibitory effects on MMP-1 secretion and increased collagen secretion in comparison to quercetin-3-glucuronide (8). Additional in vivo studies are required to fully understand the activity of P. chinensis leaf extracts and potentilloside A as an active ingredient. Nevertheless, our study demonstrates that potentilloside A is a representative active ingredient in P. chinensis leaves that inhibits skin damage, including aging. Furthermore, potentilloside A not only holds potential as a candidate for addressing skin aging but also shows potential applications in other inflammatory skin diseases such as atopic dermatitis.Consequently, potentilloside A has a potential protective activity against NHDF cell damage by decreasing MMP-1 and ROS generation and increasing COLIA1 by inhibiting the phosphorylation of MAPK/AP-1. In particular, potentilloside A . Comparing the three experimental results of each isolate, potentilloside A (1) showed the strongest skin cell protection effect among the isolates. Furthermore, potentilloside A suppressed the phosphorylation of MAPKs (ERK and JNK) and c-Jun. Thus, potentilloside A suppressed NHDF damage by regulating oxidative stress and collagenase MMP-1 activity under TNF-α-stimulated conditions. Although further investigations are required to understand the impact of potentilloside A, it nevertheless represents a candidate compound with the potential to mitigate skin damage.The protective effects of flavonol and flavone glucuronides from


Allergic disease is common. The aim of this study was to look at the change in asthma and rhinitis over time and to characterise factors contributing to remission and persistence of disease.ENO), eosinophil cationic protein (ECP) and eosinophil‐derived neurotoxin (EDN).This cohort study included 255 individuals with or without asthma and or rhinitis that participated in a population survey and a follow‐up 10 years later. The participants were tested for allergic sensitisation, total IgE, multiplex allergen component analysis and type‐2 inflammatory markers: exhaled nitric oxide , compared with those that remained healthy. Subjects with persistent rhinitis were more likely to be sensitised to grass pollen and had higher total IgE levels than those that went into remission. Individuals with persistent asthma were more likely to be sensitised to tree pollen and furry animals than those with only persistent rhinitis .Of the 132 healthy individuals, 112 remained healthy, 16 developed rhinitis, 4 asthma and rhinitis over the 10 years. Out of 82 subjects with rhinitis, 26 went into remission, 53 remained unchanged and 3 developed asthma in addition to rhinitis. None of the 41 participants with asthma and rhinitis went into remission. Subjects with persistent rhinitis and asthma had higher levels of total IgE  at baseline and after 10 years, and FIgE sensitisation and total IgE levels are associated with the persistence of rhinitis and asthma. Participants with persistent allergic disease had higher levels of allergen sensitisation and type 2 inflammation markers at baseline than those who remained healthy. Higher total immunoglobulin E (IgE) levels have previously been associated with elevated levels of positive specific IgE tests as well as allergic disease and multimorbidity.The aim of this study was to look at the change in allergic disease over time and to better characterise factors contributing to remission or persistent disease with the help of background factors, lung function, allergic sensitisation and type‐2 inflammation markers.22.1The study was based on participants from the European Community Respiratory Health Survey (ECRHS), a multicentre study with three visits: 1991–1992, 1999–2000 and 2011–2012. The study design of ECRHS I, II and III has been published before.2.2http://www.ecrhs.org)The ECRHS II and ECRHS III main questionnaires .2.4Total serum IgE for both ECRHS II and III was measured using the Pharmacia CAP system . Total IgE was available for 470 individuals in ECRHS II and 351 in ECRHS III. Of the participants in our study 248 had results available in ECRHS II and 217 in ECHRS III.ENO measurements were made using an exhalation flow rate of 50 ml/s.ENO was measured using an electrochemical analyser .ENO was measured in 288 people in ECRHS II and 361 in ECRHS III. In the current study, 156 participants had available results in ECRHS II and 223 in ECRHS III.FBlood samples were collected for ECP. Samples were kept at 24°C for 60 min before centrifugation. In ECRHS II, the concentration of serum ECP was assayed with Pharmacia ECP RIA (Pharmacia Diagnostics) and in ECRHS III, with ImmunoCAP ECP (Thermo Fischer Scientific). ECRHS III also included blood samples for serum EDN, which were measured using an ImmunoCAP research assay.2.51) was measured in ECRHS II using a dry rolling‐seal spirometer . Up to five technically acceptable manoeuvres were measured. American Thoracic Society recommendations were followed.1 were calculated on the basis of European Coal and Steel Union reference values.Forced expiratory volume in one second , and analysis of variance (ANOVA) with Bonferroni correction were used when comparing groups. Logistic regression and multinominal logistic regression were used in the multivariable analysis. A p‐value of <0.05 was considered statistically significant.Statistical analyses were performed using STATA . Non‐normally distributed variables, F2.7The study was approved by the Regional Ethical Review Board in Uppsala, Sweden . Informed consent was obtained from all individual participants included in the study.3Over 10 years, comparing ECRHS II and ECRHS III, we found that of the 132 healthy individuals at baseline, 112 remained healthy, 16 developed rhinitis and 4 asthma and rhinitis  that also provided financial support for this study. Kjell Alving received research material from Hemocue AB, Thermo Fisker Scientific, NIOX Group Plc. No other author has reported any conflict of interest.Supporting Information S1Click here for additional data file.Supporting Information S1Click here for additional data file.


This Special Issue compiles papers submitted by the Editorial Board Members of the Vehicular Sensing Section and outstanding scholars in this field. Our purpose is to present papers that typify the most insightful and influential original research and review the field’s key topics.The scope of the Special Issue is quite broad, covering all ground transportation (such as road and rail), water transportation and air transportation. The presented topics range from physical models and components to advanced control systems for automated vehicles, including driver monitoring, comfort considerations, communication and cooperative systems, perception sensors and solutions to issues related to electric vehicles.This Special Issue includes sixteen papers. The first and most extensive group focuses on road vehicles, and its papers can be classified into a number of subgroups.First, Refs. ,2,3 presA relevant issue regarding autonomous vehicles is the behavior of the driver when the road scenario causes a change in vehicle control. Ref.  presentsThe development and deployment of electric vehicles involve several considerations relating to conventional vehicles and traffic, and some uncertainties are proposed in ,6. For eFurthermore, electric vehicle charging demand and charging station availability forecast challenging problems that could hinder the efficient deployment of these vehicles. Many existing deep learning methods have been proposed to address this issue; however, due to complex road network structures and complex external factors, commonly used algorithms can only extract information on the historical use of roads. To enhance prediction accuracy and interpretability, Ref.  proposesA particularly relevant subgroup of papers focused on vehicular communications in order to provide additional information about the transportation system. A comprehensive review of connected vehicle technologies is provided in , includiFurthermore, the authors of  developeTwo rail transportation issues are discussed in ,12. Ref.Meanwhile, Ref.  presentsThe second group of papers is related to air transportation and focuses on unmanned aerial vehicles. A vision-based guidance technique with a log polynomial closing velocity controller is presented in . The goaFinally, the final group of papers considers water applications. Ref.  provides