Effect of vitamin D supplementation on total and allergen-specific IgE in children with asthma and low vitamin D levels.

BACKGROUND: Observational studies have yielded inconsistent findings for the relation between vitamin D level and total IgE or allergic sensitization. OBJECTIVE: To determine whether vitamin D supplementation reduces levels of total IgE and IgE to each of 2 common indoor allergens in children with asthma and low vitamin D levels. METHODS: Total IgE, IgE to Dermatophagoides pteronyssinus, and IgE to Blattella germanica were measured at the randomization and exit visits for 174 participants in the Vitamin D Kids Asthma Study, a multicenter, double-blind, randomized placebo-controlled trial of vitamin D3 supplementation (4000 IU/d) to prevent severe exacerbations in children with persistent asthma and vitamin D levels less than 30 ng/mL. Multivariable linear regression was used for the analysis of the effect of vitamin D supplementation on change in each IgE measure. RESULTS: Participants were followed for an average of 316 days. At the exit visit, more subjects in the vitamin D arm achieved a vitamin D level equal to or more than 30 ng/mL compared with those in the placebo arm (87% vs 30%; P < .001). In a multivariable analysis, vitamin D3 supplementation had no significant effect on change in total IgE, IgE to Dermatophagoides pteronyssinus, or IgE to Blattella germanica between the exit and randomization visits (eg, for log10 total IgE, ß = 0.007; 95% CI, -0.061 to 0.074; P = .85). CONCLUSIONS: Vitamin D supplementation, compared with placebo, has no significant effect on serum levels of total IgE, IgE to dust mite, or IgE to cockroach in children with asthma and low vitamin D levels.

Alpinia officinarum water extract inhibits the atopic dermatitis-like responses in NC/Nga mice by regulation of inflammatory chemokine production.

Alpinia officinarum (AO) has been traditionally used in Asia as an herbal medicine to treat inflammatory and internal diseases. However, the therapeutic effect of AO on atopic dermatitis (AD) is unclear. Therefore, we examined whether Alpinia officinarum water extract (AOWex) affects AD in vivo and in vitro. Oral administration of AOWex to NC/Nga mice with Dermatophagoies farina extract (DfE)-induced AD-like symptoms significantly reduced the severity of clinical dermatitis, epidermal thickness, and mast cell infiltration into the skin and ear tissue. Decreased total serum IgE, macrophage-derived chemokine (MDC), and regulated on activation, normal T-cell expressed and secreted (RANTES) levels were observed in DfE-induced NC/Nga mice in the AOWex-treated group. These effects were confirmed in vitro using HaCaT cells. Treatment with AOWex inhibited the expression of proinflammatory chemokines such as MDC, RANTES, IP-10 and I-TAC in interferon-γ and tumor necrosis factor-α-stimulated HaCaT cells. The anti-inflammatory effects of AOWex were due to its inhibitory action on MAPK phosphorylation (ERK and JNK), NF-κB, and STAT1. Furthermore, galangin, protocatechuic acid, and epicatechin from AOWex were identified as candidate anti-AD compounds. These results suggest that AOWex exerts therapeutic effects against AD by alleviating AD-like skin lesions, suppressing inflammatory mediators, and inhibiting major signaling molecules.

Evaluation on two types of paramyosin vaccines for the control of Haemaphysalis longicornis infestations in rabbits.

BACKGROUND: Haemaphysalis longicornis is an obligate hematophagous ectoparasite that transmits a variety of pathogens causing life-threatening diseases in humans and animals. Paramyosin (Pmy) is not only an invertebrate-specific myofibrillar protein but also an important immunomodulatory protein. Therefore, it is one of the ideal candidate antigens for vaccines. METHODS: We conducted two vaccine trials to evaluate the protective efficacy of Pmy recombinant protein (rPmy) and peptide vaccine (KLH-LEE). Each rabbit was immunized with three doses of rPmy or KLH-LEE adjuvanted with Freund's complete/incomplete at 500 µg/dose at 2-week intervals before challenge with 40 female H. longicornis/rabbit. PBS plus adjuvant, Trx or KLH was used as control group. The antibodies of rabbits were detected by ELISA. Then, female ticks were fed on the rabbits until detachment. RESULTS: ELISA results showed that both vaccines induced rabbits to produce antibodies. Compared with the Trx group, the engorgement weight, oviposition and hatchability of the rPmy group decreased by 8.87%, 26.83% and 38.86%, respectively. On the other hand, engorgement weight, oviposition and hatchability of female ticks in the KLH-LEE group correspondingly resulted in 27.03%, 53.15% and 38.40% reduction compared with that of the KLH group. Considering the cumulative effect of vaccination on the evaluated parameters, results showed 60.37% efficacy of the rPmy vaccine formulation and 70.86% efficacy in the KLH-LEE group. CONCLUSIONS: Pmy and particularly epitope LEE have potential for further development of an effective candidate vaccine to protect the host against tick infection. GRAPHIC ABSTARCT.

Interference of Dermatophagoides pteronyssinus sensitization in grass pollen allergy.

Summary: Background. Climate conditions in the northwest of Spain are from the rest of the country, and the pollen sensitisation rates and allergens involved are different. The present study aimed to investigate the sensitisation profile of patients with grass pollen allergy and the interference of other sensitisations in respiratory symptoms. Methods. A total of 959 Spanish patients with seasonal respiratory symptoms and a positive skin prick test (SPT) to Phleum pratense pollen were studied. Patients were classified as having rhinitis and/or bronchial asthma. A battery of SPTs, including common weeds and tree pollens, profilin, polcalcin, moulds, Dermatophagoides pteronyssinus, Lepidoglyphus destructor, and cat and dog dander were performed. Serum specific IgE (sIgE) to Phl p 1 and Phl p 5, adding sIgE to Phl p 7, Phl p 12 and house dust mites (HDMs) or other pollens in selected cases were measured. Results.The majority (89.8%) of the patients were polysensitised according to SPT. HDM co-sensitisation was the most prevalent (62.3%). Profilin and polcalcin rendered a positive result in 25.9% and 18.7% of the patients, respectively. A higher proportion of patients recognized sIgE to Phl p 1 (88.7%) with respect to Phl p 5 (59%). Phl p 1-sIgE levels were higher than Phl p 5-sIgE levels, and no differences were found in patients with rhinitis and/or asthma. However, total serum IgE was higher in patients with asthma. Multivariate regression analyses revealed that only sIgE to Dermatophagoides pteronyssinus (after adjusting by sIgE to Phl p 1, Phl p 5 and Lepidoglyphus destructor) was associated with a greater risk of asthma. Conclusions. Phl p 1 is the most relevant allergen in patients with grass pollen allergy in the northwest of Spain. Sensitisation rates against panallergens are low. Even in patients with grass pollen allergy, HDM sensitisation plays a relevant role in asthma.

Extracellular vesicles and post-translational protein deimination signatures in haemolymph of the American lobster (Homarus americanus).

The American lobster (Homarus americanus) is a commercially important crustacean with an unusual long life span up to 100 years and a comparative animal model of longevity. Therefore, research into its immune system and physiology is of considerable importance both for industry and comparative immunology studies. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family that catalyses post-translational protein deimination via the conversion of arginine to citrulline. This can lead to structural and functional protein changes, sometimes contributing to protein moonlighting, in health and disease. PADs also regulate the cellular release of extracellular vesicles (EVs), which is an important part of cellular communication, both in normal physiology and in immune responses. Hitherto, studies on EVs in Crustacea are limited and neither PADs nor associated protein deimination have been studied in a Crustacean species. The current study assessed EV and deimination signatures in haemolymph of the American lobster. Lobster EVs were found to be a poly-dispersed population in the 10-500 nm size range, with the majority of smaller EVs, which fell within 22-115 nm. In lobster haemolymph, 9 key immune and metabolic proteins were identified to be post-translationally deiminated, while further 41 deiminated protein hits were identified when searching against a Crustacean database. KEGG (Kyoto encyclopedia of genes and genomes) and GO (gene ontology) enrichment analysis of these deiminated proteins revealed KEGG and GO pathways relating to a number of immune, including anti-pathogenic (viral, bacterial, fungal) and host-pathogen interactions, as well as metabolic pathways, regulation of vesicle and exosome release, mitochondrial function, ATP generation, gene regulation, telomerase homeostasis and developmental processes. The characterisation of EVs, and post-translational deimination signatures, reported in lobster in the current study, and the first time in Crustacea, provides insights into protein moonlighting functions of both species-specific and phylogenetically conserved proteins and EV-mediated communication in this long-lived crustacean. The current study furthermore lays foundation for novel biomarker discovery for lobster aquaculture.

The feeding of dietary Codonopsis pilosula polysaccharide enhances the immune responses, the expression of immune-related genes and the growth performance of red swamp crayfish (Procambarus clarkii).

Polysaccharides have many functions in aquatic animals and are widely used as immunopotentiators. However, despite the emergence of serious diseases, few studies have explored the effects of Codonopsis pilosula polysaccharide (CPP) on crustaceans. We studied the effects of CPP on the growth performance, nonspecific immunity, antioxidant activity and disease resistance of red swamp crayfish (Procambarus clarkii). Healthy crayfish (5.80 ± 0.1 g) were fed diets supplemented with 0% (control), 0.05%, 0.1%, 0.15%, 0.20%, and 0.30% CPP for 8 weeks. At the end of the 8-week feeding trial, the optimal final body weight (FBW), weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) were observed in the crayfish fed the diets with 0.15% and 0.20% CPP, followed by those fed the diet with 0.30% CPP and then those fed the diet with 0.10% CPP, whereas the values of these parameters were obtained with the control crayfish (P < 0.05). The crayfish fed the diets with 0.15% and 0.20% CPP exhibited a significantly higher total hemocyte count (THC) and significantly increased phenoloxidase (PO), lysozyme (LZM), hemocyte (Hc), acid phosphatase (ACP) and alkaline phosphatase (AKP) compared with those belonging to the other groups (P < 0.05). The crayfish fed the diets with 0.15% and 0.2% CPP exhibited significantly higher total superoxide dismutase (T-SOD) and glutathione peroxidase (GPx) activities, a significantly increased total antioxidant capacity (T-AOC) and a significantly lower malondialdehyde (MDA) content compared with the other groups (P < 0.05), which indicated that antioxidant capacity was significantly induced by the CPP-supplemented diets. Significantly upregulated expression of immune-related genes (anti-lipopolysaccharide factors (alf), peroxiredoxin (prx5), cathepsin B (ctsb), mitochondrial manganese superoxide dismutase (mtMnsod), cyclophilin A (cypa), glutathione peroxidase (gpx), Toll-like receptor 3 (tlr3), and heat shock protein 70 (hsp70)) was detected in the crayfish fed the diets supplemented with 0.15% and 0.20% CPP diet compared with the levels observed in the control crayfish. These results showed that dietary CPP supplementation greatly improved the growth, immunity and antioxidant capacities of crayfish, and according to the observed results, 0.15%-0.2% is the recommended optimal level of CPP dietary supplementation for crayfish.

Oligosaccharides derived from dragon fruit modulate gut microbiota, reduce oxidative stress and stimulate toll-pathway related gene expression in freshwater crustacean Daphnia magna.

Dragon fruit oligosaccharide (DFO) is an indigestible prebiotic. In this study, we aimed to investigate the effects of DFO on gut microbiota, oxidative stress and immune-related gene expression in Daphnia magna. The 10-day-old D. magna were treated with 0, 9, and 27 mg l-1 DFO for 85 h. The gut bacterial communities, superoxide dismutase (SOD) activity, lipid peroxidation and the expressions of genes in Toll signaling pathway were observed. The results showed that D. magna treated with 9 and 27 mg l-1 DFO altered gut microbiota composition by increasing Limnohabitans and Lactobacillus, and significantly increased SOD activity and reduced lipid peroxidation. Moreover, the expressions of Toll2, Toll3, Toll5, Toll7 and Pelle genes were significantly increased in D. magna treated with 9 and 27 mg l-1 DFO. Our results suggested that DFO changed the composition of the gut microbiota of D. magna by increasing the beneficial bacteria. DFO also had the ability to stimulate innate immunity in D. magna by increasing SOD activity, reducing lipid peroxidation, and increasing the expression of immune-related genes.

Molecular profiling of allergen-specific antibody responses may enhance success of specific immunotherapy.

BACKGROUND: House dust mites (HDMs) are among the most important allergen sources containing many different allergenic molecules. Analysis of patients from a double-blind, placebo-controlled allergen-specific immunotherapy (AIT) study indicated that patients may benefit from AIT to different extents depending on their molecular sensitization profiles. OBJECTIVE: Our aim was to investigate in a real-life setting whether stratification of patients with HDM allergy according to molecular analysis may enhance AIT success. METHODS: Serum and nasal secretion samples from patients with HDM allergy (n = 24) (at baseline, 7, 15, 33, and 52 weeks) who had received 1 year of treatment with a well-defined subcutaneous AIT form (Alutard SQ 510) were tested for IgE and IgG reactivity to 15 microarrayed HDM allergen molecules with ImmunoCAP Immuno-solid-phase Allergen Chip technology. IgG subclass levels to allergens and peptides were determined by ELISA, and IgG blocking was assessed by basophil activation. In vitro parameters were related to reduction of symptoms determined by combined symptom medication score and visual analog scale score. RESULTS: Alutard SQ 510 induced protective IgG mainly against Dermatophagoides pteronyssinus (Der p) 1 and Der p 2 and to a lesser extent to Der p 23, but not to the other important allergens such as Der p 5, Der p 7, and Der p 21, showing better clinical efficacy in patients sensitized only to Der p 1 and/or Der p 2 as compared with patients having additional IgE specificities. CONCLUSION: Stratification of patients with HDM allergy according to molecular sensitization profiles and molecular monitoring of AIT-induced IgG responses may enhance the success of AIT.

Novel Mutant Phospholipase D from Hemiscorpius lepturus Acts as A Highly Immunogen in BALB/c Mice Against the Lethality of Scorpion Venom.

Hemiscorpius lepturus (H. lepturus) which belongs to the Scorpionidae family, is the deadliest scorpion in Iran. It causes pathological manifestations like dermonecrosis, hemolysis, renal failure, necrotic ulcers, and in some cases, even death. The venom of this scorpion is well-known for its cytotoxic effects in comparison with the other venomous scorpions which show significant neurotoxic effects. Due to the painless nature of the sting of this scorpion, the clinical symptoms occur in victims 24 to 72 h post-sting. In our previous studies during the last decade, we demonstrated that the medical complications are attributable to the presence of phospholipase D (PLD) as a major toxin in the venom. With the purpose of designing and constructing a vaccine against H. lepturus for humans, animal model experiments were performed. To achieve this goal, non-toxic PLD was developed by mutation of two critical catalytic residues-His12 and His48-into alanines and the product was then denominated mut-rPLD1. The in-vivo tests showed that the mice immunized with interval doses of 10 µg of mut-rPLD1, were completely protected against 10× the LD100 of the venom. In conclusion, this mutant may be an effective vaccine candidate against scorpion envenomation by H. lepturus in future clinical studies.

Phloroglucinol Treatment Induces Transgenerational Epigenetic Inherited Resistance Against Vibrio Infections and Thermal Stress in a Brine Shrimp (Artemia franciscana) Model.

Emerging, infectious diseases in shrimp like acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus and mortality caused by other Vibrio species such as Vibrio harveyi are worldwide related to huge economic losses in industrial shrimp production. As a strategy to prevent disease outbreaks, a plant-based phenolic compound could be used as a biocontrol agent. Here, using the brine shrimp (Artemia franciscana) as a model system, we showed that phloroglucinol treatment of the parental animals at early life stages resulted in transgenerational inherited increased resistance in their progeny against biotic stress, i.e., bacteria (V. parahaemolyticus AHPND strain and V. harveyi) and abiotic stress, i.e., lethal heat shock. Increased resistance was recorded in three subsequent generations. Innate immune-related gene expression profiles and potential epigenetic mechanisms were studied to discover the underlying protective mechanisms. Our results showed that phloroglucinol treatment of the brine shrimp parents significantly (P < 0.05) enhanced the expression of a core set of innate immune genes (DSCAM, proPO, PXN, HSP90, HSP70, and LGBP) in subsequent generations. We also demonstrated that epigenetic mechanisms such as DNA methylation, m6A RNA methylation, and histone acetylation and methylation (active chromatin marker i.e., H3K4Me3, H3K4me1, H3K27me1, H3 hyperacetylation, H3K14ac and repression marker, i.e., H3K27me3, H4 hypoacetylation) might play a role in regulation of gene expression leading toward the observed transgenerational inheritance of the resistant brine shrimp progenies. To our knowledge, this is the first report on transgenerational inheritance of a compound-induced robust protected phenotype in brine shrimp, particularly protected against AHPND caused by V. parahaemolyticus and vibriosis caused by V. harveyi. Results showed that epigenetic reprogramming is likely to play a role in the underlying mechanism.

Alginate from Sargassum siliquosum Simultaneously Stimulates Innate Immunity, Upregulates Immune Genes, and Enhances Resistance of Pacific White Shrimp (Litopenaeus vannamei) Against White Spot Syndrome Virus (WSSV).

Although alginate is known as an immunostimulant in shrimp, the comprehensive and simultaneous study on its activity to resolve the relationship of the hematological parameters, upregulation of immune-related gene expression, and resistance to pathogen has not been found in shrimp. We performed experiments to evaluate the effect and mechanism of alginate from S. siliquosum on Pacific white shrimp immune system. Hematological parameters were examined after oral administration of Na alginate in the shrimp. White spot syndrome virus (WSSV) was injected to the shrimp at 14 days, and its copy number was examined quantitatively (qRT-PCR). Immune-related gene expression was evaluated by qRT-PCR. Alginate increased some hematological immune parameters of shrimp. Before WSSV infection, expression levels of Toll and lectin genes were upregulated. The lectin gene were upregulated post infection, and the Toll gene in all the treatments were downregulated, except the shrimps fed with alginate at 6.0 g kg-1 at 48 h post infection (hpi). The shrimps fed with alginate at 6.0 g kg-1 were the most resistant and gave the least WSSV copy number at 48 hpi. Resistance of shrimps fed the alginate-supplemented diets against WSSV was significantly higher compared to that of the control treatment with 56% and 10% of survival rates, respectively. Oral administration of alginate did not affect the growth and total protein plasma. At 120 h post challenge, alginate treatment at 6.0 g kg-1 exhibited the highest survival rate. It is concluded that oral administration of alginate enhanced the innate immunity by upregulating immune-related gene expression. Consequently, the enhancement of the shrimp innate immunity improves the resistance against WSSV infection.

Involvement of a novel protein kinase C (nPKC) in immunocompetence in hemocytes of white shrimp, Litopenaeus vannamei.

Complementary (c)DNA encoding novel protein kinase C (PKC) messenger (m)RNA of the white shrimp Litopenaeus vannamei, consisted of 2454-bp cDNA containing an open reading frame (ORF) of 2232 bp, belonging to the novel (n)PKC family of proteins characterized by their containing two phorbol ester/diacylglycerol-binding domains (C1 domain), a C2 domain, and a catalytic domain of the serine/threonine kinase, designated LvnPKC. A comparison of amino acid sequences showed that LvnPKC was closely related to arthropod nPKC. LvnPKC cDNA was detected in all tested tissues with a real-time PCR including the hepatopancreas, gills, muscles, subcuticular epithelium, abdominal nerve, thoracic nerve, brain, the stomach, heart, and especially in hemocytes and the intestines. Moreover, significantly upregulated LvnPKC expression was only observed in the eyestalk, brain, and hepatopancreas of shrimp transferred from 28 °C to 18 °C for 30 min. Induction of LvnPKC expression in hemocytes of L. vannamei injected with Vibrio alginolyticus at 105 cfu shrimp-1 was detected earlier than in those injected with 103 cfu shrimp-1. Shrimp received LvnPKC-dsRNA for 1 days specifically depleted the expression of LvnPKC mRNA in hemocytes compared those of diethylpyrocarbonate water treatment. After that, significantly decreased expressions of lipopolysaccharide - and ß-1,3-glucan-binding protein, prophenoloxidase-activating enzyme, peroxinectin, prophenoloxidase I, and prophenoloxidase II in the prophenoloxidase-activating system; lysozyme and cytosolic manganese superoxide dismutase and mitochondrial manganese superoxide dismutase in the antioxidant system were observed. We therefore concluded that LvnPKC is involved in immune defense of L. vannamei exposed to hypothermal stress or infected with V. alginolyticus.

Dietary replacement of fish-meal impaired protein synthesis and immune response of juvenile Pacific white shrimp, Litopenaeus vannamei at low salinity.

An 8-week feeding trial was conducted to evaluate the effect of fish-meal replacement on growth performance, protein synthesis and immune response of juvenile Pacific white shrimp, Litopenaeus vannamei reared at low salinity (7‰). Five isonitrogenous and isolipidic diets were formulated to contain graded levels (25, 20, 15, 10 and 5%) of fish-meal. High quality alternative solutions were performed, crystalline amino acids, phytase, mannan oligosaccharides and some micro-nutrients were supplemented in the low fish-meal diets. Each diet was randomly assigned to triplicate tanks, each tank with 30 shrimp (mean weight 0.3 g), the shrimp were fed 3 times a day. Weight gain and survival were decreased with the decreasing dietary fish meal levels. When dietary fish-meal decreased, the gene expression of TOR, Raptor and eIF4E2 in hepatopancreas were decreased with the decreasing fish meal levels, eIF4E2 in intestine was decreased while 4E-BP was increased with the decreasing fish meal levels. The mRNA level of SOD in hepatopancreas decreased, and the expression of GPx and CAT increased with the decreasing FM levels. The Toll pathway was affected by dietary FM levels, the expression of Toll2, TNFSF, MyD88, Rho and p38 in intestine were increased with the decreasing FM levels. The results indicated that at low salinity condition, fish meal level lower than 15% would inhibit the protein synthesis and harm to the health of shrimp.

Frequency and Pattern of IgE-mediated Sensitization to Aero and Food Allergens in Ahvaz, Province of Khuzestan in Southwestern Iran.

Efficient diagnosis of allergy and proper treatment need identification of the causative allergens eliciting clinical symptoms. The present study was performed to identify the most common aero- and food allergens and determine the pattern of sensitization among people of Ahvaz (southwestern Iran), one of the most polluted cities worldwide. Based on the physical examination and medical records, patients were referred to the Allergy laboratory for "in vitro" IgE determination. Specific and total IgE was determined by the ImmunoCAP system (Thermo Fisher-Phadia, Uppsala, Sweden). A total of 666 consecutive patients (51.1% female) were tested for 202 different allergens. The majority of requests (57%) belonged to food allergens. Sensitization to at least one allergen was found in 47.6% of patients. In a selected group of allergens for which specific IgE had been tested in at least 100 patients, the most common sensitizing aeroallergens were Russian thistle, grass pollen, and willow; while wheat, honey, and shrimp were the most frequent food allergens, respectively. Sensitization profiles based on measurement of specific IgE indicated that Russian thistle, grasses, and wheat were the most prevalent allergens in people with allergic symptoms living in Ahvaz.

Jin Gui Shen Qi Wan, a traditional Chinese medicine, alleviated allergic airway hypersensitivity and inflammatory cell infiltration in a chronic asthma mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: In Asia, Jin Gui Shen Qi Wan (JGSQW) has been used for hundreds of years to treat asthma. AIM OF THE STUDY: The traditional Chinese medicine JGSQW is composed of Rehmannia glutinosa, Dioscorea opposita, Cornus officinalis, Poria cocos, Paeonia suffruticosa, Alisma orientalis, Aconitum carmichaelii and Cinnamomum cassia. However, the immunological mechanism underlying the effect of JGSQW treatment on asthma remains unclear. This study examined whether JGSQW has the potential to reduce asthma symptoms in mice with chronic asthma induced by recurrent Dermatophagoides pteronyssinus (Der p) stimulation, as well as its immunoregulatory mechanisms. MATERIALS AND METHODS: The airways of BALB/c mice were stimulated with Der p (i.t.) once per week (50 µL, 1 mg/mL) for 6 consecutive weeks, and the mice were fed JGSQW (1 g/kg) 30 min prior to the Der p stimulation. Three days after the last stimulation, the mice were sacrificed to evaluate the airway remodelling, infiltration of inflammatory cells, lung histological features, and total IgE in the blood. Additionally, after A549 cells were treated with JGSQW, loganin, or paeoniflorin for 30 min, 10 ng/mL IL-1ß was added to stimulate the A549 cells to evaluate the effect of the medicine on the ICAM-1 gene expression after IL-1ß stimulation. RESULTS: JGSQW significantly reduced the Der p-induced infiltration of inflammatory cells into airways and decreased the total IgE and Der p-specific IgG1 in serum. Collagen assays and histopathological examinations showed that JGSQW reduced lung airway remodelling. Additionally, an electrophoretic mobility shift assay and immunohistochemical staining verified that JGSQW inhibited the NF-kB expression in airway epithelial cell nuclei. JGSQW, loganin, and paeoniflorin inhibited the ICAM-1 gene expression caused by the IL-1ß stimulation of A549 cells, and loganin and paeoniflorin had the maximum inhibitory effect when mixed according to the combination of doses in JGSQW. CONCLUSION: These results indicated that in the chronic asthma mouse model, JGSQW inhibits the infiltration of inflammatory cells into the airways and airway remodelling and exhibits specific immunoregulatory effects. JGSQW with certain doses of loganin and paeoniflorin inhibited ICAM-1 gene expression in epithelial cells.

The function of Eriocheir sinensis transferrin and iron in Spiroplasma eriocheiris infection.

Transferrin, a member of the iron binding superfamily protein, plays an extremely important role in the transport of iron in the biological process of cells. The result of preliminary proteomic study on E. sinensis hemocytes infected Spiroplasma eriocheiris showed the expression of transferrin (EsTF) and ferrin (EsFe) significantly changed. In addition, other reports have confirmed that transferrin, ferritin and iron are involved in the immune response of hosts. In order to validate the immune function of EsTF, the whole length of EsTF was successfully amplified by the gene cloning and RACE technique. The results showed that the full-length cDNA of the EsTF gene was 2748 bp, including a 2193 bp open reading frame which encodes 730 amino acids. The result of bioinformatics analysis showed EsTF contains two highly conserved TR_FER domains. Evolutionary analysis showed that EsTF has a close genetic relationship with other TFs of invertebrates. In addition, EsTF mRNA was highly transcripted in nerve and intestine tissues, followed by hemocytes. The expression of EsTF, EsFe1 and EsFe2 increased after exogenous supplemental of iron under the concentration of 100 nmol/L in water. After exogenous supplement of iron and injection with S. eriocheiris, these three gene transcription of mRNA levels were higher than that of PBS group, while lower than the S. eriocheiris group and the iron group. Besides, the copy number of S. eriocheiris in the experimental group was significantly reduced, and the death rate decreased. As can be seen, iron made transferrin and ferritin return to normal levels during the infection of S. eriocheiris and help the host maintain normal immunity levels to resist S. eriocheiris. These results further demonstrated that EsTF, EsFe1, EsFe2 and iron play a role in the immune defense mechanism of the crabs to resist S. eriocheiris infection.

FmLC6: An ultimate dual-CRD C-type lectin from Fenneropenaeus merguiensis mediated its roles in shrimp defense immunity towards bacteria and virus.

C-type lectins are a member of pattern recognition receptors (PRRs) that can interact with pathogen-associated molecular patterns of invading microorganisms by using their conserved motifs in carbohydrate recognition domain (CRD). The binding can trigger various immune responses in both direct and indirect mechanisms. Hereby, an ultimate C-type lectin with dual CRDs each of which containing a different motif was identified from hepatopancreas of Fenneropenaeus merguiensis (mentioned as FmLC6). The full-length cDNA of FmLC6 consisted of 1148 bp comprising one 1005 bp open reading frame (ORF) encoding a signal peptide and a mature protein of 317 residues. FmLC6 was composed of two CRDs with a highly conserved QPD (Gln-Pro-Asp) motif and one variant EPQ (Glu-Pro-Gln) motif for illustrating the carbohydrate binding affinity. The transcription of FmLC6 was detected only in hepatopancreas of normal shrimp. After injection with pathogens or immunostimulants, the expression of FmLC6 was significantly up-regulated and reached the highest level at 12 h post-injection except with lipoteichoic acid challenge. The FmLC6 expression was severely suppressed by knockdown based-silencing. This gene silencing with co-injection by Vibrio parahaemolyticus caused increasing in cumulative mortality and reduction of the median lethal time. Purified recombinant proteins of an entire ORF and two individual CRDs of FmLC6 produced in Escherichia coli could induce a broad spectrum of microbial agglutination with calcium dependence. The agglutination induced by rFmLC6, rCRD1 and rCRD2 was suppressed by galactose plus mannose, galactose and mannose, respectively which this event was confirmed by the inhibition of hemagglutination. All three recombinant proteins possessed ability to inhibit the bacterial growth with a dose-response. Purified rFmLC6 could bind directly to white spot syndrome virus particles and also its recombinant proteins including VP15, VP39A and VP28 with different affinity. Altogether, these results indicate that FmLC6 acts as a PRR to recognize invading microorganisms and leads to mediating the immune response to cooperation in pathogenic elimination via the binding, agglutination and antimicrobial activity.

Evaluation of vaccine potential of 2-Cys peroxiredoxin from the hard tick Haemaphysalis longicornis.

Ticks require blood feeding on vertebrate animals throughout their life cycle, and also concentrate the iron-containing blood, resulting in a high concentration of hydrogen peroxide (H2O2). High concentrations of H2O2 are harmful to organisms, due to their serious damage of macromolecules. Ticks have antioxidant enzymes, such as peroxiredoxins (Prxs), that scavenge H2O2. Prxs may have important roles in regulating the H2O2 concentration in ticks during blood feeding and oviposition. Moreover, Prxs are considered potential vaccine candidates in other parasites, such as Leishmania and Fasciola. In the present study, the efficacy of a tick Prx (HlPrx2) as a vaccine candidate antigen was evaluated. First, recombinant HlPrx2 (rHlPrx2) was expressed in Escherichia coli, and then, its purity and endotoxin levels were confirmed prior to administration. The rHlPrx2 proteins were of high purity with acceptably low endotoxin levels. Second, the ability of rHlPrx2 administration to stimulate mouse immunity was evaluated. The rHlPrx2 protein, with or without an adjuvant, could stimulate immunity in mice, especially the IgG1 of Th2 immune response. Using Western blot analysis, we also observed whether rHlPrx2-immunized mice sera could recognize native HlPrx2 protein in crude tick midgut proteins. Western blot analysis demonstrated that rHlPrx2-administrated mouse sera could detect the native HlPrx2. Finally, the effects of rHlPrx2 immunization in mice were studied using nymphal ticks. Although the challenged ticks were not affected by rHlPrx2 immunization, rHlPrx2 still might be considered as a vaccine candidate against ticks because of its high immunogenicity.

The 1st step initiation essential for allergen-specific IgE antibody production upon the 2nd step: Induction of non-specific IgE+ small B cells containing secondly-sensitized allergen-specific ones in mice firstly-sensitized with an allergen.

There was a significant amount of non-specific, but not of allergen (e.g., papain, mite feces and four kinds of pollen)-specific, IgE antibodies (Abs) in the sera of normal mice. An i.n. injection of each allergen without adjuvant into mice caused an increase in total IgE Ab titers with a similar time course in the serum. However, the stage of initiation of allergy varied from allergen to allergen. Submandibular lymph node cells from normal mice contained papain-, but not mite feces- or pollen-specific IgE+ cells and an i.n. injection of papain induced papain-specific IgE Abs in the serum. In contrast, one (i.n.) or two (i.n. and s.c) injections of mite feces induced neither mite feces-specific IgE+ cells in the lymph nodes nor mite feces-specific IgE Abs in the serum. I.n. sensitization with cedar pollen induced cedar pollen-specific IgE+ small B cells in the lymph nodes on Day 10, when non-specific IgE Ab titers reached a peak in the serum, implying induction of related allergen-specific IgE+ small cells as well. In fact, a second (s.c.) injection of ragweed (or cedar) pollen into mice sensitized i.n. once with cedar (or ragweed) pollen, but not with mite feces, induced a large amount of ragweed (or cedar) pollen-specific IgE Abs in the serum. These results indicate that when firstly-sensitized non-specific IgE+ small B cells in mouse lymph nodes include some secondly-sensitized allergen-specific ones, mice produce IgE Abs specific for the secondly-injected allergen.

Characterization of a glycine-rich protein from Rhipicephalus microplus: tissue expression, gene silencing and immune recognition.

Salivary molecules, as glycine-rich proteins (GRPs), are essential to tick attachment and feeding on the host and are suggested to be involved in the host's immune system evasion, therefore representing natural candidates in the search for protective vaccine antigens. This work shows the molecular characterization of a GRP from Rhipicephalus microplus (RmGRP). The cDNA and putative amino acid sequences were analysed, as well as the transcription level in tick tissues/developmental stages, showing the highest levels of gene expression in 1-day-old larvae and salivary glands of fully engorged females. RmGRP gene silencing resulted in a lower hatching rate of larvae from treated females. In addition, recombinant RmGRP (rRmGRP) was recognized by sera from naturally and experimentally infested bovines, displaying considerable differences among the individuals tested. rRmGRP was recognized by anti-saliva and anti-salivary glands sera, while anti-rRmGRP serum recognized RmGRP in saliva and salivary glands, indicating its secretion into the host. The data collected indicate that RmGRP may present roles other than in the tick-host relationship, especially in embryo development. In addition, the high expression in adult females, antigenicity and presence of shared characteristics with other tick protective GRPs turns RmGRP a potential candidate to compose an anti-tick vaccine cocktail.