Quantification of Allergic Crustacean Tropomyosin Using Shared Signature Peptides in Processed Foods with a Mass Spectrometry-Based Proteomic Strategy.

Crustacean shellfish are major allergens in East Asia. In the present study, a major allergic protein in crustaceans, tropomyosin, was detected accurately using multiple reaction monitoring mode-based mass spectrometry, with shared signature peptides identified through proteomic analysis. The peptides were deliberately screened through thermal stability and enzymatic digestion efficiency to improve the suitability and accuracy of the developed method. Finally, the proposed method demonstrated a linear range of 0.15 to 30 mgTM/kgfood (R2 > 0.99), with a limit of detection of 0.15 mgTM/kg food and a limit of quantification of 0.5mgTM/kgfood and successfully applied to commercially processed foods, such as potato chips, biscuits, surimi, and hot pot seasonings, which evidenced the applicability of proteomics-based methodology for food allergen analysis.

Identification of toll-like receptor family and the immune function of new Sptlr-6 gene of Scylla paramamosain.

As a crucial member of pattern-recognition receptors (PRRs), the Tolls/Toll-like receptors (TLRs) gene family has been proven to be involved in innate immunity in crustaceans. In this study, nine members of TLR gene family were identified from the mud crab (Scylla paramamosain) transcriptome, and the structure and phylogeny of different SpTLRs were analyzed. It was found that different SpTLRs possessed three conserved structures in the TIR domain. Meanwhile, the expression patterns of different Sptlr genes in examined tissues detected by qRT-PCR had wide differences. Compared with other Sptlr genes, Sptlr-6 gene was significantly highly expressed in the hepatopancreas and less expressed in other tissues. Therefore, the function of Sptlr-6 was further investigated. The expression of the Sptlr-6 gene was up-regulated by Poly I: C, PGN stimulation and Vibrio parahaemolyticus infection. In addition, the silencing of Sptlr-6 in hepatopancreas mediated by RNAi technology resulted in the significant decrease of several conserved genes involved in innate immunity in mud crab after V. parahaemolyticus infection, including relish, myd88, dorsal, anti-lipopolysaccharide factor (ALF), anti-lipopolysaccharide factor 2 (ALF-2) and glycine-rich antimicrobial peptide (glyamp). This study provided new knowledge for the role of the Sptlr-6 gene in defense against V. parahaemolyticus infection in S. paramamosain.

[In Silico analysis of molecular mimic between Der p 23 against allergenic sources]. / Análisis in silico del mimetismo molecular entre Der p 23 contra fuentes alergénicas.

OBJECTIVE: To identify through In Silico analysis the possible molecular mimicry between Der p 23 and antigens from allergenic sources. METHODS: Identity was sought between Der p 23 and proteins from the mite families Pyroglyphidae, Acaridae, Chortoglyphidae and Echimyopodidae, through PSI-BLAST and They used PRALINE and EMBOSS for the alignments. Antigens with resolved experimental structure were obtained from Protein Data Bank and those not reported were generated using Swiss Model server and ALPHAFOLD 2. Epitope prediction was carried out with the Ellipro server and Pymol 2.3 was used to visualize the 3D models. RESULTS: The analysis between Pyroglyphidae allergens and Der p 23 showed identity with the endochitinase-like protein of D. pteronyssinus, and the type 2 chitin binding domain of D. farinae, with identities between 85 and 100%, with coverage of 100%, and 75% respectively. The allergens Der f 23 and Der p 23 of D. farinae and D. pteronyssinus had 100% coverage with identities of 85.42% and 79.59%, respectively. Among the allergens of Tyrophagus putrescentiae, binding to chitin, oviduct-specific glycoprotein and Cda4p were included, which had identity values corresponding to 40%, 42.22% and 34.78%, with coverage values that did not exceed the 55%. No results were found for Chortoglyphidae and Echimyopodidae. CONCLUSION: There is molecular mimicry and structural homology between Der P 23 and allergens from allergic sources of the Pyroglyphidae and Acaridae families. Potential epitopes were identified in Der p 23, which could present cross-reactivity with the proteins of the allergenic sources studied, which must be demonstrated in In vitro and In vivo studies. In vitro and in vivo work is needed to demonstrate the results obtained in the In Silico analysis.

OBJETIVO: Identificar, a través de análisis In Silico, el posible mimetismo molecular entre Der p 23 y antígenos de fuentes alergénicas. MÉTODOS: Se buscó identidad entre Der p 23 y proteínas de las familias de ácaros Pyroglyphidae, Acaridae, Chortoglyphidae y Echimyopodidae, a través de PSI-BLAST, y se utilizaron PRALINE y EMBOSS para los alineamientos. Los antígenos con estructura experimental resuelta se obtuvieron de Protein Data Bank, y aquellos no informados, se generaron mediante Swiss Model Server y ALPHAFOLD 2. La predicción de epítopes se realizó con el servidor Ellipro y para la visualización de los modelos en 3D, se utilizó Pymol 2.3. RESULTADOS: El análisis entre alérgenos de Pyroglyphidae y Der p 23, mostró identidad con la proteína parecida a endoquitinasa de D. pteronyssinus, y el dominio de unión a quitina tipo 2 de D. farinae, con identidades entre 85 y 100%, con coberturas de 100% y 75%, respectivamente. Los alérgenos Der f 23 y Der p 23 de D. farinae y D. pteronyssinu,s tuvieron una cobertura del 100% con identidades del 85,42% y 79,59%, respectivamente. Entre los alérgenos de Tyrophagus putrescentiae, se incluyeron la unión a quitina, glicoproteína específica del oviducto y Cda4p, las cuales tuvieron valores de identidad correspondientes al 40%, 42,22% y 34,78%, con valores de cobertura que no superan el 55%. No se encontraron resultados para Chortoglyphidae y Echimyopodidae. CONCLUSIÓN: Existe mimetismo molecular y homología estructural entre Der P 23 y alérgenos de fuentes alérgicas de las familias Pyroglyphidae y Acaridae. Se identificaron potenciales epítopes en Der p 23, los cuales podrían presentar reactividad cruzada con las proteínas de las fuentes alergénicas estudiadas, lo cual debe ser demostrado en estudios In Vitro e In Vivo. Se necesitan trabajos In Vitro e In Vivo que demuestren los resultados obtenidos en el análisis In Silico.

A novel exoskeletal-derived C-type lectin facilitates phagocytosis of hemocytes in the oriental river prawn Macrobrachium nipponense.

C-type lectins (CTLs) execute critical functions in multiple immune responses of crustaceans as a member of pattern recognition receptors (PRRs) family. In this study, a novel CTL was identified from the exoskeleton of the oriental river prawn Macrobrachium nipponense (MnLec3). The full-length cDNA of MnLec3 was 1150 bp with an open reading frame of 723 bp, encoding 240 amino acids. MnLec3 protein contained a signal peptide and one single carbohydrate-recognition domain (CRD). MnLec3 transcripts were widely distributed at the exoskeleton all over the body. Significant up-regulation of MnLec3 in exoskeleton after Aeromonas hydrophila challenged suggested the involvement of MnLec3 as well as the possible function of the exoskeleton in immune response. In vitro tests with recombinant MnLec3 protein (rMnLec3) manifested that it had polysaccharide binding activity, a wide spectrum of bacterial binding activity and agglutination activity only for tested Gram-negative bacteria (Escherichia coli, Vibrio anguillarum and A. hydrophila). Moreover, rMnLec3 significantly promoted phagocytic ability of hemocytes against A. hydrophila in vivo. What's more, MnLec3 interference remarkably impaired the survivability of the prawns when infected with A. hydrophila. Collectively, these results ascertained that MnLec3 derived from exoskeleton took an essential part in immune defense of the prawns against invading bacteria as a PRR.

Molecular characterization of a short-chained pentraxin gene from kuruma shrimp Marsupenaeus japonicus hemocytes.

Pentraxins (PTXs) are a family of pattern recognition proteins (PRPs) that play a role in pathogen recognition during infection via pathogen-associated molecular patterns (PAMPs). Here, we characterized a short-chained pentraxin isolated from kuruma shrimp (Marsupenaeus japonicus) hemocytes (MjPTX). MjPTX contains the pentraxin signature HxCxS/TWxS (where x can be any amino acid), although the second conserved residue of this signature differed slightly (L instead of C). In the phylogenetic analysis, MjPTX clustered closely with predicted sequences from crustaceans (shrimp, lobster, and crayfish) displaying high sequence identities exceeding 52.67 %. In contrast, MjPTX showed minimal sequence identity when compared to functionally similar proteins in other animals, with sequence identities ranging from 20.42 % (mouse) to 28.14 % (horseshoe crab). MjPTX mRNA transcript levels increased significantly after artificial infection with Vibrio parahaemolyticus (48 h), White Spot Syndrome Virus (72 h) and Yellow Head Virus (24 and 48 h). Assays done in vitro revealed that recombinant MjPTX (rMjPTX) has an ability to agglutinate Gram-negative and Gram-positive bacteria and to bind microbial polysaccharides and bacterial suspensions in the presence of Ca2+. Taken together, our results suggest that MjPTX functions as a classical pattern recognition protein in the presence of calcium ions, that is capable of binding to specific moieties present on the surface of microorganisms and facilitating their clearance.

Genome-wide identification of toll-like receptors in Octopus sinensis and expression analysis in response to different PAMPs stimulation.

Toll-like receptors (TLRs) are one of the extensively studied pattern recognition receptors (PRRs) and play crucial roles in the immune responses of vertebrates and invertebrates. In this study, 14 TLR genes were identified from the genome-wide data of Octopus sinensis. Protein structural domain analysis showed that most TLR proteins had three main structural domains: extracellular leucine-rich repeats (LRR), transmembrane structural domains, and intracellular Toll/IL-1 receptor domain (TIR). The results of subcellular localization prediction showed that the TLRs of O. sinensis were mainly located on the plasma membrane. The results of quantitative real-time PCR (qPCR) showed that the detected TLR genes were differentially expressed in the hemolymph, white bodies, hepatopancreas, gills, gill heart, intestine, kidney, and salivary gland of O. sinensis. Furthermore, the present study investigated the expression changes of O. sinensis TLR genes in hemolymph, white bodies, gills, and hepatopancreas in different phases (6 h, 12 h, 24 h, 48 h) after stimulation with PGN, poly(I: C) and Vibrio parahaemolyticus. The expression of most of the TLR genes was upregulated at different time points after infection with pathogens or stimulation with PAMPs, a few genes were unchanged or even down-regulated, and many of the TLR genes were much higher after V. parahaemolyticus infection than after PGN and poly(I:C) stimulation. The results of this study contribute to a better understanding of the molecular immune mechanisms of O. sinensis TLRs genes in resistance to pathogen stimulation.

Novel monoclonal antibodies against house dust mite allergen Der p 21 and their application to analyze allergen extracts.

Background: Allergen extracts and recombinant allergens are used in allergy diagnostics and immunotherapy. Since allergen extracts from different manufacturers lack proper standardization regarding their composition, monoclonal antibodies (MAbs) against specific allergen components can be used for their identification and quantification in allergen extracts. This study aimed to generate MAbs against allergen Der p 21 of Dermatophagoides pteronyssinus for the analysis of allergen extracts. Methods: Recombinant Der p 21 was expressed in E. coli and purified using affinity chromatography. MAbs against Der p 21 were generated using hybridoma technology. House dust mite (HDM) allergen extracts were analyzed using the newly developed sandwich enzyme-linked immunosorbent assay, Western blotting and microarray immunoassay. Results: MAbs raised against recombinant Der p 21 were characterized in detail and proven to be reactive with natural Der p 21. Highly specific sandwich enzyme-linked immunosorbent assay for the quantification of Der p 21 was developed and optimized. The allergen was detected and its concentration was determined in only three of six analyzed HDM allergen extracts from different manufacturers. Conclusion: HDM analysis by MAb-based immunoassays shows their differences in allergen composition. The results demonstrate the importance of allergen-specific MAbs as a tool for the characterization of allergen extracts and the need for their appropriate standardization before their use for allergy diagnostics or immunotherapy.

Accurate determination of house dust mite sensitization in asthma and allergic rhinitis through cytometric detection of Der p 1 and Der p 2 binding on basophils (CytoBas).

BACKGROUND: House dust mite (HDM) is the most common allergen trigger globally for allergic rhinitis and atopic asthma. OBJECTIVES: To expedite accurate confirmation of allergen sensitization, we designed fluorescent allergen tetramers to directly stain specific IgE on basophils to detect specific allergen sensitization using the flow cytometric CytoBas assay. METHODS: Recombinant proteins of major HDM allergens (component), Der f 1, Der p 1, and Der p 2 were biotinylated and conjugated with fluorochrome streptavidins as tetramers. Blood samples from 64 patients who are HDM-allergic and 26 controls that are non-HDM-sensitized were incubated with allergen tetramers for evaluation of basophil binding (CytoBas) and activation (BAT) with flow cytometry. RESULTS: The tetramers effectively bound and activated basophils from patients who are allergic but not from controls who are nonsensitized. CytoBas with Der p 1 as a single allergen had comparable sensitivity and specificity (92% and 100%) to BAT (91% and 100%) in detecting allergen sensitization, as did CytoBas with Der p 2 (95% and 96%) to BAT (95% and 87%). A positive staining for Der p 1 and/or Der p 2 in CytoBas was 100% sensitive and 96% specific for HDM allergy. CONCLUSIONS: CytoBas has diagnostic accuracy for group 1 and group 2 HDM allergens that is comparable to BAT, but with additional advantages of multiple allergen components in a single tube and no requirement for in vitro basophil activation. These findings endorse a single, multiplex CytoBas assay for accurate and component-resolved diagnosis of aeroallergen sensitization in patients with allergic asthma and/or rhinitis.

Immunostimulation of Parasteatoda tepidariorum (Araneae: Theridiidae) in juvenile and adult stages. Immunity reactions to injury with foreign body and Bacillus subtilis infection.

To assess the immune potential of spiders, in the present study juvenile and adult females of Parasteatoda tepidariorum were exposed to Bacillus subtilis infection, injury by a nylon monofilament and a combination of both. The expression level of selected immune-related genes: defensin 1 (PtDEF1), lysozyme 1 (PtLYS1), lysozyme C (PtLYSC), lysozyme M1 (PtLYSM1), autophagy-related protein 101 (PtATG101), dynamin (PtDYN) and heat shock proteins (HSP70) (PtHSPB, PtHSPB2A, PtHSPB2B), production of lysozyme and HSP70 proteins, and hemocytes viability were measured. The obtained results indicated expression of the lysozyme, autophagy-related protein and HSP70 genes in both ontogenetic stages of P. tepidariorum. It has been also shown that the simultaneous action of mechanical and biological factors causes higher level of lysozyme and HSP70, cell apoptosis intensity and lower level of hemocytes viability than in the case of exposure to a single immunostimulant. Moreover, mature females showed stronger early immune responses compared to juveniles.

New insight of variable lymphocyte receptor-likes in anti-bacteria activity from Eriocheir sinensis.

The Chinese mitten crab, Eriocheir sinensis, is a vital freshwater aquaculture species in China, however, is also facing various crab disease threats. In the present study, we identify three novel variable lymphocyte receptor-like (VLR-like) genes-VLR-like1, VLR-like3 and VLR-like4-from E. sinensis, which play vital roles in adaptive immune system of agnathan vertebrates. The bacterial challenge, bacterial binding and antibacterial-activity experiments were applied to study immune functions of VLR-likes, and the transcriptomic data from previous E. sinensis bacterial challenge experiments were analyzed to speculate the possible signaling pathway. VLR-like1 and VLR-like4 can respond to Staphylococcus aureus challenge and inhibit S. aureus specifically. VLR-like1 and VLR-like4 possess broad-spectrum bacteria-binding ability whereas VLR-like3 do not. VLR-likes in E. sinensis could associate with the Toll-like receptor (TLR) signaling pathway. The above results suggest that VLR-likes defend against bacteria invasion though exerting anti-bacteria activity, and probably connect with the TLR signaling pathway. Furthermore, studying the immune functions of these VLR-likes will provide a new insight into the disease control strategy of crustacean culture.

Dermatophagoides pteronyssinus allergen Der p 22: Cloning, expression, IgE-binding in asthmatic children, and immunogenicity.

BACKGROUND: Dust mite extract contains multiple components that, while useful in clinical allergy diagnosis and treatment, can cause serious side effects. Defining components of dust mite extract is important their contributions to allergic disease. This study aimed to characterize a novel dust mite allergen, Der p 22. METHODS: We amplified the cDNA encoding Der p 22 from total RNA of the mite Dermatophagoides pteronyssinus, and inserted it into an expression construct for transformation to competent cells. Purified recombinant (r) Der p 22 was tested for IgE-binding reactivity in sera obtained from children with allergic asthma by the Affiliated Wuxi Children's Hospital of Nanjing Medical University (Jiangsu, China). rDer p 22 also was used to challenge BALB/c mice to assess effects on T helper cells and cytokine levels and applied to cultured lung epithelial cells to evaluate apoptosis and cytokine secretion. RESULTS: rDer p 22 bound to IgE in 93.75% of sera from pediatric allergic asthma patients. Mice challenged with rDer p 22 had altered Th1/Th2 ratios in spleen and lymph, and lower levels of cytokines IFN-γ but higher levels of IL-4 and IL-10 in alveolar lavage fluid compared with controls (p < .05). Cultured lung epithelial cells had greater apoptosis rates and exhibited higher levels of IL-6, IL-8, and GM-CSF when treated with rDer p 22 compared with control treatment (p < .05). CONCLUSIONS: Recombinant Der p 22 exhibited high IgE-binding rates in allergic children, indicating the activity of the recombinant protein and suggesting this novel allergen may be appropriate for inclusion in an allergy diagnostic workup. This finding is supported by in vitro and mouse in vivo studies showing rDer p 22 induced strong allergenic reactivity and apoptosis.

Characterization of the Dual Functions of LvCrustinVII from Litopenaeus vannamei as Antimicrobial Peptide and Opsonin.

Crustin are a family of antimicrobial peptides that play an important role in protecting against pathogens infection in the innate immune system of crustaceans. Previously, we identified several novel types of crustins, including type VI and type VII crustins. However, their immune functions were still unclear. In the present study, the immune function of type VII crustin LvCrustinVII were investigated in Litopenaeus vannamei. LvCrustinVII was wildly expressed in all tested tissues, with relatively high expression levels in hepatopancreas, epidermis and lymphoid organ. Upon Vibrio parahaemolyticus infection, LvCrustinVII was significantly upregulated in hepatopancreas. Recombinant LvCrustinVII (rLvCrustinVII) showed strong inhibitory activities against Gram-negative bacteria Vibrio harveyi and V. parahaemolyticus, while weak activities against the Gram-positive bacteria Staphylococcus aureus. Binding assay showed that rLvCrustinVII could bind strongly to V. harveyi and V. parahaemolyticus, as well as the cell wall components Glu, LPS and PGN. In the presence of Ca2+, rLvCrustinVII could agglutinate V. parahaemolyticus and enhance hemocyte phagocytosis. The present data partially illustrate the immune function of LvCrustinVII, which enrich our understanding on the functional mechanisms of crustins and provide useful information for application of this kind of antimicrobial peptides.

Yorkie Negatively Regulates the Expression of Antimicrobial Proteins by Inducing Cactus Transcription in Prawns Macrobrachium nipponense.

The Hippo signaling pathway controls organ size and immune system in Drosophila and mammals. Yorkie acts as a transcriptional co-activator in the Hippo pathway and cross-talks with other essential pathways. In this study, a Yorkie gene and two Cactus isoforms (designated as MnYorkie, MnCactus-a, and MnCactus-b, respectively) were isolated and characterized from oriental river prawns (Macrobrachium nipponense). Results showed that MnYorkie includes 1620 bp open reading frame and encodes a protein of 539 amino acids (aa). MnCactus-a (377 aa) and MnCactus-b (471 aa) were produced by alternative splicing. MnYorkie and MnCactus were continuously expressed in all selected tissues. Upon Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Vibrio parahaemolyticus stimulation, the mRNA levels of MnYorkie and MnCactus in hemocytes and intestines underwent time-dependent enhancement. RNA interference studies showed that MnYorkie silencing remarkably downregulated the transcription of MnCactus but upregulated the expression of seven immune-related genes. In addition, MnYorkie silencing in vivo decreased the susceptibility of prawns to bacterial challenge. After S. aureus and V. parahaemolyticus infection, the survival rate of prawns increased significantly from 2 to 6 days, which corresponded to the period of MnYorkie knockdown. All these findings suggested that MnYorkie in the Hippo pathway might exhibit remarkable biological roles in the immune defense of M. nipponense by negatively regulating the expression of immune-related genes and promoting the transcription of MnCactus.

Identification of arginine kinase as an allergen of brown crab, Callinectes bellicosus, and in silico analysis of IgE-binding epitopes.

In recent years there has been an increase in the prevalence of allergic reactions to contact with/or consumption of crustaceans by immune responses mediated by IgE antibodies. Arginine kinase (AK) is considered one of the main allergens present in marine invertebrates. Currently, the allergenic potential of the brown crab (Callinectes bellicosus), which is a crustacean of great economic importance, has not been studied. Therefore, the aim of this work was to identify C. bellicosus AK as an allergen and to predict IgE-binding epitopes through immunobioinformatic analysis. AK was purified by precipitation with ammonium sulfate and ion- exchange chromatography. AK allergenicity was evaluated by IgE reactivity against sera from crustacean-allergic and non-allergic patients in both native and denaturing conditions. Additionally, a homology model was built based on the deduced amino acid sequence. A single band (~40 kDa) was found in SDS-PAGE, which was identified as an AK by mass spectrometry. AK showed immunoreactivity against crab-allergenic sera in both native and denaturing conditions with 70% and 80% positive reactions, respectively. Additionally, a 1073 bp ORF was obtained which codes for a deduced sequence of 357 amino acids corresponding to AK with > 90% identity with other AKs. Structural homology model of AK showed two main domains with conserved / folding of phospho-guanidine kinases. BediPred and Discotope were used for epitope prediction analysis, which suggests eight possible linear epitopes and seven conformational epitopes, respectively; and shows to be similar to other crustaceans AKs. C. bellicosus AK was identified as an allergenic protein by IgE reactivity and immunobioinformatic analysis indicates that both linear and conformational epitopes could be located in the surface of C. bellicosus AK structure.

Shoc2 recognizes bacterial flagellin and mediates antibacterial Erk/Stat signaling in an invertebrate.

Flagellin is a key bacterial virulence factor that can stimulate molecular immune signaling in both animals and plants. The detailed mechanisms of recognizing flagellin and mounting an efficient immune response have been uncovered in vertebrates; however, whether invertebrates can discriminate flagellin remains largely unknown. In the present study, the homolog of human SHOC2 leucine rich repeat scaffold protein in kuruma shrimp (Marsupenaeus japonicus), designated MjShoc2, was found to interact with Vibrio anguillarum flagellin A (FlaA) using yeast two-hybrid and pull-down assays. MjShoc2 plays a role in antibacterial response by mediating the FlaA-induced expression of certain antibacterial effectors, including lectin and antimicrobial peptide. FlaA challenge, via MjShoc2, led to phosphorylation of extracellular regulated kinase (Erk), and the subsequent activation of signal transducer and activator of transcription (Stat), ultimately inducing the expression of effectors. Therefore, by establishing the FlaA/MjShoc2/Erk/Stat signaling axis, this study revealed a new antibacterial strategy in shrimp, and provides insights into the flagellin sensing mechanism in invertebrates.

Molecular characteristics and immune function of ubiquitin C-terminal hydrolase-L3 in Macrobrachium nipponense.

Ubiquitin C-terminal hydrolase-L3 (UCHL3) is a deubiquitinating enzyme involved in the repair mechanism of homologous recombinations of DNA double strand breaks (DBS). However, the role of UCHL3 in crustacean immune regulation has not been studied. In this experiment, we cloned and analyzed the expression profile of the UCHL3 gene from Macrobrachium nipponense (MnUCHL3). The obtained full-length cDNA of the MnUCHL3 transcript was 1192 bp, and it had a 687 bp open reading frame encoding a 228 amino acid protein, and the structure of UCHL3 is highly similar to that of other invertebrates. Real-time PCR results indicated that MnUCHL3 was expressed in all detected tissues, with the highest expression levels in the hepatopancreas, and the expression of MnUCHL3 in the gill and hepatopancreas was downregulated to different degrees within 48 h after the infection of viruses and bacteria. Furthermore, knockdown of MnUCHL3 expression by double-stranded RNA (dsRNA) injection in Aeromonas hydrophila-infected prawns increased prawn mortality and bacterial growth. In addition, overexpression of MnUCHL3 in HEK293T cells in vitro suggested that MnUCHL3 could activate the NF-κB signal path and the expression levels of NF-κB signaling cascade members and AMPs, exhibiting remarkable downregulation in the MnUCHL3-silenced group. The above experimental conclusions revealed that UCHL3 gene might be involved in the innate immune response to bacterial infection by regulating the synthesis of a series of AMPs, and these results might provide new insights into UCHL3 in invertebrates.

Immunomodulatory role of crustacean cardioactive peptide in the mud crab Scylla paramamosain.

Crustacean cardioactive peptide (CCAP) is a pleiotropic neuropeptide, but its immunomodulatory role is not clear. Herein, the mud crab Scylla paramamosain provides a primitive model to study crosstalk between the neuroendocrine and immune systems. In this study, in situ hybridization showed that Sp-CCAP positive signal localized in multiple cells in the nervous tissue, while its conjugate receptor (Sp-CCAPR) positive signal mainly localized in the semigranular cells of hemocytes. The Sp-CCAP mRNA expression level in the thoracic ganglion was significantly up-regulated after lipopolysaccharide (LPS) stimulation, but the Sp-CCAP mRNA expression level was up-regulated firstly and then down-regulated after the stimulation of polyriboinosinic polyribocytidylic acid [Poly (I:C)]. After the injection of Sp-CCAP synthesis peptide, the phagocytosis ability of hemocytes was significantly higher than that of synchronous control group. Simultaneously, the mRNA expression of phagocytosis related gene (Sp-Rab5), nuclear transcription factor NF-κB homologues (Sp-Relish), C-type lectin (Sp-CTL-B), prophenoloxidase (Sp-proPO), pro-inflammatory cytokines factor (Sp-TNFSF, Sp-IL16) and antimicrobial peptides (Sp-ALF1 and Sp-ALF5) in the hemocytes were also significantly up-regulated at different time points after the injection of Sp-CCAP synthetic peptide, but Sp-TNFSF, Sp-ALF1 and Sp-ALF5 were down-regulated significantly at 24h. In addition, RNA interference of Sp-CCAP suppressed the phagocytic activity of hemocytes and inhibited the mRNA expression of Sp-Rab5, Sp-Relish, Sp-CTL-B, Sp-TNFSF, Sp-IL16 and Sp-ALF5 in the hemocytes, and ultimately weakened the ability of hemolymph bacteria clearance of mud crab. Taken together, these results revealed that CCAP induced innate immune and increased the anti-infection ability in the mud crab.

Anti-lipopolysaccharide factors regulated by Stat, Dorsal, and Relish are involved in anti-WSSV innate immune defense in Macrobrachium nipponense.

Anti-lipopolysaccharide factors (ALF) is an important antimicrobial peptide and critical effector molecule with a broad spectrum of antimicrobial activities in crustaceans. In addition to the previously reported five ALFs (MnALF1-5), another three ALFs [MnALF1, which is different from MnALF1 (ALF02818) that has been reported; MnALF6; and MnALF7] and an isoform of MnALF4 (MnALF4-isoform2) were newly identified from Macrobrachium nipponense in this study. MnALF6 has 134 amino acids and one single nucleotide polymorphism (SNP) in MnALF6 resulted in the change of 107th amino acid from E to D. Intron 1 retention produced longer transcript of MnALF6. The full length of MnALF7 has 691 bp with a 363 bp ORF encoding 120 amino acid protein. Three SNPs in MnALF2 resulted in the conversion of amino acids at positions 70, 73, and 91 from T70I73P91 to K70L73S91. The deletion of 13 bp in MnALF4 resulted in early termination of ORF, resulting in MnALF4-isoform2 with only 98 amino acids. The gDNAs of MnALF1, MnALF2, MnALF5, and MnALF6 contain three exons and two introns, while those of MnALF3 and MnALF7 contain three exons, one known intron, and one unknown intron. The MnALF1-7 in M. nipponense were widely distributed in multiple tissues. After white spot syndrome virus (WSSV) stimulation, the expression levels of MnALF1-7 changed. Knockdown of MnALF1-7 could evidently increase the expression of the envelope protein VP28 and the copy number of WSSV during viral infection. Further studies found that silencing of three transcription factors (Stat, Dorsal, and Relish) in M. nipponense significantly inhibit the synthesis of MnALF1-7 during the process of WSSV challenge. This study adds to the knowledge about the roles of ALFs in the innate immune responses to WSSV infection in M. nipponense.

Structure and function of a family of tick-derived complement inhibitors targeting properdin.

Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches.

The Expression Profile of Protease Inhibitors in the Airway Epithelial Cells after Allergen (Der p 1) Stimulation.

BACKGROUND: Airway epithelial cells are constantly exposed to intracellular and extracellular proteases that play a pivotal role in several airway diseases. Dermatophagoides pteronyssinus (Der p) 1 derived from house dust mite has protease activity that causes epithelial barrier defect and inflammatory response. Protease inhibitors released against proteases are involved in the maintenance of homeostasis. A disruption of the balance between proteases and protease inhibitors can lead to distortion of the cellular structures and cellular activities and thus culminate in disease processes. Although the effects of Der p 1 allergen on epithelial barrier integrity and inflammatory response are well-established, its contribution to protease inhibitor production is highly limited. OBJECTIVE: This study aimed to determine the profile of the protease inhibitor response to Der p 1 allergen in human airway epithelial cells, A549 and BEAS-2B. METHODS: Differentiated cells by the air-liquid interface were exposed to Der p 1 with or without Th2 type cytokines (IL-4 and IL-13). Gene expression of protease inhibitors was determined by qPCR at 2 different time points. RESULTS: We found that the effect of allergen exposure on the protease inhibitor profile can vary depending on the antigen concentration, treatment duration, and the presence or absence of type 2 cytokines. Gene expressions of serine protease inhibitor (SERPIN)B3 and SERPINB4 were increased following Th2 cytokine stimulation in both cell types at both time points, whereas SERPINB2 and TFPI-2 expressions were induced by 24-h Der p 1 stimulation in both cells. CONCLUSIONS: Our study suggests that Der p 1 exposure of the airway epithelium may have consequences related to its protease activity in the presence as well as in the absence of Th2 cytokines in the microenvironment.