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Cephalopods are economically and ecologically important species across the world, yet information linking physiological stress and associated immunological responses is limited in the current literature. Here, the effects of exhaustive exercise in a holobenthic octopus species, Octopus pallidus, were examined by evaluating immunological parameters. In whole haemolymph, the pH and refractive index were measured. To assess the cellular function of the haemolymph, the total count, cell vitality and phagocytosis capacity of the haemocytes were also measured. To assess enzymatic function, activities of the phenoloxidase system and lysozyme were quantified in the plasma and cellular components of the haemolymph. Overall, exhaustive exercise led to rapid changes in the haemolymph with a significant decrease in the pH and phagocytosis capacity though the number of cells and cell vitality were not affected. Exercise also triggered the increase of activated phenoloxidase (PO-like) activity and the decrease of the inactive zymogen prophenoloxidase (ProPO-like), total PO-like and lysozyme activity in plasma and an increase in total PO-like activity in the hemocyte compartment. These responses indicated that a realistic energetic demand had substantial, rapid impact on immune function. These results also provide an important baseline to understand the immune physiology of cephalopods that will further efforts to identify the mechanisms underlying the impacts of stressors., (Copyright © 2024. Published by Elsevier Ltd.)

Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine-antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds.

Lysozymes are hydrolytic enzymes, and they are ubiquitous among all living organisms. They are mostly associated with antibacterial properties through their muramidase activity, while other properties such as iso-peptidase activity are also common. Invertebrate-type (i-type) lysozymes include the enzyme Destabilase, which is present in the salivary secretions of the medicinal leach Hirundo medicinalis. Destabilase has the ability to hydrolyse the ε-(γ-glutamyl)-lysine iso-peptide bonds formed by transglutaminase in fibrin of vertebrate blood, thereby destabilising blood clots. We have identified an i-type lysozyme from the hemocytes of the freshwater crayfish Pacifastacus leniusculus, which was found to be upregulated at the protein level in response to an injection of the β-1,3-glucan laminarin. Based on its sequence we predicted that this lysozyme would lack muramidase activity, and therefore we decided to determine its putative immune function. The P. leniusculus i-type lysozyme (Pl-ilys), is a protein with 159 amino acid residues, including a 29 residue signal peptide, with a predicted molecular weight of 16 kDa and a predicted pI of 5.6. It is expressed primarily in the hemocytes and to a lesser extent in the hematopoietic tissue. A recombinant mature Pl-ilys using an E. coli expression system was produced, and we could ascertain that this enzyme was deficient of muramidase activity. Moreover, no iso-peptidase activity could be detected against the substrate l-γ-glutamine-p-nitroanilide. Analysis of the conserved domains in Pl-ilys showed a putative destabilase domain, and thus we tested the clot dissolving activity of this enzyme. We could show that the purified P. leniusculus clotting protein which had been coagulated and clotted with transglutaminase was dissolved by the addition of Pl-ilys. Taken together our results indicate that Pl-ilys has a clot dissolving or destabilising activity in crustacean blood., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Over the past two decades, therapeutic antibodies have emerged as a rapidly expanding domain within the field of biologics. In silico tools that can streamline the process of antibody discovery and optimization are critical to support a pipeline that is growing more numerous and complex every year. High-quality structural information remains critical for the antibody optimization process, but antibody-antigen complex structures are often unavailable and in silico antibody docking methods are still unreliable. In this study, DeepAb, a deep learning model for predicting antibody Fv structure directly from sequence, was used in conjunction with single-point experimental deep mutational scanning (DMS) enrichment data to design 200 potentially optimized variants of an anti-hen egg lysozyme (HEL) antibody. We sought to determine whether DeepAb-designed variants containing combinations of beneficial mutations from the DMS exhibit enhanced thermostability and whether this optimization affected their developability profile. The 200 variants were produced through a robust high-throughput method and tested for thermal and colloidal stability (T onset , T m , T agg ), affinity (K D ) relative to the parental antibody, and for developability parameters (nonspecific binding, aggregation propensity, self-association). Of the designed clones, 91% and 94% exhibited increased thermal and colloidal stability and affinity, respectively. Of these, 10% showed a significantly increased affinity for HEL (5- to 21-fold increase) and thermostability (>2.5C increase in T m1 ), with most clones retaining the favorable developability profile of the parental antibody. Additional in silico tests suggest that these methods would enrich for binding affinity even without first collecting experimental DMS measurements. These data open the possibility of in silico antibody optimization without the need to predict the antibody-antigen interface, which is notoriously difficult in the absence of crystal structures.

A substantial body of work has been carried out describing the structural features of the complex between single-domain antibodies (VHHs) and antigens, and the preeminence for epitopes located at concave surfaces of the antigen. However, the thermodynamic basis of binding is far less clear. Here, we have analysed the energetic profiles of five VHHs binding to the catalytic cleft or to a noncleft epitope of hen egg lysozyme. Various binding energetic profiles with distinctive enthalpic/entropic contributions and structural distribution of critical residues were found in the five antibodies analysed. Collectively, we suggest that from an energetic point of view the binding mechanism is influenced by the shape of the epitope. This information may be beneficial for the design of tailored epitopes for VHHs and their practical use., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Vaccination is the most effective, safe, and environmentally friendly method to prevent the outbreak of Photobacterium damselae subsp. piscicida (Phdp), a dangerous pathogen in aquaculture worldwide. Here, recombinant proteins of catalase, superoxide dismutase, isocitrate dehydrogenase, fructose 1,6-bisphosphate aldolase (Fba), and a mixture of all four proteins were investigated for their immunoprotective effects against photobacteriosis in Asian sea bass (Lates calcarifer). After immunization, experimental fish showed an increase in specific antibody levels and lysozyme activities, especially the Fba group. After a lethal challenge with Phdp strain AOD105021, the Fba group achieved the highest relative percentage of survival rate (70.21%) and a significantly lower bacterial load in the spleens than other groups 3 days after infection. The results suggest that Fba is a good candidate for subunit vaccine development against photobacteriosis in fish., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The association of vaccines with immunostimulants such as β-glucan, promote the production of cytokines, competent immune cells and antibodies. However, differences between β-glucan types and trials make it difficult to understand β-glucan's mechanism of action. In this study, three trials were carried out with control and fish fed β-glucan, the first trial occurred at 15 days; the second trial occurred at 30 days when we associated β-glucan and vaccine; and the third trial occurred at 15 days post-challenge with Streptococcus agalactiae in tilapia (O. niloticus) in order to investigate immune-related gene expression in the head kidney and spleen using real-time qPCR. We found increases in HSP70, IL-6, IL-1β, TNF-α, IL-10, Lys and C3 predominantly in the head kidney, except for IgM expression, which prevailed in the spleen, under vaccinated + β-glucan action. This demonstrates the trade-off presented by the head kidney and spleen after immunostimulation in order to produce acquired immunity, as well as an increase in HSP70 expression in vaccinated + β-glucan fish. The results suggest that β-glucan stimulates the immune response through damage-associated molecular patterns (DAMPs) recognition. Therefore, these dynamics of the immune response promote a more robust defense against disease., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This study investigated the immunomodulatory effects of Sargassum polycystum extract administration in rainbow trout (Oncorhynchus mykiss). S. polycystum methanolic extract was administered orally using feeding needles to individual rainbow trout at the dose of 0 (control), 1 (S1), 3 (S3) and 5 (S5) mg/100 μl/per fish twice a day for 7 days. On 1st, 5th, 3rd and 7th day, blood and tissues were collected from the fish and changes in humoral immune responses and immune-related gene expressions were determined. The result of oxidative radical production showed no difference during early stage of the experiment and was lately decreased (P < 0.05). Lysozyme activity increased on 3rd and 7th day of the study in S5 fish group and on 5th day in S3 group compared to control (P < 0.05). Myeloperoxidase activity had an increased level on the 1st and 3rd day in S1, S5 and S5 fish groups, respectively. IL-1β gene was significantly up-regulated in kidney and intestine in all experimental groups (except on the 1st day, in the intestine of S5 fish group) compared to control (P < 0.05). IL-8 gene expression was elevated on 1st and 3rd day in kidney of all experimental fish groups. IL-6 transcript enhanced in a dose-dependent manner on 3rd and 7th day. IL-10 and IL-12 genes were also up-regulated. Survival in all treated fish groups challenged with Aeromonas hydrophila was significantly increased compared to that of control. The highest survival rate was recorded in S5 fish group (83.65%) followed by S3 fish group (82.62%). Our results suggest that S. polycystum aqueous methanolic extract is an effective immunostimulant and provide protection against A. hydrophila infection in rainbow trout at a dose of 3-10 mg/20 g body weight/day., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This study aims to assess and determine the oral-administration of probiotic, Lactobacillus pentosus BD6 on growth performance, immunity and disease resistance of white shrimp, Litopenaeus vannamei. Lac. pentosus BD6 effectively inhibited the growth of aquatic pathogens, which was used in the test. Shrimp were fed with the control diet (without probiotic supplement) for 60 days and the probiotic-containing diets at 10 7 , 10 8 , 10 9 , and 10 10  cfu kg -1 , respectively. Shrimp fed with the diet containing probiotic at the doses of 10 9-10  cfu kg -1 showed significant increase in growth performance as well as feed efficiency than that of the control. After a challenge test with Vibrio alginolyticus, shrimp fed with a probiotic diet at a dose of 10 10  cfu kg -1 showed a significantly lower mortality as compared to the control and that of shrimp fed the diet containing probiotic at the levels up to 10 7-8  cfu kg -1 . In addition, a therapeutic potential of Lac. pentosus BD6 was discovered because the cumulative mortalities of shrimp fed with probiotic and pathogen V. parahaemolyticus simultaneously were significantly lower when compared to control shrimp. Probiotic in diet at a dose of 10 9-10  cfu kg -1 significantly increased PO activity of shrimp, while shrimp receiving probiotic at the doses of 10 8-10  cfu kg -1 showed significant increase in lysozyme activity and phagocytic activity. Shrimp fed with the diet containing probiotic at the level of 10 10  cfu kg -1 also indicated higher gene expression of prophenoloxidase (proPO) I, but not proPO II, lipopolysaccharide and β-1,3-glucan-binding protein and penaeidin 4. Analysis of the bacterial microbiota of the shrimp intestine revealed that oral administration of probiotic increased the relative abundance of beneficial bacteria and reduced the abundance of harmful pathogenic bacteria in the gut flora of shrimp. Despite no statistically significant difference, an analysis of microbial diversity recorded higher species richness, Shannon-Weaver diversity index and evenness in the probiotic group, compared to the control group. It was concluded that Lac. pentosus BD6 has great antibacterial ability against a wide range of pathogens and has therapeutic potential to reduce the mortality of shrimp infected with V. parahaemolyticus. Additionally, dietary Lac. pentosus BD6 at the level of 10 10  cfu kg -1 was recommended to improve growth performance, immunity and disease resistance of shrimp against V. alginolyticus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The effect of cinnamaldehyde (CM) enriched diet on immunity and cytokine gene expression in Channa striatus against Aphanomyces invadans is reported. C. striatus was uniformly divided into eight groups (n = 25 fish each) and fed with formulated diets with 0, 5, 10, and 15 mg kg -1  CM enriched diet. In healthy and infected groups fed with 5 mg kg -1 diet the leukocytes count increased significantly after 4th week; with 10 mg kg -1  CM diet the increase manifested after 6th week, but with 15 mg kg -1 not even after 8th week. In both groups, 5 mg kg -1  CM diet resulted in a significant increase in the serum total protein, albumin, and globulin levels after 4th week, whereas with other diets this effect was observed only after 6th week. Similarly, with any enriched diet the lysozyme activity increased significantly, but with 15 mg kg -1  CM diet only after 6th week. In both groups the complement activity and lymphocyte production increased significantly when fed with 5 mg kg -1  CM diet after 4th week while with other enriched diets only after 6th week. The phagocytic activity increased significantly in both groups fed with 5 mg kg -1  CM diet after 6th week, whereas the SOD activity increased after 4th week. The IgM production increased significantly in both groups fed with 5 mg kg -1  CM diet after 2nd week, while with 5 and 10 mg kg -1  CM diet after 4th week. In both groups, the expression of CXCR3α was significant on 4th week when fed with 10 mg kg -1  CM diet, while in the healthy group fed with 15 mg kg -1  CM diet the expression manifested earlier than 4th week. However, when fed with 10 and 15 mg kg -1  CM diets the increase was observed on 6th week; whereas, the expression of MHC-I reached the maximum on 6th week with any enriched diet. The results indicate that in C. striatus the innate immunity and expression of cytokine and immune related genes were significantly modulated when fed with 5 mg kg -1  CM diet on 4th week against A. invadans., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Fermentation strategy is well documented to improve the nutritional value of agricultural waste by-products such olive cake (OC), which, in turn, provides healthy, safe, and affordable feedstuff. This study assessed the combined impact of Aspergillus oryzae-fermented OC (AFOC) on the growth performance, intestinal morphometry, blood biochemistry, lysozyme activity, gut immune-related genes, and flesh quality of Nile tilapia. We divided 225 fish into five groups and further subdivided into three replicates (n = 15 each) and fed them five diets (Control, AFOC5, AFOC10, AFOC15, AFOC20) to determine AFOC nutritional value and its optimized incorporation level in the diet. The trial continued for 3 months. The crude protein content of OC improved by 7.77% after A. oryzae fermentation, while lipid content decreased by 14.19%. In addition, growth and feed utilization significantly improved at (10.8-11.2) % AFOC dietary level. High-density lipoprotein (HDL) significantly improved, and the serum lysozyme level was significantly higher in the AFOC10 group compared to other groups. Interestingly, gut-related inflammatory cytokines tumor necrosis factor alpha (TNF- α) and interleukin 1 beta (IL-1β) revealed higher relative mRNA expression in the AFOC10 group compared to other groups. The histomorphometric parameters was greatly influenced by the AFOC incorporation level (10%-20%). These findings suggested that A. orzae fermentation modifies the nutritional quality of OC, as seen through its positive impact on the growth performance, local and systemic immunity, and intestinal absorptive capacity of Nile tilapia. The recommended dose for dietary AFOC was around 11., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The waste recycling of lemon peel, as a functional feed additive in aquafeed was evaluated by estimating the effects of fermented lemon peel (FLP) supplementation in diet on growth performance, innate immune responses, and susceptibility to Photobacterium damselae of grouper, Epinephelus coioides. A basal diet was added FLP at 0%, 1%, 3%, and 5%. Four tested diets were each fed to juvenile grouper (initial weight: 15.89 ± 0.10 g, triplicate groups) in a recirculation rearing system for eight weeks. Fish fed diets with 0%-3% FLP exhibited higher (p < 0.05) final weight, weight gain, and feed efficiency than fish fed the 5% FLP-diet. After challenge test, fish fed the 3% FLP-diet appeared the lowest mortality, followed by fish fed the 1% FLP-diet, and lowest in fish fed 0% and 5% FLP-diets. Plasma lysozyme activities were higher in fish fed diets with FLP than in fish fed the FLP-free control diet before challenge test. After challenge, fish fed diets with 1% and 3% FLP showed highest lysozyme activities, followed by fish fed the diet with 5% FLP, and lowest in fish fed the control diet. Hepatic malondialdehyde content was higher in fish fed the control diet than in fish fed diets with 1%-3% FLP. Results found that diets supplemented with 1%-3% fermented lemon peel can enhance lysozyme activity and resistance to pathogen P. damselae of grouper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The sea urchin Lytechinus variegatus is considered a good candidate for aquaculture, but bacterial diseases are a major challenge in culture conditions. The innate immunological defenses of L. variegatus to bacterial challenges were assessed through hematology parameters, in vitro phagocytosis, lysozyme activity and total plasma protein concentrations in cell-free coelomic fluid. Adult sea urchins were inoculated with Microccocus lysodeikticus, Escherichia coli and Vibrio parahaemolyticus in the cavity coelomic. Filtrated and sterile seawater (FSW) injected and non-injected sea urchins were used as control groups. Righting time, external aspects and behavior of sea urchins were evaluated. Twenty-four hours post-inoculation, we found an increase in the population of colorless spherule cells (CLS), phagocytosis, and humoral responses in sea urchins challenged by bacterial inoculations. Righting time was not affected by the treatments and apparent external signs of disease were not observed at least during 96h post-inoculation. The immunological system of L. variegatus quickly eliminated pathogenic microorganisms. CLS and lysozyme activity cooperate in the immune defenses of L. variegatus, showing an extraordinary efficiency for adjusting the immune defenses under stress caused by microbes. We recommend that the cellular and humoral markers serve as routine tests to monitor health status in sea urchins., (Copyright © 2021. Published by Elsevier Ltd.)

The aim of this study was to investigate the effects of dietary bile acids (BAs) on intestinal healthy status of tongue sole in terms of immunity, antioxidant status, digestive ability, mucosal barrier-related genes expression and microbiota. Three experimental diets were prepared with BA levels at 0 mg/kg (CT), 300 mg/kg (BA1) and 900 mg/kg (BA2) in a commercial basal diet. Each diet was fed to three replicates with 120 fish (10.87 ± 0.32 g) in each tank. After an 8-week feeding trial, growth parameters were significantly enhanced in both BAs supplementary groups (P < 0.05), and compared with CT group, survival rate in BA2 group was significantly improved (P < 0.05). Intestinal lysozyme activity and contents of immunoglobulin M and complement 3 were significantly increased in both BAs supplementary groups (P < 0.05), suggesting an enhancement effect on the non-specific immune response. BAs inclusion also significantly improved intestinal antioxidant capabilities by increasing antioxidase activities and decreasing malondialdehyde levels. In addition, compared with CT group, intestinal digestive ability was substantially enhanced as indicated by the significantly increased lipase activity in BA2 group (P < 0.05) and significantly increased amylase activity in BA1 and BA2 groups (P < 0.05). Coincidentally, BAs inclusion significantly upregulated the relative expression of intestinal mucosal barrier-related genes (P < 0.05). Further, dietary BAs distinctly remodeled intestinal microbiota by decreased the abundance of some potential pathogenic bacteria. In conclusion, dietary BAs supplementation is an effective way to improve the intestinal healthy status of tongue sole., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Medicinal herbs are used for growth promotion, disease control and other health benefits in aquaculture industry. Here, we examined the effect of dietary laurel-leaf cistus (Cistus laurifolius) ethanolic extract on growth performance, digestive enzyme activity, haematological profile and nonspecific immune responses in common carp (Cyprinus carpio). In addition, resistance against Aeromonas hydrophila infection was examined. Common carp was fed diets containing 0 (Control), 0.1 (CL0.1), 0.5 (CL0.5) and 1 (CL1) g kg -1 laurel-leaf cistus extract for 45 days. After 30 days, superoxide anion production (SAP) increased in CL0.1 and CL0.5 fish groups and at the end of the study all experimental fish groups had higher SAP compared to that of the control (P ˂ 0.05). Lysozyme activity (LA) was elevated in CL0.5 and CL1 treated groups on 30th day (P < 0.05), and this increase was only observed in C0.1 fish group at the end of study compared to control (P ˂ 0.05). Myeloperoxidase activity was significantly increased in CL0.5 and CL1 fish groups at the end of study. IL-1βgene expression was significantly increased in treated fish in a dose-depended manner. Similar results were observed for transcription of IL-6 and IL-8 (P < 0.05). Anti-inflammatory cytokines, IL-10 and TGF-β were highly up-regulated in the intestine and head kidney of CL treated fish groups compared to control (P < 0.05). At the end of experiment, significantly higher final body weight, weight gain, and specific growth rate were obtained in CL0.1 treated fish group compared to control. However, growth was negatively affected in CL1 fish group (P < 0.05). CL1 fish group had also a significantly higher FCR. Amylase activity was significantly increased in all experimental fish groups compared to control (P ˂ 0.05). Trypsin activity was decreased in CL0.1 and CL1 fish groups (P ˂ 0.05). WBC and RBC were significantly increased (P ˂ 0.05) in CL0.5 and CL1 fish groups, whereas haemoglobin, haematocrit, mean cell, mean cell haemoglobin contents were no significantly changed among control and treatment groups. Result of challenge test with A. hydrophila exhibited that survival rate in all treatment groups was significantly higher than that of control. These findings demonstrated that laurel-leaf cistus at 0.1 g kg -1 can be a suitable candidate for growth promotion, immune system induction and infection control in fish., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The efficiency of laccase-catalyzed protein cross-linking can be impacted by substrate protein structure and competing reactions. In this study, chemical grafting of ferulic acid (FA) on protein surface was applied to modulate the cross-linking of two inflexible globular proteins, lysozyme (LZM) and ovalbumin (OVA). The extent of FA-grafting was positively correlated with protein cross-linking extent, and determined the molecular weight profile and structures of the cross-linked product. While laccase-catalyzed reactions (with or without free FA mediator) did not lead to evident cross-linking of the native proteins, oligomeric (up to 16.4%), polymeric (up to 30.6%) FA-LZMs and oligomeric FA-OVA (5.1-31.1%) were obtained upon the enzymatic treatments. The cross-linking on the grafted FA sites occurred mainly through the formation of 8-5'-noncyclic-dehydro-diferulic linkages. The effects of investigated cross-linking approach on the emulsifying, foaming properties and the immunoglobulin E (IgE) binding capacity of LZM and OVA were also evaluated in relation to the structural properties of cross-linked proteins., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

An eight-week investigation was conducted to access the potential impact of dietary watermelon rind powder (WMRP) and L. plantarum CR1T5 (LP) administered individually or in combination on immunity, disease resistance, and growth rate of Nile tilapia fingerlings cultured in a biofloc system. Three hundred twenty fish (average weight 16.57 ± 0.14 g) were distributed into 16 tanks at a rate of 20 fish per tank. The fish were fed different diets: Diet 1 (0 g kg -1 WMRP and 0 CFU g -1 L. plantarum) (control), Diet 2 (40 g kg -1 WMRP), Diet 3 (10 8  CFU g -1 LP), and Diet 4 (40 g kg -1 WMRP + 10 8  CFU g -1 LP) for eight weeks. A completely randomized design (CRD) with four replications was applied. Skin mucus, serum immunity, and growth parameters were analyzed every 4 weeks, and a challenge study against S. agalactiae was conducted at the end of the experiment. The findings showed that the inclusion of WMRP + LP, administrated individually or in a mixture, significantly (P<0.05) stimulated growth, skin mucus, and serum immune parameters of Nile tilapia fingerlings compared with the control. The highest values were detected in fish fed the combination of WMRP and LP, as opposed to individual administration of either WMRP or LP, in which no significant differences were detected. Within the challenge study, the relative percent survival (RPS) in Diet 2, Diet 3, and Diet 4 was 48.0%, 52.0%, and 68.0%, respectively. Fish fed 40 g kg -1 WMRP + LP produced significantly higher RPS and protection against S. agalactiae than the other treated groups. Current results suggest that the dual administration of WMRP and LP maybe an effective feed additive for Nile tilapia grown in an indoor biofloc system, capable of improving growth parameters and increasing resistance to S. agalactiae infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The gut microbiota affects the physiology and metabolism of animals and its alteration can lead to diseases such as gut dysplasia or metabolic disorders. Several reports have shown that the immune system plays an important role in shaping both bacterial community composition and abundance in Drosophila , and that immune deficit, especially during aging, negatively affects microbiota richness and diversity. However, there has been little study at the effector level to demonstrate how immune pathways regulate the microbiota. A key set of Drosophila immune effectors are the antimicrobial peptides (AMPs), which confer defense upon systemic infection. AMPs and lysozymes, a group of digestive enzymes with antimicrobial properties, are expressed in the gut and are good candidates for microbiota regulation. Here, we take advantage of the model organism Drosophila melanogaster to investigate the role of AMPs and lysozymes in regulation of gut microbiota structure and diversity. Using flies lacking AMPs and newly generated lysozyme mutants, we colonized gnotobiotic flies with a defined set of commensal bacteria and analyzed changes in microbiota composition and abundance in vertical transmission and aging contexts through 16S rRNA gene amplicon sequencing. Our study shows that AMPs and, to a lesser extent, lysozymes are necessary to regulate the total and relative abundance of bacteria in the gut microbiota. We also decouple the direct function of AMPs from the immune deficiency (IMD) signaling pathway that regulates AMPs but also many other processes, more narrowly defining the role of these effectors in the microbial dysbiosis observed in IMD-deficient flies upon aging. IMPORTANCE This study advances current knowledge in the field of host-microbe interactions by demonstrating that the two families of immune effectors, antimicrobial peptides and lysozymes, actively regulate the gut microbiota composition and abundance. Consequences of the loss of these antimicrobial peptides and lysozymes are exacerbated during aging, and their loss contributes to increased microbiota abundance and shifted composition in old flies. This work shows that immune effectors, typically associated with resistance to pathogenic infections, also help shape the beneficial gut community, consistent with the idea that host-symbiont interactions use the same "language" typically associated with pathogenesis.

In the present study, antioxidant activity, immune responses, and growth performance of rainbow trout (Onchorhynchus mykiss) juveniles fed with diets supplemented with dandelion (Taraxacum officinalis) and lichen (Usnea barbata) extracts were assessed. Four different concentrations of aqueous methanolic extract of the plants (0% (control), 0.1%, 0.5%, and 1% (D, dandelion; L, lichen) were added to the diets, and fish were fed for 75 days. On the 15 th , 45 th , and 75 th day of the study, liver antioxidant enzyme activities were determined, and immune responses were determined every 15 th day. The results showed that SOD activity increased in the fish group of 0.1% D on the 15 th and 45 th day compared to control; however, it was lower in all the lichen extract-treated groups than in control at almost all sampling times, except on the 15 th day in the 0.1% L group. CAT activity showed an increased value (P < 0.05) in 0.5% L and 1% L treated fish groups on the 15 th day, in fish of 1% D and 1% L groups on 45 th and on 75 th day in 0.1% D group. GPX activity increased on the 15 th day of the study in fish of 0.1% D group, on the 45 th day in 1% D and 1% L groups and on the 75 th day in fish of 0.5% D, 0.1% D, and 0.5% L groups (P < 0.05). G6PDH enhanced in all treatment groups compared to control on the 15 th day, except in 0.1% L and 0.5% L groups. An elevated G6PDH activity was also observed on the 75 th day of the study in 0.5% D, 1% D, and 0.5% L fish groups. An increase on lipid peroxidation (LP) was observed in all L groups on the 45 th day of the study. Lysozyme activity was determined to be the highest in 0.5% and 1% L on the 45 th day, in 0.1% L on the 60 th day and in the 0.5% L fish group on the 75 th day compared to control (P < 0.05). Myeloperoxidase was found to be the highest at the end of the study in 1% L fish group compared to the control (P < 0.05). In conclusion, we suggest the use of dandelion to combat oxidative stress and to lower FCR and the use of lichen to modulate the immune response in rainbow trout. The use of such products will be economical for aquaculture and harmless for the environment., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The degree of antigen adsorption to adjuvants in subunit vaccines may significantly influence the immune responses they induce upon vaccination. Commonly used approaches for studying how the level of adsorption affects the induction of antigen-specific immune responses include (i) using adjuvants with different abilities to adsorb antigens, and (ii) comparing different antigens selected based on their ability to adsorb to the adjuvant. A weakness of these approaches is that not only the antigen adsorption level is varied, but also other important functional factors such as adjuvant composition and/or the B/T cell epitopes, which may affect immunogenicity. Hence, we investigated how changing the adsorption capabilities of a single antigen to an adjuvant influenced the vaccine-induced immune responses. The model antigen lysozyme, which displays a positive net charge at physiological pH due to an isoelectric point (pI) of 11, was succinylated to different extents, resulting in a reduction of the pI value to 4.4-5.9, depending on the degree of succinylation. A pronounced inverse correlation was found between the pI value of the succinylated lysozyme analogues and the degree of adsorption to a cationic liposomal adjuvant consisting of dimethyldioctadecylammonium bromide (DDA) and trehalose dibehenate (TDB) (CAF®01). Furthermore, increased adsorption to this adjuvant correlated directly with the magnitude of lysozyme-specific Th1/Th17 immune responses induced by the vaccine in mice, while there was an inverse correlation with antibody induction. However, high lysozyme-specific antibody titers were induced with an increased antigen dose, even upon vaccination with a strongly adsorbed succinylated lysozyme analogue. Hence, these data illustrate that the degree of lysozyme adsorption to CAF®01 strongly affects the quality of the resulting immune responses., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Despite the significant adverse clinical consequences of RBC alloimmunization, our understanding of the signals that induce immune responses to transfused RBCs remains incomplete. Though RBC storage has been shown to enhance alloimmunization in the hen egg lysozyme, ovalbumin, and human Duffy (HOD) RBC alloantigen mouse model, the molecular signals leading to immune activation in this system remain unclear. Given that the nonclassical major histocompatibility complex (MHC) Class I molecule CD1D can bind to multiple different lysophospholipids and direct immune activation, we hypothesized that storage of RBCs increases lysophospholipids known to bind CD1D, and further that recipient CD1D recognition of these altered lipids mediates storage-induced alloimmunization responses., Study Design and Methods: We used a mass spectrometry-based approach to analyze the changes in lysophospholipids that are induced during storage of mouse RBCs. CD1D knockout (CD1D-KO) and wild-type (WT) control mice were transfused with stored HOD RBCs to measure the impact of CD1D deficiency on RBC alloimmunization., Results: RBC storage results in alterations in multiple lysophospholipid species known to bind to CD1D and activate the immune system. Prior to transfusion, CD1D-deficient mice had lower baseline levels of polyclonal immunoglobulin (IgG) relative to WT mice. In response to stored RBC transfusion, CD1D-deficient mice generated similar levels of anti-HOD IgM and anti-HOD IgG., Conclusion: Although storage of RBCs leads to alteration of several lysophospholipids known to be capable of binding CD1D, storage-induced RBC alloimmunization responses are not impacted by recipient CD1D deficiency., (© 2021 AABB.)

The complement fragment C5a is closely associated with adaptive immune induction in the mucosa. However, the mechanisms that control CD8 + T cell responses by C5a have not been extensively explored. This study reveals that C5/C5a in the Peyer's patch (PP) subepithelial dome increases upon oral Listeria infection. We hypothesize that C5aR + PP cells play an important role in the induction of antigen-specific T cell immunity. Using single-cell RNA sequencing, we identify C5aR- and lysozyme-expressing dendritic cells (C5aR + LysoDCs) in PP and examine their role in CD8 + T cell immune induction. Stimulation of C5aR + LysoDCs by C5a increases reactive oxygen species levels, leading to efficient antigen cross-presentation, which elicits an antigen-specific CD8 + T cell response. In C5-deficient mice, oral co-administration of C5a and Listeria enhances Listeria-specific cytotoxic T cell levels. Collectively, these findings suggest a role of the complement system in intestinal T cell immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mesoporous silica nanoparticles (MSNs) have been used in the field of biomedicine as antigen carriers and adjuvants for protective antigens. In the present study, an oral nanovaccine against Vibrio alginolyticus was prepared employing MSNs as carriers. The uptake of the dihydrolipoamide dehydrogenase (DLDH) antigens in the intestine of large yellow croaker was evaluated using an immunohistochemistry assay. Additionally, the effects of the nanovaccine on the early immune response in large yellow croaker were investigated via oral vaccination. The presence of the antigens was detected in the mucosa and lamina propria of the foregut, midgut, and hindgut of large yellow croaker at 3 h following oral immunization. The expression levels of cytokines (i.e., lysozyme, IFN-γ, IFITM, TNF-α, IL-1β, IL-2, IL-4, IL-10, and IL-13) in the intestine, spleen, and head kidney tissues of large yellow croaker before and after the immune challenge were determined via RT-qPCR assay. The obtained results revealed that the expression levels of lysozyme, IFN-γ, IFITM, TNF-α, IL-1β, IL-2, IL-4, IL-10, and IL-13 in the intestine and head kidney of the vaccinated large yellow croaker, as well as the expression of lysozyme, IL-1β, and IL-10 in the spleen, exhibited time-dependent oscillation regulation patterns. Notably, the nanovaccine immunization could induce early (6 h) and high expression of IFN-γ in the spleen and kidney tissues after the bacterial infection. The current study supplements the available data on the early immune response to fish nanovaccines. It also provides a valuable theoretical basis for the future development of large yellow croaker oral vaccines., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction and Objectives: It was urgent to explain the role of egg yolk allergen sensitization to the egg allergic population and we would evaluate the diagnostic value of allergen components in whole eggs, including egg white and egg yolk., Materials and Methods: Firstly, we collected 99 positive and 21 negative sera against egg allergy. Then we used modified enzyme linked immunosorbent assay (ELISA) to survey specific IgE (sIgE) to all-proven and single component in eggs, Ovomucoid (Gal d 1), Ovalbumin (Gal d 2), Ovotransferrin (Gal d 3), Lysozyme C (Gal d 4), Serum Albumin (Gal d 5), and YGP42(Gal d 6) in allergic and non-allergic populations. Last but not least, we studied the sIgE reactivities to egg allergen components by receiver operating characteristic (ROC) analysis., Results: Among egg-allergic individuals, nearly 10% were sensitized to five of six egg allergen components, and the cross-reaction frequency between two egg yolk allergens with Gal d 1 was about 30% in the groups diagnosed with egg allergy or non-allergy. The best component-combination diagnosis in egg allergy of Gal d 1+ Gal d 6 demonstrated the largest area under curve (AUC) of 0.994., Conclusions: Our results suggested that there were individual differences in allergenicity of different egg allergen components, especially in the samples negative to egg allergy diagnosed but sensitive to egg yolk components. It was indicated that component resolved diagnosis of egg yolk improved the value for egg allergy management indispensably.

A completely randomized experimental design carried out to investigate the effects of different levels of Pediococcus acidilactici (PA) including 0 (basal diet as a control diet), 1 × 10 6 , 2 × 10 6 , 4 × 10 6 , and 8 × 10 6 colony-forming unit (CFU) per gram of the diet for 60 days on the mucosal immunity responses, growth, and reproductive performance, in zebrafish, Danio rerio (with mean weigh ± SE: 120 ± 10 mg). The obtained results revealed that the best growth and reproduction indices were related to the concentration of 4 × 10 6  CFU PA g -1 diet (P < 0.05). The maximum activities of mucosal immune responses including total protein, alternative complement system, IgM, and lysozyme were observed in the fish fed with 4 × 10 6  CFU PA g -1 diet (P < 0.05). Furthermore, the maximum alkaline phosphatase activity of skin mucus was recorded in the fish fed with 8 × 10 6  CFU PA g -1 diet (P < 0.05). Fish fed with 4 × 10 6  CFU PA g -1 diet had the highest villus length and width of the intestine (P < 0.05). Supplementing the diet with 4 × 10 6  CFU PA g -1 diet more significantly enhanced Cyp19a gene expression in comparison with this in other groups. Hence, PA with a concentration of 4 × 10 6  CFU g -1 diet can be considered as a proper level of probiotic for improving the health, growth, and reproductive performance of the D. rerio.

Accumulating evidence has suggested a correlation of tumor infiltrating B cells (TiBcs) and a good prognosis of cancer diseases. In some cases, TiBcs appear to have experienced antigen stimulation since they have undergone class-switching and somatic hypermutation and formed tertiary lymphoid structures around tumors together with T cells. Assuming TiBcs include those that recognize some tumor antigens, we sought to investigate their possible usefulness for cell-mediated immunotherapies. To expand usually small number of TiBcs in vitro, we modified our B cell culture system: we transduced B cells with ERT2-Bach2 so that they grow unlimitedly provided with tamoxifen, IL-21 and our original feeder cells. Such cells differentiate into plasma cells and produce antibodies upon withdrawal of tamoxifen, and further by addition of a Bach2-inhibitor in vitro. As a preliminary experiment, thus expanded splenic B cells expressing a transgenic antigen receptor/antibody against hen egg lysozyme were intravenously injected into mice pre-implanted with B16 melanoma cells expressing membrane-bound HEL in the skin, which resulted in suppression of the growth of B16 tumors and prolonged survival of the recipient mice. To test the usefulness of TiBcs for the immunotherapy, we next used APCmin/+ mice as a model that spontaneously develop intestinal tumors. We cultured TiBcs separated from the tumors of APCmin/+ mice as above and confirmed that the antibodies they produce recognize the APCmin/+ tumor. Repeated injection of such TiBcs into adult APCmin/+ mice resulted in suppression of intestinal tumor growth and elongation of the survival of the recipient mice. Serum antibody from the TiBc-recipient mice selectively bound to an antigen expressed in the tumor of APCmin/+ mice. These data suggest a possibility of the novel individualized cancer immunotherapy, in which TiBcs from surgically excised tumor tissues are expanded and infused into the donor patients., Competing Interests: The authors have declared that no competing interests exist.

In the current study, we investigated the effect of a probiotic bacterium (Lactobacillus rhamnosus ATCC 7469) microencapsulated with alginate and hi-maize starch and coated with chitosan on improving growth factors, body composition, blood chemistry, and the immune response of rainbow trout (initial weight: 18.41 ± 0.32 g). Four experimental diets were formulated to feed fish for 60 days. They were control diet without any additive (C), diet added with beads without probiotic (E), a probiotic sprayed to the diet (L.r), and encapsulated probiotic supplemented diet (E-L.r). The results indicated that feeding with E-Lr significantly improved weight gain (84.98 g) and feed conversion ratio (0.95) compared to the other groups (P < 0.05). Also, fish fed E-Lr diet had a significantly higher value of whole-body protein (17.51%), total protein in the blood (4.98 g/dL), lysozyme (30.66 U/mL), alternative complement pathway hemolytic activity (134 U/mL), superoxide dismutase (203 U/mg protein), and catalase (528.33 U/mg protein) (P < 0.05) as compared to those fed the control diet. Similarly, a higher relative expression of immune-related genes such as interleukin-1 (Il-1) and tumor necrosis factor-alpha (TNF-1α) were reported in those fed E-L.r and L.r diets respectively. Interestingly, the fish fed dietary E-L.r had a significantly lower value of lipid in the whole body (4.82%) and cholesterol in the blood (160.67%) in comparison with those fed the control diet (P < 0.05). At the end of the experiment, all groups were challenged by Yersinia ruckeri where the survival rate of rainbow trout fed dietary E-L.r (70.36%) was statistically higher than that of the others (P < 0.05). Overall, the results suggested that encapsulated probiotic Lact. rhamnosus ATCC 7469 acted better than unencapsulated probiotic and has a potential to improve growth performance, flesh quality, and the immune response of rainbow trout.

A 56-day research was performed to examine the influence of graded levels (0 (control), 0.5, 1 and 2%) of Fern (Adiantum capillus-veneris) leaves powder (FLP) in diet on immune competence and growth of common carp (Cyprinus carpio, initial weight = 20 g). The serum total immunoglobulins content and lysozyme activity in the 1 and 2% FLP groups remarkably increased compared to the other groups (P < 0.05). The skin mucosal lysozyme activity enhanced with increasing dietary FLP level in a dose-response manner. Fish fed on the FLP-supplemented diets had higher skin-mucosal superoxide dismutase activity than the control (P < 0.05). However, serum antioxidant enzymes were not affected by dietary fern (P > 0.05). The serum bactericidal activity against human and fish pathogens increased with enhancing the FLP level in diet against Staphylococcus aureus, Escherichia coli (EHEC ATCC 43895), Escherichia coli (CI), Pseudomonas aeruginosa, Klebsiella pneumonia and Aeromonas hydrophila. The serum antibacterial activity against Yersinia ruckeri in the 2% FLP group was higher than the other treatments. Furthermore, the serum bactericidal activity against P. aeruginosa (ATCC 27853) only observed in fish fed on the 1 and 2% FLP-supplemented diets. The skin mucosal bactericidal activity and inhibitory effects increased with enhancing the FLP level in diet against E. coli, K. pneumonia, Y. ruckeri and A. hydrophila in a dose response manner. Moreover, the skin mucosal bactericidal activity against S. aureus only observed in fish fed on 1 and 2% FLP-supplemented diets. The weight gain values in the 1 and 2% FLP groups were higher than the other treatments (P < 0.05). Feed conversion ratio (FCR) improved with increasing FLP level in diet in a dose-response manner (P < 0.05). By considering serum and mucosal bactericidal activities against different pathogenic bacteria, the supplementation of 2% FLP in diet is recommended for C. carpio during grow-out phase., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In this study, we isolated and characterized natural antibodies found in serum samples from Bester sturgeon (Huso huso × Acipenser ruthenus). Natural antibodies specifically detected hen egg lysozyme (HEL), keyhole limpet hemocyanin (KLH), and several species of pathogenic bacteria. Interestingly, we detected no antibodies with similar specificity in serum samples from rainbow trout (Oncorhynchus mykiss) or from Japanese flounder (Paralichthys olivaceus). Binding capacity of the sturgeon natural serum antibodies increased slightly at 7 months compared to 3 months after hatching. Antigen-specific antibodies against KLH, Aeromonas hydrophila and Streptococcus iniae were affinity-fractionated from naive sera of Bester sturgeon; specific detection of the corresponding antigens was observed. We conclude that Bester sturgeon are capable of generating unique natural antibodies including those that are pathogen-specific., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The effect of diet enriched with dried lemon (Citrus limon) peel was fed to Labeo rohita at three different levels (0, 1, 2.5, and 5 g kg -1 ) for a period of 60 days; the impact of the diet on the hematology, antioxidant activity and immunological reaction and gene expression against Aeromonas sorbia is reported. In both un-challenged and challenged groups treated with 2.5 g and 5 g kg -1 dried lemon peel diets, the enhanced significant changes are: the weight gain and specific growth rate, white blood cell and total protein content, the antioxidants: superoxide dismutase, catalase, glutathione peroxidase, and glutathione activities, the respiratory burst, alternative complement pathway, complement C3, and total immunoglobulin M levels. Similarly, the heat shock protein-70 and -90, superoxide dismutase, glutathione peroxidase, glutathione, interleukin-1β and -8, tumor necrosis factor alpha, inducible nitric oxide synthase, transforming growth factor beta, and immunoglobulin M were up-regulated significantly. Any dried lemon peel enriched diet increased the phagocytic and lysozyme activities significantly in both groups. In the un-challenged group treated with 0 g kg -1 diet or in both groups treated with 2.5 g kg -1 diet the SR was 100%. These results indicate that in both un-challenged and challenged-treated groups the 2.5 and 5 g kg -1 dried lemon peel enriched diets positively modulate growth rate, physiology, and antioxidant status, innate-adaptive immune response as well as antioxidant and immune related gene expression in L. rohita against A. sorbia., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Vaccination plays a critical role in the protection of humans and other animals from infectious diseases. However, the same vaccine often confers different protection levels among individuals due to variation in genetics and/or immunological histories. While this represents a well-recognized issue in humans, it has received little attention in fish. Here we address this knowledge gap in a proteomic study of rainbow trout ( Oncorhynchus mykiss , Walbaum), using non-lethal repeated blood sampling to establish the plasma protein response of individual fish following immunization. Six trout were immunized with adjuvanted hen egg-white lysozyme (HEL) and peripheral blood sampled at ten time points from day 0 to day 84 post-injection. We confirm that an antigen-specific antibody response to HEL was raised, showing differences in timing and magnitude among individuals. Using label-free liquid chromatography-mass spectrometry, we quantified the abundance of 278 plasma proteins across the timecourse. As part of the analysis, we show that this approach can distinguish many (but not all) duplicated plasma proteins encoded by paralogous genes retained from the salmonid-specific whole genome duplication event. Global variation in the plasma proteome was predominantly explained by individual differences among fish. However, sampling day explained a major component of variation in abundance for a statistically defined subset of 41 proteins, representing 15% of those detected. These proteins clustered into five groups showing distinct temporal responses to HEL immunization at the population level, and include classical immune (e.g. complement system members) and acute phase molecules (e.g. apolipoproteins, haptoglobins), several enzymes and other proteins supporting the immune response, in addition to evolutionarily conserved molecules that are as yet uncharacterized. Overall, this study improves our understanding of the fish plasma proteome, provides valuable marker proteins for different phases of the immune response, and has implications for vaccine development and the design of immune challenge experiments., (Copyright © 2020 Bakke, Monte, Stead, Causey, Douglas, Macqueen and Dooley.)

In this study, the effect of ammonia derived from different stocking densities on immunological, hematological, and oxidative stress parameters was analyzed in the blood or liver of red seabream. Density- and time-dependent increases in inorganic nitrogen compounds were measured for 20 days by analyzing the three major inorganic nitrogen compounds, total ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen. Three immunity parameters, alternative complement activity, lysozyme activity, and total immunoglobulin content were significantly decreased in the blood at the highest stocking density (10 kg m -3 ). The concentrations of hemoglobin and white blood cells were significantly decreased at 10 kg m -3 , while there was no significant change in red blood cells. The significant increases in cortisol level and the enzymatic activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase at 10 kg m -3 clearly supported inorganic nitrogen compounds-triggered stress. A significant elevation of lipid peroxidation value and depletion of intracellular glutathione were observed at 5 and/or 10 kg m -3 in the liver tissue. The hepatic enzymatic activities of antioxidant defense enzymes, catalase and superoxide dismutase were also significantly increased. When a protein skimmer removes the inorganic nitrogen compounds at the highest density, most parameters showed no significant change. Taken together, these results suggest that accumulated inorganic nitrogen compounds at the highest stocking density inhibit innate immunity and induce oxidative stress in red seabream. This information will be helpful to maintain homeostasis of red seabream by controlling immunity and oxidative status through inorganic nitrogen compounds removal in intensive culture condition., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Plant viruses are biologically safe for mammals and can be successfully used as a carrier/platform to present foreign epitopes in the course of creating novel putative vaccines. However, there is mounting evidence that plant viruses, their virus-like and structurally modified particles may also have an immunopotentiating effect on antigens not bound with their surface covalently. Here, we present data on the adjuvant properties of plant viruses with various shapes (Tobacco mosaic virus, TMV; Potato virus X, PVX; Cauliflower mosaic virus, CaMV; Bean mild mosaic virus, BMMV) and structurally modified TMV spherical particles (SPs). We have analysed the effectiveness of immune response to individual model antigens (ovalbumin, OVA/hen egg lysozyme, HEL) and to OVA/HEL in compositions with plant viruses/SPs, and have shown that CaMV, TMV and SPs can effectively induce total IgG titers to model antigen. Some intriguing data were obtained when analysing the immune response to the plant viruses/SPs themselves. Strong immunity was induced to CaMV, BMMV and PVX, whereas TMV and SPs stimulated considerably lower self-IgG titers. Our results provide new insights into the immunopotentiating properties of plant viruses and can be useful in devising adjuvants based on plant viruses.

Polybrominated diphenyl ethers (PBDEs) are ubiquitous in marine ecosystems and have been suggested to bioaccumulate in aquatic food webs, with potentially negative impacts on marine organism. In this study, a 21-day experiment was performed under controlled laboratory conditions, in which 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), the most biotoxic PBDE in the marine environment, was fed to rainbow trout (Oncorhynchus mykiss) at concentrations of 50 and 500 ng g -1 in the diet. BDE-47 significantly decreased the specific growth rate of O. mykiss and was highly concentrated in the liver and head kidney, as evidenced by increased bioaccumulation factor (BAF) values. Tissue observation revealed impairment of the microstructure of the head kidney. Important immune factors in the skin, blood and head kidney were significantly inhibited by BDE-47 treatment (p < 0.05), whereas the respiratory burst activity of macrophages was enhanced. Additionally, immune-related genes were strongly downregulated following BDE-47 exposure (p < 0.05). In a bacterial challenge, the treatment groups had much higher mortality than did the control group (p < 0.05). BDE-47 accumulated and impaired immune organs, and the hierarchy of immune responses was impaired, consequently reducing O. mykiss resistance to pathogen invasion., Competing Interests: Declaration of Competing Interest All the authors do not have any possible conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cadmium (Cd), a substance with one of the most critical health hazard indices, can cause damage to both the blood and kidneys and accumulates in the body at last. The present work studied the toxicological effects of Cd and the therapeutic effects of metallothionein (MT) and vitamin E (VE) on the trunk kidney and blood of freshwater grass carp (Ctenopharyngodon idellus). Grass carp were divided into three groups: Cd + phosphate-buffered saline (PBS) group, Cd + VE group, and the Cd + MT group. Fish were injected with CdCl 2 on the first day and then VE, MT, or PBS was administered 4 days post-injection. Fish not injected with Cd were used as a negative control. The blood and trunk kidney amassed Cd and suffered severe damage in the forms of organ toxicity cytotoxicity, and immunotoxicity. However, the MT reduced the Cd content in the trunk kidney and blood and partially stabilized the damaged organs. Treatment with VE, however, only demonstrated weaker protection against on Cd-induced toxicity. The results indicate that exogenous MT may play an essential role in restoring homeostasis of the Cd-poisoned urinary and circulatory system and that it may help eliminate Cd in aquatic animals.

Effects of dietary Lactobacillus plantarum (KC426951) on growth and innate responses of Nile tilapia Oreochromis niloticus were evaluated in biofloc technology system and stagnant-renewal culture system (SRCS). The 90-day-long experiment contained four treatments: SRCS without probiotic (T1), SRCS with probiotic (T2), biofloc without probiotic (T3), and biofloc with probiotic (T4). The administration dose of probiotic was 2 × 10 8  CFU kg -1 diet. At the end of experiment, the mean final weights, specific growth rates, feed conversion ratios, and total biomass were significantly (P < 0.05) better in BFT treatments, with no significant effect of probiotic on these parameters in both culture systems. Meanwhile, skin mucosal parameters including total protein (TP), lysozyme (LYZ), alkaline phosphatase (ALP), and protease (PRO) activity were significantly enhanced following probiotic supplementation. T4 treatment displayed a significantly higher LYZ and ALP activity in mucus versus other treatments. Also, serum alternative complement activity was significantly heightened in probiotic-supplemented fish. Superoxide dismutase activity in T4 was detected higher than that of SRCS groups. The results of the current study demonstrated the enhancement of some mucosal and serum innate responses of Nile tilapia in both culture systems upon L. plantarum (KC426951) supplementation.

In aquatic animals, the mucosal barrier is the first line of innate immune defence against external chemicals and pathogens. In this study, the effects of dietary Moringa oleifera leaf (MOL) supplementation on skin and gill mucosal immunity, antioxidants and stress responses were evaluated in seabream (Sparus aurata) fingerlings exposed to hydrogen peroxide (H 2 O 2 ). A total of 144 specimens (10.11 ± 0.41 g) were divided into four treatments (three replicates per treatment contained 12 specimens each) and fed a non-supplemented control diet or a 1, 2.5 or 5% MOL-supplemented diet. After three weeks of feeding, six specimens from each aquarium were sampled for blood, mucus and tissues. The other six fish in each aquarium were subjected to H 2 O 2 exposure. The results revealed that MOL did not negatively affect either cortisol or glucose levels. MOL supplementation significantly (P < 0.05) improved skin mucosal immunity-related characteristics, including phosphatase, peroxidase and lysozyme activity and IgM levels. Additionally, MOL upregulated the expression of antioxidant genes (sod and cat), an anti-inflammatory gene (tgf-β), tight junction protein genes (occludin and zo-1), c3, and igm in both the skin and gills. However, H 2 O 2 exposure significantly (P < 0.05) increased both cortisol and glucose levels and disrupted skin mucosal immune function by significantly (P < 0.05) decreasing phosphatase, peroxidase, protease, antiprotease and lysozyme activity and IgM levels. H 2 O 2 exposure severely decreased the mRNA levels of the studied genes. MOL dietary supplementation at the 5% level successfully attenuated the negative effects of H 2 O 2 on the mucosal immune response in both the skin and gills. In conclusion, dietary MOL supplementation at the 5% level is recommended to improve S. aurata mucosal immune function under both normal and stress conditions. Additionally, exposure to H 2 O 2 disrupts the mucosal immunity of fish. This contributes knowledge on the routes involved in mucosal innate immunity and could help to understand the fish resistance against chemicals exposure. Graphical abstract.

Human viruses possess very complex supramolecular structures. Both icosahedral and enveloped viruses typically display an array of viral-encoded protein antigens at varied spatial densities on the viral particle surface. The viral nucleic acid genome, on the other hand, is encapsulated inside the viral particle. Although both the surface antigen and the interior nucleic acids could independently produce immunological responses, how B cells integrate these two types of signals and respond to a typical virus particle to initiate activation is not well understood at a molecular level. The study of these fundamental biological processes would benefit from the development of viral structural mimics that are well constructed to incorporate both quantitative and qualitative viral features for presentation to B cells. These novel tools would enable researchers to systematically dissect the underlying processes. Here we report the development of such particulate antigens based on liposomes engineered to display a model protein antigen, hen egg lysozyme (HEL). We developed methods to overexpress and purify various affinity mutants of HEL from E. coli . We conjugated the purified recombinant HEL proteins onto the surface of a virion-sized liposome in an orientation-specific manner at defined spatial densities and also encapsulated nucleic acid molecules into the interior of the liposome. Both the chemical conjugation of the HEL antigen on liposome surfaces and the encapsulation of nucleic acids were stable under physiologically relevant conditions. These liposomes elicited antigen-specific B-cell responses in vitro, which validate these supramolecular structures as a novel and effective approach to mimic and systematically isolate the role of essential viral features in directing the B-cell response to particulate antigens.

Evaluation of the molecular mechanisms by which an antibody recognizes a specific antigen could help in better understanding of the protein recognition mechanisms. We previously showed that anti-hen egg lysozyme (HEL) monoclonal antibody, HyC1, recognized the structural and hydrodynamic change in HEL. Here, we generated HyC1 single-chain Fv (scFv), and characterized it using different structural and biophysical methods. Similar to HyC1 monoclonal antibody, HyC1 scFv could recognize native HEL from carboxymethylated Cys6 and Cys127 HEL (CM 6,127 -HEL). Comparison of the binding thermodynamics of HyC1 scFv between HEL and CM 6,127 -HEL showed that the binding enthalpy change was different, while the binding entropy was remained unchanged. The results indicated that the fluctuation of the residual native structure in both HEL and CM 6,127 -HEL was similar. The NMR experiments for 15 N-labeled HyC1 scFv indicated that the flexibility of HyC1 scFv decreased upon the binding to HEL.

In this study, we sequenced and characterized the goose-type lysozyme gene, termed as BsLysG, from the Chinese black sleeper (Bostrychus sinensis). The BsLysG encodes 196 amino acids and contains a soluble bacterial lytic transglycosylases domain, three catalytic residues (Glu 72 , Asp 85 and Asp 102 ) and the GLMQ motif (Gly 97 , Leu 98 , Met 99 and Gln 100 ). No signal peptide was observed in the BsLysG protein. The genomic DNA of BsLysG contains five exons and four introns. The sequence analyses showed that the BsLysG exhibits high similarity with LysG from other fishes. Phylogenetic analyses showed that the BsLysG is clustered together with its counterparts from other teleost fishes. The Real-time PCR analyses showed that the BsLysG was found to be ubiquitously expressed in ten examined organs in Chinese black sleeper, with predominant expression in spleen, followed by head kidney and peripheral blood. Expression analyses showed that the BsLysG was significantly upregulated in vivo after either pathogen Vibrio parahemolyticus infection or poly (I:C) challenge in peripheral blood, head kidney, liver and spleen organs. The purified recombinant BsLysG (rBsLysG) has optimal activity at 35 °C and pH 5.5. The rBsLysG exhibited antimicrobial activity against two Gram-positive bacteria (Micrococcus lysodeikticus and Staphylococcus aureus) and two Gram-negative bacteria (Escherichia coli and V. parahemolyticus). The Scanning electron microscope (SEM) imaging analyses showed that the rBsLysG-treated V. parahemolyticus cells displayed morphological deformation. These results indicate that the BsLysG is involved in host immune defense against bacterial infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Lysozyme is one of the most important anti-bacterial effectors in the innate immune system of animals. Besides its direct antibacterial enzymatic activity, lysozyme displays other biological properties, pointing toward a significant anti-inflammatory effect, many aspects of which are still elusive. Here we investigate the perturbation of gene expression profiles induced by lysozyme in a monocyte cell line in vitro considering a perspective as broad as the whole transcriptome profiling. The results of the RNA-seq experiment show that lysozyme induces transcriptional modulation of the TNF-α/IL-1β pathway genes in U937 monocytes. The analysis of transcriptomic profiles with IPA ® identified a simple but robust molecular network of genes, in which the regulation trends are fully consistent with the anti-inflammatory activity of lysozyme. This study provides the first evidence in support of the anti-inflammatory action of lysozyme on the basis of transcriptomic regulation data resulting from the broad perspective of a whole-transcriptome profiling. Such important effects can be achieved with the supplementation of relatively low concentrations of lysozyme, for a short time of exposure. These new insights allow the potential of lysozyme in pharmacological applications to be better exploited.

Single-domain antibodies (VHHs or nanobodies), developed from heavy chain-only antibodies of camelids, are gaining attention as next-generation therapeutic agents. Despite their small size, the high affinity and specificity displayed by VHHs for antigen molecules rival those of IgGs. How such small antibodies achieve that level of performance? Structural studies have revealed that VHHs tend to recognize concave surfaces of their antigens with high shape-complementarity. However, the energetic contribution of individual residues located at the binding interface has not been addressed in detail, obscuring the actual mechanism by which VHHs target the concave surfaces of proteins. Herein, we show that a VHH specific for hen egg lysozyme, D3-L11, not only displayed the characteristic binding of VHHs to a concave region of the surface of the antigen, but also exhibited a distribution of energetic hot-spots like those of IgGs and conventional protein-protein complexes. The highly preorganized and energetically compact interface of D3-L11 recognizes the concave epitope with high shape complementarity by the classical lock-and-key mechanism. Our results shed light on the fundamental basis by which a particular VHH accommodate to the concave surface of an antigens with high affinity in a specific manner, enriching the mechanistic landscape of VHHs.

Cell surface display is a useful platform to examine the interactions between two proteins of interest, such as immune receptors and ligands. This technique is also useful for studies on the immune receptors of lower vertebrates and invertebrates. However, in many cases, the commonly used cell culture temperature is relatively high for proteins from such organisms. Since insect cells can be cultured at lower temperatures than many other cells, and since they are equipped with "quality control" system, which is advantageous for the presentation of properly folded proteins, we anticipated that the insect cell surface display system could be more suitable for that type of research. In the present study, multiple cloning site of the commercially available expression vector pIB/V5-His was modified, and whether this vector could be useful to present fish immune-related membrane proteins was investigated. Using this plasmid, fugu's CD8α and CC chemokine receptor 7 could be presented on the cell surface. The clones of the lamprey variable lymphocyte receptors obtained previously by the yeast surface display (YSD) system as hen's egg lysozyme (HEL) binders also could be presented on the cell surface and bound to HEL. These results suggest that functional immune-related membrane proteins can be presented on the insect cell surface, indicating that this system is useful for immunological studies on exothermal animals., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As an important economic mollusk in coastal areas, Octopus ocellatus dependents on innate immune system to resist the invasion of microorganisms. Lysozyme is a crucial effector owing to its significant lytic activity against bacterial pathogens during the immune responses. In this study, characteristic and immune function of an I-type lysozyme from O. ocellatus (OoLyz) was investigated. OoLyz shared a close relationship with the lysozymes from other bivalve mollusks. The mRNA of OoLyz exhibited a broad transcript in different tissues/organs, and with the greatest expression in hepatopancreas. The expression of OoLyz was significantly raised when O. ocellatus was infected by Vibrio anguillarum or Micrococcus luteus, suggesting OoLyz participated in innate immune response of host. Prokaryotic recombinant OoLyz (rOoLyz) exhibited obvious bacteriolysis ability towards both gram-negative bacteria V. anguillarum and Escherichia coli, and gram-positive bacteria M. luteus and Staphylococcus aureus. The bacteriolysis activities of rOoLyz towards gram-negative but not gram-positive bacteria was heat stable, indicating that OoLyz might clear gram-positive bacterium by enzyme-dependent mechanisms, but eliminate gram-negative microbe via enzymatic activity independent way. Scanning electron microscopy analysis showed that rOoLyz destroyed microbes by damaging cell wall. More importantly, the fact that rOoLyz could directly degrade the peptidoglycan, further revealed its bactericidal mechanism as a muramidase. Our results revealed the essential role of I-type lysozyme in the innate immunity of O. ocellatus, and shed new light to understand the mechanism of immune defense of mollusks., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Lysozyme is an important immune protein involved in the first line of defense for crustaceans. In the present study, a c-type lysozyme gene (SpLyzC) was cloned and characterized from the mud crab, Scylla paramamosain. The full-length cDNA was 849 bp with an open reading frame of 669 bp, and encoded a polypeptide of 223 amino acids with a calculated molecular mass of 23.7 kDa and an isoelectric point of 8.90. SpLyzC shared conserved active sites with c-type lysozymes from other species, detected in all tested tissues and had higher expression levels in hepatopancreas and gill tissues. The expression of SpLyzC was up-regulated in hepatopancreas and gill after infection with Vibrio parahaemolyticus and Staphylococcus aureus. The density of bacteria in the hemolymph and the mortality of crabs increased following infection with V. parahaemolyticus after SpLyzC expression was silenced by injecting double-strand RNA of SpLyzC. The recombinant protein of the S. paramamosain c-type lysozyme (rSpLyzC) exhibited antibacterial activities against Micrococcus lysodeikticus, S. aureus, Vibrio harveyi and V. parahaemolyticus. These results indicate that SpLyzC could help eliminate bacteria in S. paramamosain and may play an important role in resistance to bacterial infection., (Copyright © 2019. Published by Elsevier Ltd.)

Rationale: Recently a frequent exacerbator phenotype has been described in bronchiectasis, but the underlying biological mechanisms are unknown. Antimicrobial peptides (AMPs) are important in host defence against microbes but can be proinflammatory in chronic lung disease., Objectives: To determine pulmonary and systemic levels of AMP and their relationship with disease severity and future risk of exacerbations in bronchiectasis., Methods: A total of 135 adults with bronchiectasis were prospectively enrolled at three European centres. Levels of cathelicidin LL-37, lactoferrin, lysozyme and secretory leucocyte protease inhibitor (SLPI) in serum and sputum were determined at baseline by ELISA. Patients were followed up for 12 months. We examined the ability of sputum AMP to predict future exacerbation risk., Measurements and Main Results: AMP levels were higher in sputum than in serum, suggesting local AMP release. Patients with more severe disease at baseline had dysregulation of airway AMP. Higher LL-37 and lower SLPI levels were associated with Bronchiectasis Severity Index, lower FEV 1 (forced expiratory volume in 1 s) and Pseudomonas aeruginosa infection. Low SLPI levels were also associated with the exacerbation frequency at baseline. During follow-up, higher LL-37 and lower SLPI levels were associated with a shorter time to the next exacerbation, whereas LL-37 alone predicted exacerbation frequency over the next 12 months., Conclusions: Patients with bronchiectasis showed dysregulated sputum AMP levels, characterised by elevated LL-37 and reduced SLPI levels in the frequent exacerbator phenotype. Elevated LL-37 and reduced SLPI levels are associated with Pseudomonas aeruginosa infection and can predict future risk of exacerbations in bronchiectasis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

This study was conducted to investigate the effects of dietary dried Rocket Leaves meal (DRLM) supplementation on growth performance, immune response, and antioxidant capacity of Oreochromis niloticus reared under different water temperature. For this purpose, five hundred and forty apparently healthy O. niloticus were allocated into nine groups fed three DRLM-supplemented diets (0,1, and 3%) and reared at three water temperature (18, 24, and 32°C) in a 3 × 3 total randomized factorial design. The results revealed that exposure of fish to low (18°C) or high (32°C) temperatures for 30 days evoked significant growth retardation, depleted antioxidant enzymes activities (Catalase; CAT and super oxide dismutase; SOD), lipid peroxidation (malondialdehyde; MDA), immunosuppression, altered cortisol level compared to those reared at 24°C. Moreover, a marked down-regulation of oxidative stress related genes with up-regulation of interleukin 1β gene were apparent. In contrast, DRLM incorporation, particularly at 3%, in heat or cold stressed fish diets significantly enhanced growth, restored IgM and lysozymes levels, and SOD and CAT activities. Also, both the MDA and cortisol levels were significantly depressed. Furthermore, both antioxidant and immune-related genes expression were significantly corrected. Conclusively, 3% DRLM dietary supplementation in tilapia diet could be a promising strategy to alleviate the temperature stress-induced negative impacts on fish health and performance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Grating-coupled surface plasmon field-enhanced fluorescence spectroscopy (GC-SPFS) with optical bound/free (B/F) separation technique was developed by employing a highly directional fluorescence with polarization of surface plasmon-coupled emission (SPCE) to realize highly sensitive immunoassay regardless of the ligand affinity. A highly sensitive immunoassay system with GC-SPFS was constructed using a plastic sensor chip reproducibly fabricated in-house by nanoimprinting and applied to the quantitative detection of an anti-lysozyme single-domain antibody (sdAb), to compare conventional washing B/F separation with optical B/F separation. Differences in the affinity of the anti-lysozyme sdAb, induced by artificial mutation of only one amino acid residue in the variable domain were attributed to higher sensitivity than that of the conventional Biacore surface plasmon resonance (SPR) system. The detection limit (LOD; means of six replicates of the zero standard plus three standard deviations) of the GC-SPFS immunoassay with optical B/F separation, was estimated to be 1.2 ng/ml with the low-affinity ligand (mutant sdAb Y52A: KD level was of the order of 10-7 ~ 10-6 M) and was clearly improved as compared to that (LOD: 9.4 ng/ml) obtained with the conventional washing B/F separation. These results indicate that GC-SPFS with the optical B/F separation technique offers opportunities to re-evaluate low-affinity biomaterials that are neither fully utilized nor widespread, and could facilitate the creation of novel and innovative methods in drug and diagnostic development., Competing Interests: We have the following interests: T.K., K.N., Y.N. are employed by Konica Minolta, Inc‥ The patent related to this research is disclosed as follow. “Surface plasmon enhanced fluorescence measurement device and surface plasmon enhanced fluorescence measurement method.” PCT/JP2014/002009, Priority date: 8 Apr. 2014. There are no further patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Immune homeostasis in the gut associated lymphoid tissues (GALT) is critical to prevent the development of inadvertent pathologies. B cells as the producers of antibodies and cytokines plays an important role in maintaining the GALT homeostasis. However, the mechanism by which B cells specifically direct their responses towards non-self-antigens and become ignorant to self-antigens in the GALT is not known. Therefore, we developed a novel mouse model by expressing Duck Egg Lysozyme (DEL) in gut epithelial cells in presence of HEL reactive B cells. Notably, we observed a transient activation and rapid deletion of self-reactive B cells in Peyers Patches and Mesenteric lymph nodes upon self-antigen exposure. The survival of self-reactive B cells upon exposure to their self-antigen was partially rescued by blocking receptor editing but could be completely rescued by stronger survival signal like ectopic expression of BCL2. Importantly, rescuing the self-reactive B cells promoted production of auto-antibodies and gut inflammation. Mechanistically, we identify a specific activation of TGFβ signaling in self-reactive B cells in the gut and a critical role of this pathway in maintaining peripheral tolerance. Collectively, our studies describe functional consequences and fate of self-reactive B cells in GALT and provide novel mechanistic insights governing self-tolerance of B cells in the gut.

Lysozymes are important immune effectors present in phylogenetically diverse organisms. They play vital roles in bacterial elimination during early immune responses. In the present study, a second invertebrate-type (i-type) lysozyme gene from razor clam Sinonovacula constricta (denoted as ScLYZ-2) was cloned by RACE and nested PCR methods. The full-length cDNA sequences of ScLYZ-2 were 1558 bp, including a 5' untranslated region (UTR) of 375 bp, an open reading frame of 426 bp, and a 3'-UTR of 757 bp with polyadenylation signal sequence (AATAAA) located upstream of the poly(A) tail. SMART analysis showed that ScLYZ-2 contains a signal peptide in the first 16 amino acid (AA) sequences and a destabilase domain located from 24 to 134 AA sequences. The deduced AA sequences of ScLYZ-2 were highly similar (42%-58%) to other known lysozyme genes of bivalve species. Multiple alignments of AA sequences showed that ScLYZ-2 possesses the classical i-type lysozyme family signature of two motifs ["MDVGSLSCGP(Y/F)QIK" and "CL(E/L/R/H)C(I/M)C"] and two catalytic residues (Glu 35 and Asp 46 ). Moreover, phylogenetic analysis showed that ScLYZ-2 is a new member of the i-type lysozyme family. In healthy razor clams, ScLYZ-2 was highly expressed in the hepatopancreas, followed by the gills, water pipes, and abdominal foot. Lysozyme activity and ScLYZ-2 expression levels were significantly upregulated in the hepatopancreas and gills after being infected with V. splendidus, V. harveyi, V. parahaemolyticus and S. aureus and M. luteus. Moreover, the recombinant ScLYZ-2 had strong antimicrobial activities against V. splendidus, V. harveyi, and V. parahaemolyticus. Furthermore, the minimal inhibitory concentration of the recombinant ScLYZ-2 against V. parahaemolyticus was 7.2 μmol/mL. Taken together, our results show that ScLYZ-2 plays an important role in the immune defense of razor clam by eliminating pathogenic microorganisms., (Copyright © 2019 Elsevier Ltd. All rights reserved.)