Gut mucosal immune responses and protective efficacy of oral yeast Cyprinid herpesvirus 2 (CyHV-2) vaccine in Carassius auratus gibelio.

Cyprinid herpesvirus 2 (CyHV-2) causes herpesviral hematopoietic necrosis (HVHN) disease outbreaks in farmed Cyprinid fish, which leads to serious economic losses worldwide. Although oral vaccination is considered the most suitable strategy for preventing infectious diseases in farmed fish, so far there is no commercial oral vaccine available for controlling HVNN in gibel carp (C. auratus gibelio). In the present study, we developed for the first time an oral vaccine against CyHV-2 by using yeast cell surface display technology and then investigated the effect of this vaccine in gibel carp. Furthermore, the protective efficacy was evaluated by comparing the immune response of a single vaccination with that of a booster vaccination (booster-vaccinated once 2 weeks after the initial vaccination). Critically, the activities of immune-related enzymes and genes expression in vaccine group, especially in the booster vaccine group, were higher than those in the control group. Moreover, strong innate and adaptive immune responses could be elicited in both mucosal and systemic tissues after receipt of the oral yeast vaccine. To further understand the protective efficacy of this vaccine in gibel carp, we successfully developed the challenge model with CyHV-2. Our results showed the relative percent survival was 66.7% in the booster vaccine group, indicating this oral yeast vaccine is a promising vaccine for controlling CyHV-2 disease in gibel carp aquaculture.

Conserved Role of mTORC1 Signaling in B Cell Immunity in Teleost Fish.

Mammalian studies have demonstrated that B cell immune responses are regulated by mechanistic target of rapamycin complex 1 (mTORC1) signaling. Teleost fish represent the oldest living bony vertebrates that contain bona fide B cells. So far, whether the regulatory mechanism of mTORC1 signaling in B cells occurred in teleost fish is still unknown. In this study, we developed a fish model by using rapamycin (RAPA) treatment to inhibit mTORC1 signaling and demonstrated the role of mTORC1 signaling in teleost B cells. In support, we found inhibition of mTORC1 signaling by RAPA decreased the phagocytic capacity, proliferation, and Ig production of B cells. Critically, Flavobacterium columnare induced specific IgM binding in serum, and these titers were significantly inhibited by RAPA treatment, thus decreasing Ab-mediated agglutination of F. columnare and significantly increasing the susceptibility of fish upon F. columnare reinfection. Collectively, our findings elucidated that the mTORC1 pathway is evolutionarily conserved in regulating B cell responses, thus providing a new point for understanding the B cells functions in teleost fish.

Molecular cloning and expression analysis of CD79a and CD79b in rainbow trout (Oncorhynchus mykiss) after bacterial, parasitic, and viral infection.

CD79a and CD79b heterodimers are important components that consist of B cell receptor compound, which play a crucial role in transduction activation signal of the antigen binding BCR, and B cell development and antibody production. In order to investigate the characters and potential functions of CD79a and CD79b in rainbow trout (Oncorhynchus mykiss), we firstly cloned and analyzed the expression of CD79a and CD79b and found that the cDNA sequences of CD79a and CD79b both contained open reading frame of 711 and 645 bp in length for encoding the protein of 237 and 215 amino acid residues, respectively. The predicted amino acid sequences from trout were highly conserved with those of other teleost fishes in structure. Phylogenetic tree was constructed to analyze the evolutionary relationship between the trout and other known species, the result indicated that CD79a and CD79b of trout clustered at high bootstrap values with Salmo salar. Moreover, three trout infection models with F. columnare G4, I. multifiliis and infectious hematopoietic necrosis virus (IHNV) were constructed, which resulted in morphological changes and serious lesions in skin and gills. Importantly, the high expression of CD79a and CD79b occurred in skin, gills, and followed by head kidney in response to bacterial, parasitic, and viral infection, as its expression was closely related to that of Igs. Our findings indicated that CD79a and CD79b play vital roles in both systemic and mucosal immune responses of rainbow trout during bacterial, parasitic, and viral infection, which will contribute to explore the roles of CD79 subunits in B cell signaling during ontogeny and disease.

Molecular Characterization and Expression Analysis of Intercellular Adhesion Molecule-1 (ICAM-1) Genes in Rainbow Trout (Oncorhynchus mykiss) in Response to Viral, Bacterial and Parasitic Challenge.

The intercellular adhesion molecule-1 (ICAM-1), known as CD54, is a transmembrane cell surface glycoprotein that interacts with two integrins (i.e., LFA-1 and Mac-l) important for trans-endothelial migration of leukocytes. The level of ICAM-1 expression is upregulated in response to some inflammatory stimulations, including pathogen infection and proinflammatory cytokines. Yet, to date, our knowledge regarding the functional role of ICAM-1 in teleost fish remains largely unknown. In this study, we cloned and characterized the sequence of ICAM-1 in rainbow trout (Oncorhynchus mykiss) for the first time, which exhibited that the molecular features of ICAM-1 in fishes were relatively conserved compared with human ICAM-1. The transcriptional level of ICAM-1 was detected in 12 different tissues, and we found high expression of this gene in the head kidney, spleen, gills, skin, nose, and pharynx. Moreover, upon stimulation with infectious hematopoietic necrosis virus (IHNV), Flavobacterium columnare G4 (F. columnare), and Ichthyophthirius multifiliis (Ich) in rainbow trout, the morphological changes were observed in the skin and gills, and enhanced expression of ICAM-1 mRNA was detected both in the systemic and mucosal tissues. These results indicate that ICAM-1 may be implicated in the mucosal immune responses to viral, bacterial, and parasitic infections in teleost fish, meaning that ICAM-1 emerges as a master regulator of mucosal immune responses against pathogen infections in teleost fish.

Prevailing Role of Mucosal Igs and B Cells in Teleost Skin Immune Responses to Bacterial Infection.

The skin of vertebrates is the outermost organ of the body and serves as the first line of defense against external aggressions. In contrast to mammalian skin, that of teleost fish lacks keratinization and has evolved to operate as a mucosal surface containing a skin-associated lymphoid tissue (SALT). Thus far, IgT representing the prevalent Ig in SALT have only been reported upon infection with a parasite. However, very little is known about the types of B cells and Igs responding to bacterial infection in the teleost skin mucosa, as well as the inductive or effector role of the SALT in such responses. To address these questions, in this study, we analyzed the immune response of trout skin upon infection with one of the most widespread fish skin bacterial pathogens, Flavobacterium columnare This pathogen induced strong skin innate immune and inflammatory responses at the initial phases of infection. More critically, we found that the skin mucus of fish having survived the infection contained significant IgT- but not IgM- or IgD-specific titers against the bacteria. Moreover, we demonstrate the local proliferation and production of IgT+ B cells and specific IgT titers, respectively, within the SALT upon bacterial infection. Thus, our findings represent the first demonstration that IgT is the main Ig isotype induced by the skin mucosa upon bacterial infection and that, because of the large surface of the skin, its SALT probably represents a prominent IgT-inductive site in fish.

Pharyngeal Immunity in Early Vertebrates Provides Functional and Evolutionary Insight into Mucosal Homeostasis.

The pharyngeal organ is located at the crossroad of the respiratory and digestive tracts in vertebrate, and it is continuously challenged by varying Ags during breathing and feeding. In mammals, the pharyngeal mucosa (PM) is a critical first line of defense. However, the evolutionary origins and ancient roles of immune defense and microbiota homeostasis of PM are still unknown. In this study, to our knowledge, we are the first to find that diffuse MALT is present in PM of rainbow trout, an early vertebrate. Importantly, following parasitic infection, we detect that strong parasite-specific mucosal IgT and dominant proliferation of IgT+ B cell immune responses occurs in trout PM, providing, to our knowledge, the first demonstration of local mucosal Ig responses against pathogens in pharyngeal organ of a nonmammal species. Moreover, we show that the trout PM microbiota is prevalently coated with secretory IgT and, to a much lesser degree, by IgM and IgD, suggesting the key role of mucosal Igs in the immune exclusion of teleost pharyngeal bacteria. Overall, to our knowledge, our findings provide the first evidence that pharyngeal mucosal immunity appear earlier than tetrapods.

Convergent Evolution of Mucosal Immune Responses at the Buccal Cavity of Teleost Fish.

The buccal mucosa (BM) is a critical first line of defense in terrestrial animals. To gain further insights into the evolutionary origins and primordial roles of BM in teleosts here we show that rainbow trout, a teleost fish, contains a diffuse mucosal associated lymphoid tissue (MALT) within its buccal cavity. Upon parasite infection, a fish immunoglobulin specialized in mucosal immunity (sIgT) was induced to a high degree, and parasite-specific sIgT responses were mainly detected in the buccal mucus. Moreover, we show that the trout buccal microbiota is prevalently coated with sIgT. Overall our findings revealed that the MALT is present in the BM of a non-tetrapod species. As fish IgT and mucus-producing cells are evolutionarily unrelated to mammalian IgA and salivary glands, respectively, our findings indicate that mucosal immune responses in the BM of teleost fish and tetrapods evolved through a process of convergent evolution.

A study of the damage of the intestinal mucosa barrier structure and function of Ctenopharyngodon idella with Aeromonas hydrophila.

The aim of this study is to explore the effect of Aeromonas hydrophila on the intestinal mucosal barrier structure and intestinal permeability in grass carp (Ctenopharyngodon idella). Histopathological examinations showed that A. hydrophila induced severe intestinal lesions, including inflammatory cell infiltration and intestinal villus fusion and swelling. Messenger RNA (mRNA) expression of the inflammatory cytokines TNF-α, IL-1ß, IL-8, IL-10 and MyD88 was significantly increased after infection with A. hydrophila. The permeability of intestinal mucosa was determined using Evans blue (EB) and D-lactic acid. The results indicated that the levels of EB and serum D-lactic acid were significantly increased after infection with A. hydrophila (p < 0.05). Our results also indicated that the intestinal mucosal barrier injury induced by A. hydrophila infection was closely associated with the expression of the tight junction (TJ) protein zonula occludens-1 (ZO-1), occludin, claudin b and claudin c as well as the activity of Na+, K+-ATPase and Ca2+, Mg2+-ATPase. Lower mRNA levels of occludin and lower Na+, K+-ATPase and Ca2+, Mg2+-ATPase activity in the intestines were observed after challenge. ZO-1 and claudin c were significantly increased 24 h after infection with A. hydrophila. The most interesting finding was that claudin b also significantly increased 24 h after challenge and then decreased to lower levels at 72, 120 and 168 h post-infection compared to the PBS-treated control group. The results demonstrated that grass carp infection with A. hydrophila induced intestinal inflammation and impaired the structure and function of the intestinal mucosal barrier.

Silver salts and DBU cooperatively catalyzed nucleophilic addition/cyclization of propargylic alcohols with trifluoromethyl ketones.

A general and efficient synthesis of trifluoromethyl substituted 5-alkylidene-1,3-dioxolane using a silver salt and DBU cooperatively catalyzed nucleophilic addition/cyclization of propargylic alcohols and trifluoromethyl ketones is described. The procedure produced 5-alkylidene-1,3-dioxolane derivatives with good to excellent yields and can be used with a broad range of substrates.

[Effects of acupuncture combined with medicine on expression of matrix metalloproteinase-2 in the rat of endometriosis].

OBJECTIVE: To probe into an effective method for treatment of endometriosis (EMs) and the mechanism. METHODS: Rat EMs model was established and they were randomly divided into a model group, an acup-moxibustion group, a TCM group, an acupuncture and medicine group, with a control group set. The acup-moxibustion group were treated with electroacupuncture at "Xuehai (SP 10)", "Sanyinjiao (SP 6)" and moxibustion at "Guanyuan (CV 4)"; the TCM group were treated with stomach perfusion of modified Mojie Tablet in normal saline; the acupuncture and medicine group were treated with the above two methods; both the control group and the model group were bound and treated with stomach perfusion of saline. After treatment of 35 days, the greatest diameter of the ectopic tissue was measured in the rats, pathological observation of the ectopic tissue was made and matrix metalloproteinase-2 (MMP-2) expression in the ectopic tissue was determined. RESULTS: The greatest diameter of ectopic tissue and MMP-2 expression in the ectopic tissue in the acupuncture and medicine group were significantly lower than those in the model group, the acup-moxibustion group and the TCM group (P < 0.05); and the ectopic endometrium trended to atrophy, and with necrosis of some epithelial cells. CONCLUSION: Combination of acupuncture with medicine has a better therapeutic effect on endometriosis and down-regulates the abnormal increase of MMP-2 level to inhibit the invasion of ectopic tissue to extracellular matrix, so as to reduce the ectopic tissue, hence cure of endometriosis.