An In Vitro System Mimics the Intestinal Microbiota of Striped Beakfish (Oplegnathus fasciatus) and Inhibits Vibrio alginolyticus by Limosilactobacillus reuteri-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are functional substances secreted by microbes and host cells, and it has been discovered that they participate in the interactions between different microorganisms. Our recent findings indicate that Limosilactobacillus reuteri-derived EVs have the potential to improve the intestinal microbiota of Oplegnathus fasciatus fish and inhibit pathogenic bacteria. Previous research has reported that the host intestinal cells play a regulatory role in the intestinal microbiota. This suggested that to investigate the mechanisms through which L. reuteri-derived EVs regulate the intestinal microbiota, a system that excludes interference from host intestinal cells should be established. In this study, an in vitro cultured intestinal bacteria system, without host factors, was used to simulate the intestinal microbiota of O. fasciatus fish. After adding L. reuteri-derived EVs to the system, the changes in the microbiota were analyzed. The results showed that L. reuteri-derived EVs effectively reduced the abundance of Vibrio spp. In the results of the in vitro experiments, it was also observed that L. reuteri-derived EVs have the ability to inhibit Vibrio alginolyticus. We further sequenced the small RNA contained in L. reuteri-derived EVs and found that these small RNAs can interfere with genes (LysR, pirin, MIpA/OmpV, CatB, and aspartate-semialdehyde dehydrogenase) related to the growth of V. alginolyticus. Taken together, the results indicate that in the absence of host involvement, the small RNAs present in L. reuteri-derived EVs have the function of inhibiting pathogenic bacteria and exhibit the potential to regulate the intestinal microbiota.

Effects of Taiwanese indigenous cinnamon (Cinnamomum osmophloeum) leaf hot-water extract on nonspecific immune responses, resistance against Vibrio parahaemolyticus, nonviable cells, and haemocyte subpopulations in white shrimp (Penaeusvannamei).

This study investigated the effects of Cinnamomum osmophloeum leaf hot-water extract (CLWE) on nonspecific immune responses and resistance to Vibrio parahaemolyticus in white shrimp (Penaeus vannamei). Firstly, a cell viability assay demonstrated that the CLWE is safe to white shrimp heamocytes in the concentration of 0-500 mg L-1. Haemocytes incubated in vitro with 10 and 50 mg L-1 of CLWE showed significantly higher response in superoxide anion production, PO activity, and phagocytic activity. In the in vivo trials, white shrimp were fed with 0, 0.5, 1, 5, and 10 g kg-1 CLWE supplemented feeds (designated as CLWE 0, CLWE 0.5, CLWE 1, CLWE 5, and CLWE 10, respectively) over a period of 28 days. In vivo experiments demonstrated that CLWE 0.5 feeding group resulted in the highest total haemocyte count, superoxide anion production, phenoloxidase activity, and phagocytic activity. Moreover, CLWE 0.5 supplemented feed significantly upregulated the clotting system, antimicrobial peptides, pattern recognition receptors, pattern recognition proteins, and antioxidant defences in white shrimp. Furthermore, the shrimp were infected with V. parahaemolyticus injections after 14 days of feeding as challenge test. Based on the challenge test result, both CLWE 0.5 and CLWE 5 demonstrated a strong resistance to V. parahaemolyticus. These two dosages effectively reduced the number of nonviable cells and activated different haemocyte subpopulations. These findings indicated that treatment with CLWE 0.5 could promote nonspecific immune responses, immune-related gene expression, and resistance to V. parahaemolyticus in white shrimp.

Assessment of fish protein hydrolysate as a substitute for fish meal in white shrimp diets: Impact on growth, immune response, and resistance against Vibrio parahaemolyticus infection.

This study investigated the effects of fish protein hydrolysate derived from barramundi on growth performance, muscle composition, immune response, disease resistance, histology and gene expression in white shrimp (Penaeus vannamei). In vitro studies demonstrated FPH enhanced mRNA expressions of key immune-related genes and stimulated reactive oxygen species (ROS) production and phagocytic activity in shrimp hemocytes. To evaluate the effects of substituting fish meal with FPH in vivo, four isoproteic (43 %), isolipidic (6 %), and isoenergetic diets (489 kcal/100 g) were formulated with fish meal substitution levels of 0 % (control), 30 % (FPH30), 65 % (FPH65), and 100 % (FPH100). After 8-week feeding, the growth performance of FPH65 and FPH100 were significantly lower than that of control and FPH30 (p < 0.05). Similarly, the midgut histological examination revealed the wall thickness and villi height of FPH100 were significantly lower than those of control (p < 0.05). The shrimps were received the challenge of AHPND + Vibrio parahaemolyticus at week 4 and 8. All FPH-fed groups significantly enhanced resistance against Vibrio parahaemolyticus at week 4 (p < 0.05). However, this protective effect diminished after long-period feeding. No significant difference of survival rate was observed among all groups at week 8 (p > 0.05). The expressions of immune-related genes were analyzed at week 4 before and after challenge. In control group, V. parahaemolyticus significantly elevated SOD in hepatopancreas and Muc 19, trypsin, Midline-fas, and GPx in foregut (p < 0.05). Moreover, hepatopancreatic SOD of FPH65 and FPH100 were significantly higher than that of control before challenge (p < 0.05). Immune parameters were measured at week 8. Compared with control, the phagocytic index of FPH 30 was significantly higher (p < 0.05). However, dietary FPH did not alter ROS production, phenoloxidase activity, phagocytic rate, and total hemocyte count (p > 0.05). These findings suggest that FPH30 holds promise as a feed without adverse impacts on growth performance while enhancing the immunological response of white shrimp.

Application of nZnO supported with nanoclay for improving shrimp immunity.

This study discloses the nanoscale silicate platelet-supported nZnO (ZnONSP) applied as novel feed additives in aquaculture. The preparation of the nanohybrid (ZnO/NSP = 15/85, w/w) was characterized by UV-visible spectroscopy, powder X-ray diffraction and transmission electron microscope. The effects of ZnONSP on growth, zinc accumulation, stress response, immunity and resistance to Vibrio alginolyticus in white shrimp (Penaeus vannamei) were \demonstrated. To evaluate the safety of ZnONSP, shrimps (2.0 ± 0.3 g) were fed with ZnONSP containing diets (200, 400 and 800 mg/kg) for 56 days. Dietary ZnONSP did not affect the weight gain, specific growth rate, feed conversion ratio, survival rate, zinc accumulation, and the expression of heat shock protein 70 in tested shrimps. To examine the immunomodulatory effect of ZnONSP, shrimps (16.6 ± 2.4 g) were fed with the same experimental diets for 28 days. Dietary ZnONSP improved the immune responses of haemocyte in tested shrimps, including phagocytic rate, phagocytic index, respiratory burst, and phenoloxidase activity, and upregulated the expression of several genes, including lipopolysaccharide, ß-1,3-glucan binding protein, peroxinectin, penaeidin 2/3/4, lysozyme, crustin, anti-lipopolysaccharide factor, superoxide dismutase, glutathione peroxidase, clotting protein and α-2-macroglobulin. In the challenge experiment, shrimps (17.2 ± 1.8 g) were fed with ZnONSP containing diets (400 and 800 mg/kg) for 7 days and then infected with Vibrio alginolyticus. Notably, white shrimps that received ZnONSP (800 mg/kg) showed significantly improved Vibrio resistance, with a survival rate of 71.4 % at the end of 7-day observation. In conclusion, this study discovers that ZnONSP is a new type of immunomodulatory supplement that are effective on enhancing innate cellular and humoral immunities, and disease resistance in white shrimp.

Improving red-color performance, immune response and resistance to Vibrio parahaemolyticus on white shrimp Penaeus vannamei by an engineered astaxanthin yeast.

Astaxanthin (AST), a super antioxidant with coloring and medical properties, renders it a beneficial feed additive for shrimp. This study conducted a white shrimp feeding trial of 3S, 3'S isoform AST, which was derived from metabolic-engineered Kluyveromyces marxianus fermented broth (TB) and its extract (TE) compared to sources from two chemically synthetic ASTs (Carophyll Pink [CP] and Lucantin Pink [LP]), which contain 3S, 3'S, 3R, 3'S (3S, 3'R) and 3R, 3'R isoforms ratio of 1:2:1. The effects on red coloration, immune parameters and resistance to Vibrio infection were evaluated. Four AST sources were incorporated into the diets at concentrations of 0 (control), 100 mg kg-1 (TB100, TE100, CP100, and LP100), and 200 mg kg-1 (TB200, TE200, CP200, and LP200). Results revealed that in week 4, shrimps that received AST-supplemented feeds, especially TB100, TB200, and TE200, significantly increased redness (a*) values. Immune responses including phagocytosis activity, superoxide-anion production, phenoloxidase activity, and immune-related genes were examined on days 0, 1, 2, 4, 7, 14, 21, and 28. Generally, shrimps that received AST-supplemented feeds exhibited higher immune responses on days 7 and 14 than the control feed. Gene expression levels of superoxide dismutase and glutathione peroxidase were significantly upregulated on days 7 and 14 in shrimps that received AST-supplemented feeds, while genes of penaeidins, antilipopolysaccharide factor, and lysozyme were upregulated on days 4, 7, and 14, especially received TB200 and TE200. Furthermore, shrimps that received TB100, TE100, CP100, and LP100 7 days were then challenged with Vibrio parahaemolyticus and the result demonstrated higher survival rates especially TB100 at 168 h than the control feed. In conclusion, incorporating AST into the diets enhanced shrimp red coloration, immune parameters, and resistance against V. parahaemolyticus infection. The K. marxianus-derived AST exhibited higher performance than did chemical AST to be a potential feed additive in shrimp aquaculture.

Dietary of Lactobacillus paracasei and Bifidobacterium longum improve nonspecific immune responses, growth performance, and resistance against Vibrio parahaemolyticus in Penaeus vannamei.

This study investigated the effects of two probiotics, namely Lactobacillus paracasei and Bifidobacterium longum, as feed additives on growth performance, nonspecific immunity, immune-related gene expression, and disease resistance against Vibrio parahaemolyticus in Penaeus vannamei. The experimental diets were prepared using L. paracasei and B. longum at concentrations of 105 and 107 CFU/g; these diets were referred to as P5, P7, B5, and B7. After 8 weeks of the diets, regarding growth performance, the B7 group showed the highest weight gain rate (890.34 ± 103.65%), special growth rate (4.08 ± 0.19%), and feed conversion rate (1.52 ± 0.19%) compared with the other groups. Moreover, the total hemocyte counts were significantly increased (p < 0.05) in the P7 groups on day 14 during the 28-day feeding trial. The phagocytosis rate in all experimental groups was increased on day 14 and was persistently significantly activated to day 21, especially in the P7 and B5 group. The phagocytic index of the P7 group showed a significant increase on day 14 and persistent activation to day 21. In the analysis of respiratory burst activity and phenoloxidase activity, the P7 and B5 groups showed a significant increase on day 7 and persistent activation to day 21. The expression level of the immune-related genes of superoxide dismutase, clotting protein, Penaeidin2, Penaeidin3, Penaeidin4, anti-LPS factor, crustin, and lysozyme was significantly increased in the experimental groups, especially in the P7 group. Furthermore, the optimum conditions of feed additives were determined in challenge trials conducted using P7 and B5. Shrimps fed P7 and B5 showed an increased survival rate (72.73% and 66.67%) after the V. parahaemolyticus challenge. In sum, the results revealed that B. longum, as a feed additive at 107 CFU/g, enhanced growth performance. L. paracasei at 107 CFU/g and B. longum at 105 CFU/g can enhance nonspecific immune responses and immune-related gene expression, and 107 CFU/g L. paracasei has the highest resistance ability for V. parahaemolyticus. Thus, dietary supplementation with L. paracasei and B. longum may be a valuable approach in white shrimp aquaculture.

Effect of dietary supplementation with Moringa oleifera leaf extract and Lactobacillus acidophilus on growth performance, intestinal microbiota, immune response, and disease resistance in whiteleg shrimp (Penaeus vannamei).

This study investigated the effect of the moringa (Moringa oleifera) leaf extract and Lactobacillus acidophilus individually or combined on growth performance, enzyme activity, intestinal and hepatopancreatic histology, intestinal microbiota, immune response, and resistance against Vibrio alginolyticus and Vibrio parahaemolyticus in whiteleg shrimp (Penaeus vannamei). Six diets were formulated: three diets without L. acidophilus containining 0 (control, ME0), 2.5 (ME2.5), and 5.0 g/kg of moringa (ME5.0) and the same three diets containing L. acidophilus at 1 × 107 CFU/g of diet (ME0+P, ME2.5 + P, and ME5.0 + P, respectively). Growth performance was measured after 60 days of the rearing period. On the final day, the shrimp were sampled to assess enzyme activity, intestinal and hepatopancreatic histology, and gut microbiota. Shrimp hemocytes were examined on Days 0, 1, 2, 4, 7, 14, 21, and 28 to measure the immune response in terms of the total hemocyte count, phenoloxidase activity, phagocytosis, and superoxide anion production. Furthermore, the shrimp were challenged with V. alginolyticus and V. parahaemolyticus. The results revealed that ME2.5 + P significantly increased (P < 0.05) final weight, weight gain, specific growth rate, enzyme activities, and villi height compared with ME2.5 and control. Wall thickness was increased in the shrimp fed diet supplemented with moringa and L. acidophilus compared with the control shrimp. Hepatopancreatic histology revealed that R cells were more abundant in the shrimp fed diet containing moringa and L. acidophilus compared with those fed diet containing moringa alone (P < 0.05) at the same concentration. High-throughput sequencing analysis indicated that the dietary supplementation with moringa and L. acidophilus affected the gut microbiota composition. All gene functions, members of KEGG level 2, related to metabolism were increased in diet supplemented with moringa with or without L. acidophilus compared with the control group. The immune assay revealed that the total hemocyte count, phenoloxidase activity, phagocytic rate, superoxide anion production, and immune-related gene expression (including those of prophenoloxidase II, alpha-2-macroglobulin, penaeidin2, antilipopolysaccharide factor, crustin, lysozyme, glutathione peroxidase, and superoxide dismutase) were higher in the experimental groups than in the control group on several observed days; however, the increases were observed more often in the ME2.5 + P group than in the other treatment groups. Furthermore, the ME2.5 + P group exhibited a significantly higher survival rate (P < 0.05) in the challenge test against V. alginolyticus and V. parahaemolyticus. In conclusion, supplementation with dietary moringa and L. acidophilus at ME2.5 + P improved growth performance, immune system, and resistance against Vibrio in the shrimp.

Effects of Replacing Fishmeal with Defatted Black Soldier Fly (Hermetia illucens Linnaeus) Larvae Meal in Japanese Eel (Anguilla japonica) Diet on Growth Performance, Fillet Texture, Serum Biochemical Parameters, and Intestinal Histomorphology.

An 8-week feeding trial was conducted to investigate the effects of replacing fishmeal with defatted black soldier fly larvae meal (DBSFLM) in the diets of Japanese eel on their growth performance, fillet texture, serum biochemical parameters, and intestinal histomorphology. Six isoproteic (520 g kg-1), isolipidic (80 g kg-1), and isoenergetic (15 MJ kg-1) diets were formulated with fishmeal replacement levels of 0% (R0), 15% (R15), 30% (R30), 45% (R45), 60% (R60), and 75% (R75). The growth performance, feed utilization efficiency, survival rate, serum liver function enzymes, antioxidant ability, and lysozyme activity of fish were not affected (P > 0.05) by DBSFLM. However, the crude protein and cohesiveness of the fillet in groups R60 and R75 significantly decreased, and the fillet hardness significantly increased (P < 0.05). Additionally, the intestinal villus length significantly decreased in the R75 group, and the goblet cell densities were significantly lower in the R45, R60, and R75 groups (P < 0.05). Overall, high levels of DBSFLM did not affect growth performance and serum biochemical parameters but significantly altered fillet proximate composition and texture and intestinal histomorphology (P < 0.05). The optimal fishmeal replacement level is 30% with 184 g kg-1 DBSFLM.

Characterisation and analysis of IFN-gamma producing cells in rainbow trout Oncorhynchus mykiss.

IFN-γ is one of the key cytokines involved in Th1 immune responses. It is produced mainly by T cells and NK cells, which drive both innate and adaptive responses to promote protection against infections. IFN-γ orthologues have been discovered to be functionally conserved in fish, suggesting that type I immunity is present in early vertebrates. However, few studies have looked at IFN-γ protein expression in fish and its role in cell mediated immunity due to a lack of relevant tools. In this study, four monoclonal antibodies (mAbs) V27, N2, VAB3 and V91 raised against short salmonid IFN-γ peptides were developed and characterised to monitor IFN-γ expression. The results show that the IFN-γ mAbs specifically react to their peptide immunogens, recognise E. coli produced recombinant IFN-γ protein and rainbow trout IFN-γ produced in transfected HEK 293 cells. The mAb VAB3 was used further, to detect IFN-γ at the cellular level after in vitro and in vivo stimulation. In flow cytometry, a basal level of 3-5% IFN-γ secreting cells were detected in peripheral blood leucocytes (PBL), which increased significantly when stimulated in vitro with PAMPs (Aeromonas salmonicida bacterin), a mitogen (PHA) and recombinant cytokine (IL-2). Similarly, after injection of live bacteria (Aeromonas salmonicida) or poly I:C the number of IFN-γ+ cells increased in the lymphoid population of PBL, as well as in the myeloid population after infection, with the myeloid cells increasing substantially after both treatments. Immunohistochemistry was used to visualise the IFN-γ+ cells in spleen and head kidney following vaccination, which increased in intensity of staining and number relative to tissue from saline-injected control fish. These results show that several types of cells can produce IFN-γ in trout, and that they increase following infection or vaccination, and likely contribute to immune protection. Hence monitoring IFN-γ producing cells/protein secretion may be an important means to assess the effectiveness of Th1 responses and cell mediated immunity in fish.

Expansion of fish CCL20_like chemokines by genome and local gene duplication: Characterisation and expression analysis of 10 CCL20_like chemokines in rainbow trout (Oncorhynchus mykiss).

Mammalian CCL20, or macrophage inflammatory protein-3α, can function as a homeostatic and inflammatory chemokine. In relation to the latter, it is responsible for the chemoattraction of lymphocytes and dendritic cells to mucosal immune sites under inflammatory and pathological conditions. CK1, CK8A and CK8B are rainbow trout (Oncorhynchus mykiss) CC chemokines that were reported previously to be phylogenetically related to mammalian CCL20. In the current study, an additional seven CCL20_L paralogues in rainbow trout are reported, that are divided into three subgroups and have been designated here as: CCL20_L1a (also referred to as CK1), CCL20_L1b1-2, CCL20_L2a (CK8A), CCL20_L2b (CK8B), CCL20_L3a, and CCL20_L3b1-4. Multiple CCL20_L genes were also identified in other salmonids that arose from both whole genome duplication and local gene duplication. Phylogenetic tree, homology and synteny analysis support that CCL20_L1-3 found in salmonids are also present in most teleosts arose from the 3 R whole genome duplication and in some species, local gene duplication. Like mammalian CCL20, rainbow trout CCL20_L molecules possess a high positive net charge with a pI of 9.34-10.16, that is reported to be important for antimicrobial activity. Rainbow trout CCL20_L paralogues are differentially expressed and in general highly expressed in mucosal tissues, such as gills, thymus and intestine. The expression levels of rainbow trout CCL20_L paralogues are increased during development and following PAMP/cytokine stimulation. For example, in RTS-11 cells CCL20_L3b1 and CCL20_L3b2 are highly up-regulated by LPS, Poly I:C, recombinant(r) IFNa and rIL-1ß. Trout CCL20_L paralogues are also increased after Yersinia ruckeri infection or Poly I:C stimulation in vivo, with CCL20_L3b1 and CCL20_L3b2 again highly up-regulated. Overall, this is the first report of the complete CCL20 chemokine subfamily in rainbow trout, and the analysis of their expression and modulation in vitro and in vivo. These results suggest that teleosts possess divergent CCL20_L molecules that may have important roles in anti-viral/anti-bacterial defence and in mucosal immunity.

Induction of IL-22 protein and IL-22-producing cells in rainbow trout Oncorhynchus mykiss.

IL-22 is a critical cytokine which is involved in modulating tissue responses during inflammation, and is produced mainly by T cells and innate leucocytes. In mammals, IL-22 is a key component in mucosal defences, tissue repair, epithelial cell survival and proliferation. In teleosts, IL-22 has been cloned and studied in several species, and the transcript is highly expressed in mucosal tissues and induced by pathogen associated molecular patterns (PAMPs), suggesting IL-22 also functions as an important component of the innate immune response in fish. To investigate these immune responses further, we have validated and characterised two monoclonal antibodies (mAbs) which were raised against two different peptide immunogens of salmonid IL-22. Our results show that both mAbs specifically react to their own peptide immunogens and recombinant IL-22, and are able to detect the induction of native protein expression after stimulation. In flow cytometry, an increase in IL-22 positive cells was detected after stimulation in vitro with cytokines and PAMPs and in vivo after bacterial challenge. The immunohistochemistry results showed that IL-22 is highly upregulated in the gills after challenge, both in cells within the gill filaments and in the interbranchial lymphoid tissue. Such results suggest IL-22 may have a role in triggering local antimicrobial defences in fish that may facilitate efficient microbial clearance. Hence monitoring IL-22 producing cells/protein secretion may provide an alternative mean to assess the effectiveness of mucosal vaccines.

The expression of brown fat-associated proteins in colorectal cancer and the relationship of uncoupling protein 1 with prognosis.

Colorectal carcinoma is one of the most common types of malignancy and a leading cause of cancer related death. The aberrant expression of a brown fat-like phenotype in cancer cells has been previously implicated in tumour growth. Therefore, the expression of brown fat-associated proteins in colorectal cancer could be associated with tumour prognosis. Monoclonal antibodies to brown fat-associated proteins CIDEA, ELOVL3, ELOVL5, and UCP1 were developed. The antibodies were used to profile the expression of protein targets by immunohistochemistry in a discovery cohort comprising 50 normal colonic mucosa samples and 274 primary colorectal cancers and a validation cohort comprising 549 colorectal cancers. Immunostaining for UCP1 was observed in the majority of colorectal tumours while no immunostaining was observed in normal colonic mucosa (p < 0.001). The expression of UCP1 was significantly associated with better overall survival in both the discovery cohort (HR = 0.615, 95%CI = 0.416-0.909, χ2 = 6.119, p = 0.013) and the validation cohort (HR = 0.629, 95%CI = 0.480-0.825, χ2 = 11.558, p = 0.001). Furthermore, UCP1 was independently prognostic in multivariate analysis (p = 0.004). This study has identified the brown fat-like phenotype as a novel pathway associated with survival in colorectal cancer. The expression of UCP1 was identified as a significant prognostic biomarker for colorectal cancer.

Interleukin (IL)-2 Is a Key Regulator of T Helper 1 and T Helper 2 Cytokine Expression in Fish: Functional Characterization of Two Divergent IL2 Paralogs in Salmonids.

Mammalian interleukin (IL)-2 is a cytokine centrally involved in the differentiation and survival of CD4+ T helper subsets and CD4+ T regulatory cells and in activation of cytotoxic effector lymphocytes. In bony fish, IL2 orthologs have been identified with an additional divergent IL2-Like gene on the same locus present in several fish species. We report here two divergent IL2 paralogs, IL2A and IL2B, in salmonids that originated from the whole genome duplication event in this fish lineage. The salmonid IL2 paralogs differ not only in sequence but also in exon sizes. The IL-2 isoforms that are encoded have disparate pI values and may have evolved to preferentially bind specific IL-2 receptors. Rainbow trout IL2 paralogs are highly expressed in thymus, spleen, gills, kidney and intestine, important tissues/organs in fish T cell development and function. Their expression in peripheral blood leukocytes (PBL) is low constitutively but can be upregulated by the mixed leukocyte reaction, by the T cell mitogen phytohemagglutinin and by signal mimics of T cell activation (phorbol 12-myristate 13-acetate and calcium ionophore). Both trout IL-2 isoforms promoted PBL proliferation and sustained high-level expression of CD4 and CD8, suggesting that trout IL-2 isoforms are T cell growth/survival factors mainly expressed by activated T cells. The recombinant proteins for these two trout IL2 paralogs have been produced in E. coli and possess shared but also distinct bioactivities. IL-2A, but not IL-2B, induced IL12P35A1 and CXCR1 expression in PBL. IL-2B had a stronger effect on upregulation of the T helper 1 (Th1) cytokine interferon-γ (IFNγ) and could sustain CD8α and CD8ß expression levels. Nevertheless, both cytokines upregulated key Th1 (IFNγ1, IFNγ2, TNFα2 and IL12) and T helper 2 (Th2) cytokines (IL4/13B1 and IL4/13B2), cytokine and chemokine receptors and the antimicrobial peptide cathelicidin-1 but had limited effects on T helper 17 cytokines and TGFß1 in PBL. They could also enhance PBL phagocytosis. These results suggest, for the first time in fish, that IL-2 isoforms may have an important role in regulating Th1 and Th2 cell development, and innate and adaptive host defenses in fish, and shed light on lineage-specific expansion, evolution, and functional diversification of IL2 in vertebrates.

Characterisation of rainbow trout peripheral blood leucocytes prepared by hypotonic lysis of erythrocytes, and analysis of their phagocytic activity, proliferation and response to PAMPs and proinflammatory cytokines.

Rapid and high quality preparation of peripheral blood leucocytes (PBL) is important in fish immunology research and in particular for fish vaccine development, where multiple immune parameters can be monitored on the same fish over time. Fish PBL are currently prepared by density separation using Percoll or Hispaque-1.077, which is time consuming, costly and prone to erythrocyte contamination. We present here a modified PBL preparation method that includes a 20 s hypotonic lysis of erythrocytes and a subsequent separation of PBL from cell debris by a cell strainer. This method is simple, rapid and cost effective. The PBL obtained are similar in cellular composition to those prepared by density separation but have less erythrocyte contamination as demonstrated by FACS analysis and the expression of cell marker genes. Marker gene analysis also suggested that PBL prepared by hypotonic lysis are superior to those obtained by the gradient method in that some high-density cells (certain B cell types and neutrophils) might be lost using the latter. The PBL prepared in this way can proliferate in response to the T cell mitogen PHA, and both lymphoid and myeloid cells can phagocytose fluorescent beads and bacteria, with the latter enhanced by treatment with pro-inflammatory cytokines (IL-1ß and IL-6). Furthermore, the PBL can respond to stimulation with PAMPs (LPS, poly I:C) and cytokines (IL-1ß and IFNγ) in terms of upregulation of proinflammatory cytokine gene expression. Such data demonstrate the utility of this approach (hypotonic lysis of erythrocytes) for PBL isolation and will enable more studies of their role in disease protection in future immunological and vaccine development research in fish.

Dysregulation of B Cell Activity During Proliferative Kidney Disease in Rainbow Trout.

Proliferative kidney disease (PKD) is a widespread disease caused by the endoparasite Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea). Clinical disease, provoked by the proliferation of extrasporogonic parasite stages, is characterized by a chronic kidney pathology with underlying transcriptional changes indicative of altered B cell responses and dysregulated T-helper cell-like activities. Despite the relevance of PKD to European and North American salmonid aquaculture, no studies, to date, have focused on further characterizing the B cell response during the course of this disease. Thus, in this work, we have studied the behavior of diverse B cell populations in rainbow trout (Oncorhynchus mykiss) naturally infected with T. bryosalmonae at different stages of preclinical and clinical disease. Our results show a clear upregulation of all trout immunoglobulins (Igs) (IgM, IgD, and IgT) demonstrated by immunohistochemistry and Western blot analysis, suggesting the alteration of diverse B cell populations that coexist in the infected kidney. Substantial changes in IgM, IgD, and IgT repertoires were also identified throughout the course of the disease further pointing to the involvement of the three Igs in PKD through what appear to be independently regulated mechanisms. Thus, our results provide strong evidence of the involvement of IgD in the humoral response to a specific pathogen for the first time in teleosts. Nevertheless, it was IgT, a fish-specific Ig isotype thought to be specialized in mucosal immunity, which seemed to play a prevailing role in the kidney response to T. bryosalmonae. We found that IgT was the main Ig coating extrasporogonic parasite stages, IgT+ B cells were the main B cell subset that proliferated in the kidney with increasing kidney pathology, and IgT was the Ig for which more significant changes in repertoire were detected. Hence, although our results demonstrate a profound dysregulation of different B cell subsets during PKD, they point to a major involvement of IgT in the immune response to the parasite. These results provide further insights into the pathology of PKD that may facilitate the future development of control strategies.

Determining the cleavage site for the mature antimicrobial peptide of Nile tilapia ß-defensin using 2D electrophoresis, western blot, and mass spectrometry analysis.

Several proteomic techniques were used to determine the cleavage site of the mature antimicrobial peptide of Nile tilapia ß-defensin. The computer-predicted Nile tilapia ß-defensin (25ASFPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL66) composed of 42 amino acids was chemically synthesized and prepared to produce an antibody for Western blotting. Total proteins from the skin of the Nile tilapia were separated on two-dimensional electrophoresis, and the spot of Nile tilapia ß-defensin was recognized using Western blot analysis. It was then excised and extracted from the gel. The precise molecular mass of this spot was determined by LC-MS/MS spectrometry. Four major peptides were discovered, with molecular weights of 4293.2 Da, 4306.5 Da, 4678.9 Da, and 4715.0 Da. The calculated mass of the 40-amino-acid sequence (27FPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL66) of Nile tilapia ß-defensin starting from Phe27 and ending with Leu66 was 4293.18 Da, which completely matched the 4293.2 Da peptide that was obtained from the mass spectrometry analysis. This result confirmed that the cleavage site for the mature C-terminal Nile tilapia ß-defensin is at residue Ser26-Phe27, not at Ala24-25 as predicted by computer analysis. This study provides a simple but reliable model to determine the cleavage site for a mature antimicrobial peptide.

Induction of chemokine receptor CXCR4 expression by transforming growth factor-ß1 in human basal cell carcinoma cells.

BACKGROUND: Higher CXCR4 expression enhances basal cell carcinoma (BCC) invasion and angiogenesis. The underlying mechanism of increased CXCR4 expression in invasive BCC is still not well understood. OBJECTIVE: To investigate the mechanisms involved in the regulation of CXCR4 expression in invasive BCC. METHODS: We used qRT-PCR, RT-PCR, Western blot, and flow cytometric analyses to examine different CXCR4 levels among the clinical samples, co-cultured BCC cells and BCC cells treated with recombinant transforming growth factor-ß1 (TGF-ß1) and connective tissue growth factor (CTGF). Immunohistochemical studies were used to demonstrate the correlation between TGF-ß1 and CXCR4 expressions. The signal transduction pathway and transcriptional regulation were confirmed by treatments with chemical inhibitors, neutralizing antibodies, or short interfering RNAs, as well as luciferase reporter activity. RESULTS: Invasive BCC has higher TGF-ß1 and CTGF levels compared to non-invasive BCC. Non-contact dermal fibroblasts co-culture with human BCC cells also increases the expression of CXCR4 in BCC cells. Treatment with recombinant human TGF-ß1, but not CTGF, enhanced the CXCR4 levels in time- and dose-dependent manners. The protein level and surface expression of CXCR4 in human BCC cells was increased by TGF-ß1 treatment. TGF-ß1 was intensely expressed in the surrounding fibroblasts of invasive BCC and was positively correlated with the CXCR4 expression of BCC cells. The transcriptional regulation of CXCR4 by TGF-ß1 is mediated by its binding to the TGF-ß receptor II and phosphorylation of the extracellular signal-related kinase 1/2 (ERK1/2)-ETS-1 pathway. CONCLUSION: TGF-ß1 induces upregulation of CXCR4 in human BCC cells by phosphorylation of ERK1/2-ETS-1 pathway.