Phagocytic Plasma Cells in Teleost Fish Provide Insights into the Origin and Evolution of B Cells in Vertebrates.

Teleost IgM+ B cells can phagocytose, like mammalian B1 cells, and secrete Ag-specific IgM, like mammalian B2 cells. Therefore, teleost IgM+ B cells may have the functions of both mammalian B1 and B2 cells. To support this view, we initially found that grass carp (Ctenopharyngodon idella) IgM+ plasma cells (PCs) exhibit robust phagocytic ability, akin to IgM+ naive B cells. Subsequently, we sorted grass carp IgM+ PCs into two subpopulations: nonphagocytic (Pha-IgM+ PCs) and phagocytic IgM+ PCs (Pha+IgM+ PCs), both of which demonstrated the capacity to secrete natural IgM with LPS and peptidoglycan binding capacity. Remarkably, following immunization of grass carp with an Ag, we observed that both Pha-IgM+ PCs and Pha+IgM+ PCs could secrete Ag-specific IgM. Furthermore, in vitro concatenated phagocytosis experiments in which Pha-IgM+ PCs from an initial phagocytosis experiment were sorted and exposed again to beads confirmed that these cells also have phagocytic capabilities, thereby suggesting that all teleost IgM+ B cells have phagocytic potential. Additionally, we found that grass carp IgM+ PCs display classical phenotypic features of macrophages, providing support for the hypothesis that vertebrate B cells evolved from ancient phagocytes. These findings together reveal that teleost B cells are a primitive B cell type with functions reminiscent of both mammalian B1 and B2 cells, providing insights into the origin and evolution of B cells in vertebrates.

Aggregation and proliferation of B cells and T cells in MALTs upon Cryptocaryon irritans infection in large yellow croaker Larimichthys crocea.

Mucosal immunity in mucosa-associated lymphoid tissues (MALTs) plays crucial roles in resisting infection by pathogens, including parasites, bacteria and viruses. However, the mucosal immune response in the MALTs of large yellow croaker (Larimichthys crocea) upon parasitic infection remains largely unknown. In this study, we investigated the role of B cells and T cells in the MALTs of large yellow croaker following Cryptocaryon irritans infection. Upon C. irritans infection, the total IgM and IgT antibody levels were significantly increased in the skin mucus and gill mucus. Notably, parasite-specific IgM antibody level was increased in the serum, skin and gill mucus following parasitic infection, while the level of parasite-specific IgT antibody was exclusively increased in MALTs. Moreover, parasitic infection induced both local and systemic aggregation and proliferation of IgM+ B cells, suggesting that the increased levels of IgM in mucus may be derived from both systemic and mucosal immune tissues. In addition, we observed significant aggregation and proliferation of T cells in the gill, head kidney and spleen, suggesting that T cells may also be involved in the systemic and mucosal immune responses upon parasitic infection. Overall, our findings provided further insights into the role of immunoglobulins against pathogenic infection, and the simultaneous aggregation and proliferation of both B cells and T cells at mucosal surfaces suggested potential interactions between these two major lymphocyte populations during parasitic infection.

Molecular and functional characterization of two IgM subclasses in large yellow croaker (Larimichthys crocea).

As the predominant immunoglobulin (Ig) isotype, IgM plays a crucial role in the acquired immunity of vertebrates. There is only one Igµ gene in mammals, except cattle, while the number of Igµ gene varies among teleost fish. In the current study, we found two functional Igµ genes (Igµ1 and Igµ2) and a pseudo Cµ gene (ψIgµ) in large yellow croaker (Larimichthys crocea). Both Igµ1 and Igµ2 genes possessed two transcript variants, which encoded the heavy chains of secreted (sIgM1 and sIgM2) and membrane-bound IgM1 and IgM2 (mIgM1 and mIgM2), respectively. Both the heavy chains of sIgM1 and sIgM2 consisted of a variable Ig domain, four constant Ig domains (CH1, CH2, CH3 and CH4) and a secretory tail, while those of mIgM1 and mIgM2 consisted of a variable Ig domain, three constant Ig domains (CH1, CH2 and CH3), a transmembrane domain and a short cytoplasmic tail. Cysteine residues that are necessary for the formation of intrachain and interchain disulfide bonds and tryptophan residues that are important for the folding of the Ig superfamily domain were well conserved in large yellow croaker IgM1 and IgM2. Interestingly, large yellow croaker IgM2 had an extra cysteine (C94) in the CH1 domain compared with IgM1, which may cause the structural difference between IgM1 and IgM2. A liquid chromatography-tandem mass spectrometry analysis revealed that both IgM1 and IgM2 were present at the protein level in large yellow croaker serum. Both the Igµ1 and Igµ2 genes were mainly expressed in systemic immune tissues, such as head kidney and spleen, but the expression level of Igµ2 was much lower than that of Igµ1. After Pseudomonas plecoglossicida infection, the expression levels of Igµ1 and Igµ2 in both the spleen and head kidney were significantly upregulated, with a higher upregulation of Igµ2 than that of Igµ1. These results suggested that Igµ1 and Igµ2 may play a differential role in the immune response of large yellow croaker against bacterial infection.

Characterization of snakehead (Channa argus) interleukin-21: Involvement in immune defense against two pathogenic bacteria, in leukocyte proliferation, and in activation of JAK-STAT signaling pathway.

Interleukin-21 (IL-21), a crucial immune regulatory molecule, belongs to the common γ-chain family of type I cytokines, and exerts pleiotropic effects on multiple immune cell types in mammals. However, the characteristics and functions of fish IL-21 remain unclear. To further investigate the molecular mechanism of IL-21 in teleosts, we first cloned and identified the IL-21 gene (designated shIL-21) of the snakehead (Channa argus). The full-length open reading frame of shIL-21 is 438 bp in length, and encodes a predicted protein of 145 amino acid residues. A sequence analysis showed that shIL-21 has the typical structural characteristics of other IL-21 proteins, containing four α-helices and four conserved cysteine residues. In a phylogenetic analysis, shIL-21 clustered within a subgroup of IL-21 proteins from other teleost species and shared its closest evolutionary relationship with that of Lates calcarifer. The expression analysis showed that shIL-21 was ubiquitously expressed in all the healthy snakehead tissues tested, albeit at different levels. After infection with Nocardia seriolae or Aeromonas schubertii, the relative expression of shIL-21 was mainly upregulated in the head kidney and spleen in vivo. Similarly, after stimulation with the three pathogen analogues lipoteichoic acid, lipopolysaccharides, and polyinosinic-polycytidylic acid, the expression of shIL-21 was also induced in head kidney leukocytes in vitro. A recombinant shIL-21 protein was expressed and purified, and promoted the proliferation of head kidney leukocytes, induced the expression of genes encoding critical signaling molecules in the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathway, including JAK1, JAK3, STAT1, and STAT3, and induced the expression of endogenous shIL-21 and genes encoding several key proinflammatory cytokines (tumor necrosis factor-α, interferon-γ, and IL-1ß). Taken together, these preliminary findings suggest that shIL-21 is involved in the immune defense against bacterial infection, in leukocyte proliferation, and in the activation of the JAK-STAT pathway. They thus extend the functional studies of IL-21 in teleosts.

Molecular identification and functional exploration of interleukin-20 in snakehead (Channa argus) involved in bacterial invasion and the proliferation of head kidney leukocytes.

As an inflammatory cytokine of the interleukin-20 (IL-20) subfamily, IL-20 has various functions in immune defenses, inflammatory diseases, tissue regeneration, cancer, and metabolism. Although the characteristics and functions of mammalian IL-20 have been clarified, those of fish IL-20 remain unclear. In this study, the IL-20 gene from the snakehead Channa argus (shIL-20) was cloned and functionally characterized. Similar to the IL-20 homologues of other species, the shIL-20 has a five exon/four intron structure in the coding region. The open reading frame of shIL-20 consists of 528 base pairs and encodes 175 amino acids (aa), including a signal peptide (aa 1-24) and a mature peptide (aa 25-175). The mature shIL-20 protein has six conserved cysteine residues, which occur in the IL-20 proteins of all species analyzed, and an additional cysteine residue (Cys-82) found only in the IL-20 proteins of several teleosts. The modeled tertiary structure of shIL-20 is similar with that of Homo sapiens IL-20. The shIL-20 was expressed constitutively in all the tissues analyzed, and its transcription was induced in the spleen and head kidney by Aeromonas schubertii and Nocardia seriolae in vivo and in head kidney leukocytes (HKLs) by lipoteichoic acid, lipopolysaccharide, and polyinosinic-polycytidylic acid in vitro. The recombinant shIL-20 protein induced the transcription of tumor necrosis factor α1 (TNF-α1), TNF-α2, IL-1ß, and endogenous shIL-20, and promoted the proliferation of HKLs. In conclusion, these findings demonstrate that shIL-20 participates in the immune response to bacterial invasion and promotes leukocyte proliferation, offering new insights into the functions of fish IL-20 during pathogen invasion.

ATG12 is involved in the antiviral immune response in large yellow croaker (Larimichthys crocea).

ATG12, a core autophagy protein, forms a conjugate with ATG5 to promote the formation of autophagosome membrane, and plays an important role in antiviral immunity. However, little is known about the function of ATG12 in fish. Here, we cloned the open reading frame (ORF) of large yellow croaker (Larimichthys crocea) ATG12 (LcATG12), which is 354 nucleotides long and encodes a protein of 117 amino acids. The deduced LcATG12 possesses a conserved APG12 domain (residues 31 to 117), and shares 91.45% identities with ATG12 in orange-spotted grouper (Epinephelus coioides). LcATG12 was constitutively expressed in all examined tissues, with the highest level in intestine. Its transcript was also detected in primary head kidney granulocytes (PKG), primary head kidney macrophages (PKM), primary head kidney lymphocytes (PKL), and large yellow croaker head kidney (LYCK) cell line, and was significantly up-regulated by poly(I:C). LcATG12 was regularly distributed in both cytoplasm and nucleus of LYCK and epithelioma papulosum cyprinid (EPC) cells. Overexpression of LcATG12 in EPC cells significantly inhibited the replication of spring viremia of carp virus (SVCV). Further studies reveled that LcATG12 could induce the occurrence of autophagy in LYCK cells. Furthermore, overexpression of LcATG12 in LYCK cells increased the expression levels of large yellow croaker type I interferons (IFNs, IFNc, IFNd, and IFNh), IFN regulatory factors (IRF3 and IRF7), and IFN-stimulated genes (PKR, Mx, and Viperin). All these data indicated that LcATG12 plays a role in the antiviral immunity possibly by inducing both autophagy and type I IFN response in large yellow croaker.

Two types of TNF-α and their receptors in snakehead (Channa argus): Functions in antibacterial innate immunity.

Tumor necrosis factor-α (TNF-α) is a pluripotent mediator of pro-inflammatory and antimicrobial defense mechanisms and a regulator of lymphoid organ development. Although two types of TNF-α have been identified in several teleost species, their functions in pathogen infection remain largely unexplored, especially in pathogen clearance. Herein, we cloned and characterized two types of TNF-α, termed shTNF-α1 and shTNF-α2, and their receptors, shTNFR1 and shTNFR2, from snakehead (Channa argus). These genes were constitutively expressed in all tested tissues, and were induced by Aeromonas schubertii and Nocardia seriolae in head kidney and spleen in vivo, and by lipoteichoic acid (LTA), lipopolysaccharides (LPS), and Polyinosinic-polycytidylic acid [Poly (I:C)] in head kidney leukocytes (HKLs) in vitro. Moreover, recombinant shTNF-α1 and shTNF-α2 upregulated the expression of endogenous shTNF-α1, shTNF-α2, shTNFR1, and shTNFR2, and enhanced intracellular bactericidal activity, with shTNF-α1 having a greater effect than shTNF-α2. These findings suggest important roles of fish TNFα1, TNFα2, and their receptors in bacterial infection and pathogen clearance, and provide a new insight into their function in antibacterial innate immunity.

Bacteria-induced IL-1ß and its receptors in snakehead (Channa argus): Evidence for their involvement in antibacterial innate immunity.

As a central pro-inflammatory cytokine, interleukin-1ß (IL-1ß) plays critical roles in the inflammatory response, pathogen infection, and immunological challenges in mammals. Although fish IL-1ß has been confirmed to participate in inflammatory response to pathogen infection, few studies have been performed to characterize the antibacterial and bactericidal functions of fish IL-1ß. In this study, snakehead (Channa argus) IL-1ß (shIL-1ß) and its receptors, shIL-1R1 and shIL-1R2, were cloned and functionally characterized. ShIL-1ß contained the IL-1 family signature domain, and a potential cutting site at Asp96 that presented in all vertebrate IL-1ß sequences. ShIL-1R1 had three extracellular IG-like domains and one intracellular signal TIR domain, while shIL-1R2 had three extracellular IG-like domain but lacked the intracellular signal TIR domain. ShIL-1ß, shIL-1R1, and shIL-1R2 were constitutively expressed in all tested tissues, and their expressions could be induced by Aeromonas schubertii and Nocardia seriolae in the head kidney and spleen in vivo, and by LTA, LPS, and Poly (I:C) in head kidney leukocytes (HKLs) in vitro. Moreover, recombinant shIL-1ß upregulated the expression of endogenous shIL-1ß, shIL-R1, and shIL-R2 in snakehead HKLs, and enhanced intracellular bactericidal activity. Taken together, this study found that, like IL-1ß and its receptors in mammals, shIL-1ß and its receptors play crucial roles in antibacterial innate immunity. This provides new insight into the evolution of IL-1ß function in vertebrates.

Treatment with Gold Nanoparticles Using Cudrania tricuspidata Root Extract Induced Downregulation of MMP-2/-9 and PLD1 and Inhibited the Invasiveness of Human U87 Glioblastoma Cells.

In this study, we aimed to elucidate the anti-invasive effects of Cudrania tricuspidata root-gold nanoparticles (CTR-GNPs) using glioblastoma cells. We demonstrated the rapid synthesis of CTR-GNPs using UV-vis spectra. The surface morphology, crystallinity, reduction, capsulation, and stabilization of CTR-GNPs were analyzed using high resolution transmission electron microscopy (HR-TEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FT-IR). Furthermore, CTR-GNPs displayed excellent photocatalytic activity as shown by the photo-degradation of methylene blue and rhodamine B. Cell migration and invasion assays with human glioblastoma cells were performed to investigate the anti-invasive effect of CTR-GNPs on U87 cells that were treated with phorbol 12-myristate 13-acetate. The results show that CTR-GNPs can significantly inhibit both basal and phorbol 12-myristate 13-acetate (PMA)-induced migration and invasion ability. Importantly, treatment with CTR-GNPs significantly decreased the levels of metalloproteinase (MMP)-2/-9 and phospholipase D1 (PLD1) and protein but not PLD2, which is involved in the modulation of migration and the invasion of glioblastoma cells. These results present a novel mechanism showing that CTR-GNPs can attenuate the migration and invasion of glioblastoma cells induced by PMA through transcriptional and translational regulation of MMP-2/-9 and PLD1. Taken together, our results suggest that CTR-GNPs might be an excellent therapeutic alternative for wide range of glioblastomas.

Molecular and functional characterization of the indoleamine 2,3-dioxygenase in grass carp (Ctenopharyngodon idella).

Indoleamine 2,3-dioxygenase (IDO) is a kind of dioxygenase that can catalyze the degradation of levo-tryptophan (L-Trp) and plays key roles in immune tolerance. In this study, the IDO gene was cloned and functionally characterized from grass carp (gcIDO). The results showed that gcIDO overexpressed in GCO cells could catalyze the degradation of L-Trp through the L-Trp - kynurenine pathway, and this activity could be promoted by δ-aminolevulinic acid (ALA) while inhibited by levo-1-methyl tryptophan (L-1MT). Moreover, gcIDO was constitutively expressed in various tissues, and its expression could be significantly up-regulated by LPS and Poly (I:C) in peripheral blood leukocytes (PBLs). Furthermore, recombinant TGF-ß1 of grass carp could up-regulate the expression of IDO, TGF-ß1, CD25, and Foxp3 in PBLs, indicating that the TGF-ß1/IDO pathway is present in fish. In the soybean meal induced enteritis (SBMIE) model, the expression of gcIDO in the intestine was up-regulated significantly, demonstrating that gcIDO may play an immunoregulatory role in SBMIE. Taken together, these data suggest that the IDO plays multiple roles in the immunity of fish.

Oral vaccination of tilapia against Streptococcus agalactiae using Bacillus subtilis spores expressing Sip.

Streptococcus agalactiae infections are becoming an increasing problem in aquaculture because of significant morbidity and mortality, which restricts the healthy development of tilapia aquaculture. To seek safe and effective prevention measures, a Bacillus subtilis GC5 surface displayed vaccine was prepared and applied orally in tilapia. The study first showed that recombinant spores can engraft in the tilapia intestine. Then, the effect of protection and the immune responses were evaluated. The results of ELISA showed that Sip-specific antibody in the sera of GC5-Sip-immunized fish can be detected after the first oral administration when compared to the phosphate buffer saline (PBS) control group, and the levels of specific IgM gradually strengthened with boosting, so does the specific antibody against bacteria, proving that humoral immunity was induced. Quantitative real-time PCR (qRT-PCR) results showed that the immune-related gene expression of the gut and spleen exhibited a different rising trend in the GC5-Sip group, revealing that innate immune response and local as well as systemic cellular immunity were induced. The outcome of fish immunized with GC5-Sip spores provided a relative percent survival (RPS) of 41.7% against S. agalactiae and GC5 group had an RPS of 24.2%, indicating that GC5-Sip was safe and effective in protecting tilapia against bacterial infection. Our study demonstrated that the oral administration of B. subtilis spores expressing Sip could cause an effective immune response and offer good resistance to bacterial infection. Our work may lead to the development of new ideas for immunoprophylaxis against S. agalactiae infection.

Characterization of grass carp CD40 and CD154 genes and the association between their polymorphisms and resistance to grass carp reovirus.

In bony fish, CD40 and CD154 are two very important costimulatory molecules involved in T and B cell cooperation in thymus-dependent antibody production. In the current study, we identified the cDNAs of CD40 and CD154 and analyzed their genomic structures in grass carp. Quantitative real-time PCR indicated that the CD40 and CD154 were mainly expressed in immune organs. After challenge with grass carp reovirus (GCRV), these two genes were up-regulated at 72 h in head kidney and spleen. Moreover, seven and five single nucleotide polymorphisms (SNPs) were identified in the CD40 and CD154 respectively. Statistical analysis indicated that three SNPs in the coding region of the CD40 were significantly associated with the resistance of grass carp against GCRV. These results indicated that CD40 and CD154 play important roles in the responses to GCRV in grass carp. The SNP markers in the CD40 associated with the resistance to GCRV may facilitate the disease-resistant breeding of grass carp.

Comparative study of the immunoprotective effect of two DNA vaccines against grass carp reovirus.

Grass carp reovirus II (GCRV II) causes severe hemorrhagic disease with high mortality in grass carp, Cyenopharyngodon idellus. DNA vaccination has been proven to be a very effective method in conferring protection against fish viruses. However, DNA vaccines for GCRV II have not yet been conducted on grass carp. In the current work, we vaccinated grass carp with a DNA vaccine consisting of the segment 6 (pC-S6; encoding VP4) or 10 (pC-S10; encoding NS38) of GCRV II and comparatively analyzed the immune responses induced by these two vaccines. The protective efficacy of pC-S6 and pC-S10, in terms of relative percentage survival (RPS), was 59.9% and 23.1% respectively. This suggests that pC-S6 and pC-S10 DNA vaccines could increase the survival rate of grass carp against GCRV, albeit with variations in immunoprotective effect. Immunological analyses indicated the following. First, post-vaccination (pv), both pC-S6 and pC-S10 up-regulated the expression of interferon (IFN-1), Mx1, IL-1ß, and TNF-α. However, CD4 and CD8α were up-regulated in the case of pC-S6 but not pC-S10. Second, comparing non-vaccinated and pC-S10-vaccinated fish, the T cell response related genes, such as CD4, CD8α, and GATA3, were elevated in pC-S6-vaccinated fish at 48 h post-challenge (pc). Third, pC-S6 and pC-S10 induced similar patterns of specific antibody response pv. However, only anti-VP4 IgM in the sera of surviving fish infected with GCRV was significantly increased pc compared with that pre-challenge. Taken together, these results indicate that pC-S6 promotes both innate (IFN-1 and Mx1 induction) and adaptive (T cell and specific antibody response) immunity pv and that the induction of a memory state promptly primes the immune response upon later encounters with the virus, whereas pC-S10 only induces the type I IFN-related response pv and a lower inflammatory response pc.

Sequential Combination of Microwave- and Ultrasound-Assisted Extraction of Total Flavonoids from Osmanthus fragrans Lour. Flowers.

Microwave-assisted and ultrasound-assisted extraction assays were used to isolate total flavonoids (TF) from Osmanthus fragrans flowers. The effects of the solid-liquid ratio, ethanol concentration, microwave power, microwave extraction time, ultrasonic power and ultrasonic extraction time on the yield of TF were studied. A sequential combination of microwave- and ultrasound-assisted extraction (SC-MUAE) methods was developed, which was subsequently optimized by Box-Behnken design-response surface methodology (BBD-RSM). The interaction effects of the ethanol concentration (40-60%), microwave extraction time (5-7 min), ultrasonic extraction time (8-12 min) and ultrasonic power (210-430 W) on the yield of TF were investigated. The optimum operating parameters for the extraction of TF were determined to be as follows: ethanol concentration (48.15%), microwave extraction time (6.43 min), ultrasonic extraction time (10.09 min) and ultrasonic power (370.9 W). Under these conditions, the extraction yield of TF was 7.86 mg/g.

Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed.

Bacillus subtilis is widely used as probiotic species in aquaculture for water quality control, growth promoting, or immunity enhancing. The aim of this study is to find novel B. subtilis strains from fish as potential probiotics for aquaculture. Eleven B. subtilis isolates derived from the intestinal tract of grass carp were identified by gene sequencing and biochemical tests. These isolates were classified into 4 groups, and the representatives (GC-5, GC-6, GC-21 and GC-22) of each group were further investigated for antibiotic susceptibility, sporulation rate, biofilm formation, activity against pathogenic bacteria, resistance to stress conditions of intestinal tract (high percentage of bile and low pH) and high temperature, which are important for probiotics to be used as feed additives. Additionally, the adhesion properties of the 4 characterized strains were assessed using Caco-2 cell and gut mucus models. The results showed that the 4 strains differed in their capacities to adhere to intestinal epithelial cells and mucus. Furthermore, the strains GC-21 and GC-22 up-regulated the expression levels of IL-10 and TGF-ß but down-regulated IL-1ß, suggesting their potential anti-inflammatory abilities. Based on physiological properties of the 4 characterized B. subtilis strains, one or more strains may have potential to be used as probiotics in aquaculture.

A carrier-free, injectable, and self-assembling hydrogel based on carvacrol and glycyrrhizin exhibits high antibacterial activity and enhances healing of MRSA-infected wounds.

Inspired by glycyrrhizin's strong pharmacological activities and the directed self-assembly into hydrogels, we created a novel carrier-free, injectable hydrogel (CAR@glycygel) by combining glycyrrhizin with carvacrol (CAR), without any other chemical crosslinkers, to promote wound healing on bacteria-infected skin. CAR appeared to readily dissolve and load into CAR@glycygel. CAR@glycygel had a dense, porous, sponge structure and strong antioxidant characteristics. In vitro, it showed better antibacterial ability than free CAR. For methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, and Escherichia coli, the diameter of inhibition zone values of CAR@glycygel were 3.80 ± 0.04, 3.31 ± 0.20 and 3.12 ± 0.24 times greater, respectively, than those of free CAR. The MICs for CAR@glycygel was 156.25 µg/mL while it was 1250.00 µg/mL for free CAR to these three bacteria. Its antibacterial mechanism appeared to involve destruction of the integrity of the bacterial cell wall and biomembrane, leading to a leakage of AKP and inhibition of biofilm formation. In vivo, CAR@glycygel effectively stopped bleeding. When applied to skin wounds on rats infected with MRSA, CAR@glycygel had strong bactericidal activity and improved wound healing. The wound healing rates for CAR@glycygel were 49.59 ± 15.78 %, 93.02 ± 3.09 % and 99.02 ± 0.55 % on day 3, day 7, and day 11, respectively, which were much better than blank control and positive control groups. Mechanisms of CAR@glycygel accelerating wound healing involved facilitating epidermis remolding, promoting the growth of hair follicles, stimulating collagen deposition, mitigating inflammation, and promoting angiogenesis. Overall, CAR@glycygel showed great potential as wound dressing for infected skin wounds.

[Study of the association between SNP rs7903146(C/T) in TCF7L2 and metabolic syndrome in Chinese Korean and Han populations from Yanbian].

OBJECTIVE: To assess the association between a rs7903146(C/T) polymorphism of TCF7L2 gene and metabolic syndrome (MS), plasma lipoprotein, and plasma adiponectin (PA) in Chinese Korean and Han populations from Yanbian region. METHODS: Polymerase chain reaction and DNA sequencing were used to determine the genotype of rs7903146 in 310 Chinese Korean (190 in case group and 120 in control group) and 344 Chinese Han (255 in case group and 89 in control group). ELIAS was used to test serum insulin (INS) and PA. RESULTS: The frequency of T allele was higher in ethnic Han compared with ethnic Koreans (0.022 vs. 0.008), lower than that of Europeans (0.279) and Africans (0.257), but similar to those of Beijing Chinese and Japanese. For ethnic Korean Chinese, the frequencies of TT and CT genotypes as well as the T allele in patients with EH were significantly higher than those of the control group (P< 0.01), which also showed an increasing trend for both MS and T2DM groups (P=0.09 and P=0.07, respectively). By contrast, for Chinese Han, the frequencies of genotypes and particular allele in patients with MS, T2DM and EH showed no significant difference from those of the control group. For T2DM, EH, and control groups, PA level of individuals with CT or TT genotypes was significantly higher compared with that of the CC genotype (P< 0.05). The TC and LDL-C levels were significantly higher in T2DM, MS and EH groups compared with those of the control group. The PA level was lower in MS group compared with the control group. CONCLUSION: The T allele of SNP rs7903146 of TCF7L2 gene may be a risk factor for EH in Chinese Korean population from Yanbian region. The T allele also affects the PA level; lower PA is a risk factor for MS. The rs7903146 polymorphism showed a racial and ethnic difference.

Neutrophil functions can be regulated by IL-35, which is mainly expressed in IL-15Rα+ cells in grass carp (Ctenopharyngodon idella).

IL-35 plays a key role in regulatory T (Treg) and regulatory B (Breg) cell functions in mammals. CD25 has been demonstrated as one of the markers of Treg cells, and CD19+CD25hiCD71hi cells have been verified as a type of Breg cells in humans. These results indicate that there is a close relationship between IL-35 and CD25+ cells. In mammals, CD25 (alias IL-2Rα) has been identified as having high affinity and specificity for IL-2 binding, and is closely linked and structurally related to IL-15Rα, which having high affinity for IL-15 binding. In teleost, IL-15Rα can bind to both IL-2 and IL-15, with higher affinity to IL-15 than IL-2, and has been termed a CD25-like molecule in some research studies. To date, no studies of IL-35 and IL-15Rα have been documented in fish. In this work, five isoforms of IL-15Rα were cloned from grass carp, and a monoclonal antibody to the protein was developed. The results of flow cytometry and quantitative real-time PCR analyses demonstrated that grass carp IL-35 subunit genes EBI3a and IL-12p35 were mainly expressed in IL-15Rα+ cells, while the expression levels of IL-10 and TGF-ß in IL-15Rα+ and IL-15Rα- cells were insignificant. Recombinant grass carp IL-35 (rgcIL-35) could increase the proportion of IL-15Rα+ cells in leukocytes, and a certain proportion of IL-15Rα+ cells also appeared in myeloid cell subset II after stimulation with rgcIL-35. Meanwhile, the migration, phagocytic ability, and bactericidal ability of grass carp neutrophils were significantly decreased after stimulation with certain concentrations of rgcIL-35. Moreover, neutrophil apoptosis could be significantly inhibited by rgcIL-35.

MEKK3 in hybrid snakehead (Channa maculate ♀ ×Channa argus ♂): Molecular characterization and immune response to infection with Nocardia seriolae and Aeromonas schubertii.

Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) is a serine/threonine protein kinase that acts as a key regulator and is widely involved in various innate and acquired immune signaling pathways. In this study, we first cloned the complete open reading frame (ORF) of the MEKK3 gene (named CcMEKK3) in a hybrid snakehead (Channa maculate ♀ × Channa argus ♂). The full-length ORF of CcMEKK3 is 1851 bp, and encodes a putative protein of 616 amino acids containing a serine/threonine kinase catalytic (S-TKc) domain and a Phox and Bem1p (PB1) domain. A sequence alignment and phylogenetic tree analysis showed that CcMEKK3 is highly conserved relative to the MEKK3 proteins of other teleost species. CcMEKK3 was constitutively expressed in all the healthy hybrid snakehead tissues tested, with greatest expression in the immune tissues, such as the head kidney and spleen. The expression of CcMEKK3 was usually upregulated in the head kidney, spleen, and liver at different time points after infection with Nocardia seriolae or Aeromonas schubertii. Similarly, the dynamic expression levels of CcMEKK3 in head kidney leukocytes after stimulation revealed that CcMEKK3 was induced by LTA, LPS, and poly(I:C). In the subcellular localization analysis, CcMEKK3 was evenly distributed in the cytoplasm of HEK293T cells, and its overexpression significantly promoted the activities of NF-κB and AP-1. These results suggest that CcMEKK3 is involved in the immune defense against these two pathogens, and plays a crucial role in activating the NF-κB and MAPK signaling pathways.

Molecular characterization and functional analysis of IL-18 in snakehead (Channa argus) during Aeromonas schubertii and Nocardia seriolae infections.

As a proinflammatory cytokine of the interleukin-1 (IL-1) family, IL-18 plays important roles in host protection against bacterial, viral, and fungal infection. We cloned the open reading frame of snakehead (Channa argus) IL-18 (shIL-18) and found that it contained 609 base pairs and encoded 202 amino acid residues. The shIL-18 included a conserved IL-1-like family signature and two potential IL-1ß-converting enzyme cutting sites; one was conserved in all analyzed IL-18s, but the other was unique to shIL-18. Unlike other IL-18s, shIL-18 also contained a predicted signal peptide. In this study, shIL-18 was constitutively expressed in all tested tissues, and its expression was induced by Aeromonas schubertii and Nocardia seriolae in the head kidney and spleen in vivo and by lipoteichoic acid, lipopolysaccharides, and polyinosinic-polycytidylic acid in head kidney leukocytes in vitro. Moreover, recombinant shIL-18 upregulated the expression of interferon-γ, IL-1ß, and tumor necrosis factor-α1 and -α2 and promoted the proliferation of leukocytes. Taken together, these results showed that IL-18 played crucial roles in host defense against bacterial infection in fish, as it does in mammals.