Natural Killer Cell Enhancing Factor Research Articles

Transcriptional analysis of immune-relevant genes in the mucus of Labeo rohita, experimentally infected with Argulus siamensis.

The knowledge of mucosa-associated molecular events that occur during infections is scarce despite the well-established importance of mucus in fish immunity. Using qRT-PCR, we analyzed the immune gene expression patterns in mucus of Labeo rohita experimentally infected with an ectoparasite Argulus siamensis. Mucus samples were collected at 0 h, 12 h, 24 h, 3 d, 7 d, 15 d, and 30 d post challenge of L. rohita with metanauplii of A. siamensis. All interleukins studied herein (IL 6, IL 15, and IL 1β) showed significant upregulation of expression levels in mucus of A. siamensis-infected fish compared to control samples. Further, the expression levels of molecules involved in pathogen recognition, toll like receptor 22, and pathogen presentation, β2 microglobulin, were found to be significantly upregulated in experimental samples until 7 d post challenge compared to control samples. The upregulated expression of lysozyme G at all time points post infection indicated the early activation of acute phase responses in mucus of infected L. rohita. Moreover, the expression levels of natural killer cell enhancing factor B were found to be higher in infected fish than they were in the control fish. The early upregulation of the immune genes observed herein reinforces the role of mucus as the first line of defense against pathogenic assault; furthermore, it expands our understanding of mucosal-immune responses to A. siamensis infection, which can aid development of immunological interventions.

Differential proteome profile of skin mucus of gilthead seabream (Sparus aurata) after probiotic intake and/or overcrowding stress

Gilthead seabream (Sparus aurata L.) is the major cultured fish species in the Mediterranean area. High density stocking causes stress and increases the impact of diseases leading to economic losses. Probiotics could represent a solution to prevent diseases through several mechanisms such as improving the immune status and/or mucosal microbiota or competing with pathogens. The probiotic Shewanella putrefaciens, also known as Pdp11, was firstly isolated from the skin of healthy gilthead seabream. Our study focuses on the skin mucus proteome after dietary probiotic Pdp11 intake in fish maintained under normal or overcrowding conditions. 2-DE of skin mucus followed by LC–MS/MS analysis was done for each experimental group and differentially expressed proteins were identified. The results showed differentially expressed proteins especially involved in immune processes, such as lysozyme, complement C3, natural killer cell enhancing factor and nonspecific cytotoxic cell receptor protein 1, whose transcript profiles were studied by qPCR. A consistency between lysozyme protein levels in the mucus and lysozyme mRNA levels in skin was found. Further research is necessary to unravel the implications of skin mucosal immunity on fish welfare and disease. Biological significanceThe present work reveals the proteomic changes, which are taking place in the skin mucus of stressed and non-stressed gilthead seabream after Pdp11 probiotic intake. The study contributes to improving the knowledge on skin mucosal immunology of this relevant farmed fish species. Furthermore, the paper shows for the first time how a suitable proteomic methodology, in this case 2-DE followed by LC–MS/MS is useful to perform a comparative study with a non-invasive technique of skin mucus of gilthead seabream.

The immune response in rohu,Labeo rohita(Actinopterygii: Cyprinidae) toArgulus siamensis(Branchiura: Argulidae) infection: kinetics of immune gene expression and innate immune response

Argulus siamensis is a major pathogen in freshwater aquaculture. The immune responses of Indian major carp, Labeo rohita to experimental infection of A. siamensis was evaluated by quantitation of immune-relevant gene expression in head kidney and skin, and serum innate immune parameters through the course of infection. In skin of infected fish, antioxidant genes like natural killer cell enhancing factor (NKEF-B) and superoxide dismutase (MnSOD) were significantly up-regulated in addition to lysozyme G and β2 microglobulin (β2M). Both tumour necrosis factor α (TNFα) and toll-like receptor 22 (TLR22) genes were significantly down-regulated in skin during early phases of the infection. Most of the genes exhibited significant down-regulation in head kidney; immunoglobulin (IgM) and β2M genes being the exceptions which were significantly up-regulated at 12 h and 3 days post infection. Most of the innate immune parameters like serum complement activity and ceruloplasmin levels showed significant reduction in infected fish. The observed results are indicative of A. siamensis modulating the immune response of rohu by down-regulation of many immune factors which may explain the susceptibility of rohu to A. siamensis infection. The interaction of this parasite with the host need to be further explored to understand its pathogenesis.

Molecular identification and expression analysis of a natural killer cell enhancing factor (NKEF) from rock bream Oplegnathus fasciatus and the biological activity of its recombinant protein

Natural killer cell enhancing factor (NKEF) belongs to the defined peroxiredoxin (Prx) family. Rock bream NKEF cDNA was identified by expressed sequence tag (EST) analysis of rock bream liver that was stimulated with the LPS. The full-length RbNKEF cDNA (1062bp) contained an open reading frame (ORF) of 594bp encoding 198 amino acids. RbNKEF was significantly expressed in the gill, liver, and intestine. mRNA expression of NKEF in the head kidney was examined under viral and bacterial challenge via real-time RT-PCR. Experimental challenge of rock bream with Edwardsiella tarda, Streptococcus iniae, and RSIV resulted in significant increases in RbNKEF mRNA in the head kidney. To obtain a recombinant NKEF, the RbNKEF ORF was expressed in Escherichia coli BL21 (DE3), and the purified soluble protein exhibited a single band corresponding to the predicted molecular mass. When kidney leucocytes were treated with a high concentration of rRbNKEF (10μg/mL), they exhibited significantly enhanced cell proliferation and viability under oxidative stress.

Comparative study of the immune effect of an Edwardsiella tarda antigen in two forms: Subunit vaccine vs DNA vaccine

Edwardsiella tarda is a Gram-negative bacterial pathogen and the etiological agent of a systematic fish disease called edwardsiellosis, which affects a wide range of marine and freshwater fish. E. tarda vaccines in various forms have been reported by a number of research groups; however, comparative studies on the immune mechanisms of these vaccines are lacking. In this report, we identified a new E. tarda vaccine candidate, Eta2, and analyzed in a comparative manner the immune response induced by Eta2 in two different forms: purified recombinant subunit vaccine and DNA vaccine. Eta2 is a protein of 178 residues and shares high levels of sequence identities with the OmpH family of outer membrane protein chaperones of several bacterial species. Recombinant Eta2 (rEta2) purified from Escherichia coli was highly protective against E. tarda challenge in a Japanese flounder (Paralichthys olivaceus) model and produced relative percent of survival rates of 83% and 78%, respectively, at 4- and 8-week post-vaccination (p.v.). Eta2 as a DNA vaccine in the form of plasmid pCEta2 also induced strong protective immunity at 4- and 8-week p.v. Immunological analysis indicated that (i) rEta2 and pCEta2 enhanced head kidney macrophage activation at 1- and, for pCEta2, 7-day p.v.; (ii) rEta2 and pCEta2 induced similar patterns of serum antibody production, however, the antibodies induced by rEta2 were of much higher levels and afforded stronger passive immunoprotection upon naïve flounder than those induced by pCEta2; (iii) both rEta2 and pCEta2 upregulated the expression of specific and nonspecific immune factors which include, in the case of pCEta2 but not rEta2, interferon, interferon-induced Mx protein, and CD8α; however, the induction patterns effected by rEta2 and pCEta2 were different. While high levels of interleukin 1β (IL-1β), natural killer cell enhancing factor, Mx, MHC Iα, and IgM inductions were observed in pCEta2-vaccinated fish, only IL-1β, complement C3, and IgM inductions were highly induced in rEta2-vaccinated fish. Taken together, these results indicate that both rEta2 and pCEta2 induce specific and nonspecific immunities, however, pCEta2 induces both B cell and T cell responses, whereas rEta2 induces mainly humoral response.

Increased liver protein and mRNA expression of natural killer cell-enhancing factor B (NKEF-B) in ayu ( Plecoglossus altivelis) after Aeromonas hydrophila infection

Natural killer cell-enhancing factor (NKEF) may mediate cellular responses to proinflammatory molecules. The liver proteins of Aeromonas hydrophila-infected ayu ( Plecoglossus altivelis) and healthy control fish were analyzed by 2DE. A protein, which increased significantly in diseased fish, was identified as NKEF-B by MALDI-TOF-MS. A full-length cDNA clone of this protein was subsequently isolated. It contains 1092 bp with an open reading frame of 591 bp, coding for 197 amino acids with MW 21.9 kDa and p I 6.38, values similar to those determined by 2DE. Ayu NKEF-B had highest similarity (93.1% amino acid identity) to those of carp and zebrafish. Phylogenetic analysis showed that ayu NKEF-B falls into the fish NKEF-B cluster and is most closely related to that of carp and zebrafish. It was determined that ayu NKEF-B mRNA expression was significantly increased in many tissues at the early stage of bacterial infection. In conclusion, the increased NKEF-B mRNA and protein expression in ayu were closely associated with A. hydrophila infection.

Structure, organization and expression of common carp ( Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3′ and 5′ RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5′-terminal untranslated region (UTR), a 355 bp 3′-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74–96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1-k long genomic DNA of carp NKEF-B containing six exons and five introns. Real-time RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity.

Molecular identification and expression analysis of the natural killer cell enhancing factor (NKEF) gene from turbot ( Scophthalmus maximus)

Natural killer cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. It was originally isolated from human red blood cells. It has the ability to enhance the cytotoxic activity of natural killer cells, and appears most effective in the protection of DNA and protein against oxidative damage, chemotherapy agents, and inflammation-induced monocyte adhesion. However, few reports are available about the structure and function of NKEF in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding NKEF from turbot ( Scophthalmus maximus). The cDNAs of NKEF were cloned from turbot spleen cDNA library. The 904 bp full-length cDNA of the NKEF was obtained by RACE and consisted of 122 bp 5′ terminal UTR, 591 bp encoding region and 191 bp 3′ terminal UTR. The 591 bp open reading frame was found to code for a protein with 197 amino acid residues. The deduced amino acid sequence of turbot had 85.3%, 81.7%, 81.2%, 80.7%, 80.7%, 79.2%, 78.2%, 77.7%, 77.7%, 77.2% and 58.2% identity with that of zebrafish, channel catfish, flounder, Tetraodon nigroviridis, rainbow trout and mouse Prx2-1, human NKEF-A, carp and rat TSA, human NKEF-B and yeast TSA, respectively. RT-PCR indicated that NKEF transcripts were abundant in the head kidney, spleen and liver, less abundant in the kidney and intestine, and least in gill, muscle and heart. The level of NKEF mRNA in embryos gradually increases during embryogenesis from two cell stage to fry stage. Challenge of turbot with pathogenic bacteria Vibrio anguillarum, significantly elevated NKEF mRNA levels in the liver and spleen in a time-dependent fashion. The NKEF transcripts were detected in turbot embryonic cell line (TEC). Challenge of the TEC cell cultures with pathogenic bacteria, V. anguillarum, significantly elevated NKEF mRNA levels in TEC cell cultures 48 h after infection.

Cloning and characterisation of two natural killer enhancing factor genes (NKEF-A and NKEF-B) in pufferfish, Tetraodon nigroviridis

Natural killer cell enhancing factor (NKEF) belongs to a highly conserved peroxiredoxin (Prx) family. Its functions are involved in immune cytotoxicity, apoptosis, cell proliferation, differentiation and antioxidant activity. In present paper, two NKEF genes, NKEF-A and NKEF-B, were isolated and characterised in spotted green pufferfish, Tetraodon nigroviridis. Comparative genomic location analysis shows conserved syntenies within the NKEF-A and NKEF-B regions between Tetraodon and human, indicating that the Tetraodon NKEF-A and NKEF-B genes are orthologs for mammalian NKEF-A and NKEF-B encoding genes, respectively. The genomic organisation of the two genes is similar in different species, containing one non-coding exon, five coding exons and five introns. The deduced amino acids of the two genes are highly homologous with mammalian NKEF-A and NKEF-B proteins, including two well conserved Val-Cys-Pro (VCP) motifs. Tissue specific expression analysis by RT-PCR reveals that both genes are globally expressed. Stimulation with LPS inhibits both NKEF-A and NKEF-B gene expression in most tissues but enhances NKEF-A and NKEF-B transcriptions in spleen. This is the first report on the existence of a NKEF-B homologue in fish.

Genomic organisation and expression of the natural killer cell enhancing factor (NKEF) gene in channel catfish, Ictalurus punctatus (Rafinesque)

The gene encoding Natural Killer Cell Enhancing Factor (NKEF) was identified in clones from a gynogenetic channel catfish BAC library. NKEF gene sequence (5.2 kb) was obtained by direct sequencing of BAC clones. The catfish NKEF gene contains one non-coding exon, five coding exons and five introns. A putative TATA box and other transcription factor binding sites were identified in the promoter region. The NKEF amino acid sequence is highly conserved between fish and mammals. Gene expression, measured by real-time quantitative PCR, was detected in all major tissues with the highest level of expression in stomach and heart and lowest levels in gonad and pituitary gland. Catfish NKEF mRNA levels were slightly upregulated 8 and 24 h after injection of lipopolysaccharide. A TAA-repeat microsatellite was identified in a BAC clone containing the NKEF gene, and this locus contained at least 12 alleles in a random-bred catfish population. Multipoint linkage analysis in two reference families placed the NKEF gene on linkage group U18, 5.1 cM from locus IpCG0177 ( r = 0.05; LOD = 45.8), 6.0 cM from a novel immune type receptor, syntenic (40 cM) with T-cell receptor α, and not linked with MHC loci.

Identification of differentially expressed proteins between human hepatoma and normal liver cell lines by two-dimensional electrophoresis and liquid chromatography-ion trap mass spectrometry.

In the previous study, the proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 were separated by high resolution two-dimensional electrophoresis (2-DE). Image analysis revealed that 99 protein spots showed quantitative and qualitative variations that were significant (P < 0.01) and reproducible. Here we report the identification results of some of these protein spots. Protein spots excised from 2-D gels were subjected to in-gel digestion with trypsin, and the resulting peptides were measured by microbore high performance liquid chromatography - ion trap - mass spectrometry (LC-IT-MS) to obtain the tandem mass (MS/MS) spectra. Twelve protein spots were identified with high confidence using SEQUEST with uninterpreted MS/MS raw data. Besides inosine-5'-monophosphate dehydrogenase 2, heat shock 27 kDa protein, calreticulin and calmodulin, whose expression was elevated in hepatoma cells, glutathione-S-transferase P was identified from hepatoma cells in which its level was 18-fold higher compared to human liver cells. Two spots were identified as the homologs of reticulocalbin for the first time in hepatoma cells and their expression increased compared to liver cells. However, tubulin beta-1 chain and natural killer cell enhancing factor B were downregulated in hepatoma cells. A tumor suppressing serpin, maspin precursor, was identified from one spot whose quantity was much higher in the normal liver cell line. More interestingly, epidermal fatty acid-binding protein (E-FABP) and fatty acid-binding protein, adipocyte-type (A-FABP), were detected in liver cells but not in hepatoma cells. The functional implication of the identified proteins was discussed.

Pravastatin Increases Survival and Inhibits Natural Killer Cell Enhancement Factor in Liver Transplanted Rats

Pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has been shown to decrease the number of acute rejection episodes in cardiac and renal transplant patients. This study evaluates the effects of pravastatin on survival of rats following liver transplant and attempts to elucidate the mechanisms of these effects. Both survival and natural killer cell enhancing factor (NKEF) studies utilized Dark Agouti rats for donor livers transplanted into Brown Norway rats as recipients. All rats received daily low-dose cyclosporine (CsA) 2 mg/kg/day by gavage. The treated groups also received gavage doses of pravastatin, 20 mg/kg/day. Survival data were analyzed by the method of Kaplan–Meier and log-rank χ2tests for statistical significance. For NKEF evaluation, rats were sacrificed at varying time points; total RNA was extracted from the liver and hybridized with32P-radiolabeled NKEF DNA probes in the Northern blot technique. Radiographs were quantitated using densitometry. Data were analyzed by two-way ANOVA. Actuarial survival was improved (P<< 0.05) in rats treated with pravastatin in addition to low-dose CsA (n= 41, CsA alonen= 74). Less fibrosis and chronic rejection was seen on histological section in the treated animal livers,P< 0.05. NKEF was seen maximally at Days 5–15 tapering off at Day 21. NKEF-a and NKEF-b levels were significantly decreased in the animals treated with CsA and pravastatin compared to CsA alone in the group of animals < 16 days postop (P<< 0.05). Pravastatin improves survival in rats following OLT and while the mechanism is still unknown, inhibition of natural killer cell enhancement factor may represent an alteration in the overall immune response.