Non-specific Cytotoxic Cell Receptor Protein Research Articles

Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus)

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.

Molecular characteristics and functional analysis of non-specific cytotoxic cell receptor (NCCRP1) in golden pompano (Trachinotus ovatus)

Non-specific cytotoxic cells (NCCs) are cytotoxic cell population found in innate immune system of teleost, playing crucial role in immune defense. Non-specific cytotoxic cell receptor protein 1 (NCCRP1) is responsible for recognizing target cells and activating NCCs. That said, since the studies regarding NCCs’ role in fish during pathogen infection are few, it is necessary to conduct more comprehensive studies. In this study, we identified NCCRP1 from Trachinotus ovatus (ToNCCRP1). The open reading frame of ToNCCRP1 was found to be 702 bp, encoding a protein of 233 amino acids. Additionally, ToNCCRP1 contained a conserved F-box-associated domain and exhibited more than 61 % similarity to NCCRP1 in other fish species. Quantitative real-time PCR analysis showed that ToNCCRP1 mRNA was generally expressed in all tissues, with the highest level expressed in the liver. Furthermore, the expression of ToNCCRP1 was significantly upregulated following infection with Streptococcus iniae. In vitro experiments demonstrated that recombinant ToNCCRP1 possessed bacterial agglutination and binding capabilities, suggesting its antibacterial function. Additionally, we investigated the immune response of head kidney leukocytes (HKLs) to ToNCCRP1. The challenge experiments revealed that ToNCCRP1 played a role in the immune response by influencing the inflammatory response, regulating signaling pathways and apoptosis in HKLs. These findings suggest that NCCRP1 is involved in the immune defense against pathogenic infections in golden pompano, providing insights into the immune mechanisms of teleost.

Non-specific cytotoxic cell receptor protein-1 (NCCRP-1) is involved in anti-parasite innate CD8+ T cell-mediated cytotoxicity in ginbuna crucian carp.

CD8+ cytotoxic T cells (CTLs) are a main cellular component of adaptive immunity. Our previous research has shown that CD8+ cells demonstrate spontaneous cytotoxic activity against the parasite Ichthyophthirius multifiliis in ginbuna crucian carp, suggesting that CD8+ cells play an important role in innate immunity. Herein, we investigated the molecules and cellular signal pathways involved in the cytotoxic response of ginbuna crucian carp. We considered non-specific cytotoxic receptor protein-1 (NCCRP-1) as candidate molecule for parasite recognition. We detected NCCRP-1 protein in CD8+ cells and the thymus as well as in other cells and tissues. CD8+ cells expressed mRNA for NCCRP-1, Jak2, and T cell-related molecules. In addition, treatment with a peptide containing the presumed antigen recognition site of ginbuna NCCRP-1 significantly inhibited the cytotoxic activity of CD8+ cells against the parasites. The cytotoxic activity of CD8+ cells was significantly inhibited by treatment with the JAK1/2 inhibitor baricitinib. These results suggest that teleost CTLs recognize I. multifiliis through NCCRP-1 and are activated by JAK/STAT signaling.

Costimulatory Molecules CD80/86 Trigger Non-Specific Cytotoxic Cell of Nile tilapia (Oreochromis niloticus) to Kill CIK Cells

The teleost non-specific cytotoxic cell (NCC), as the evolutionary precursors of NK cells, is an important cytotoxic cell population in the innate immune system of teleost. We have recently realized that costimulatory CD80/86 have conservation in structural and interactional features with its ligand CD28 in Nile tilapia (Oreochromis niloticus). However, the ability of CD80/86 to regulate NCC activity has not been fully investigated. In the present study, we first obtained the recombinant fusion CD80/86 protein from O. niloticus (rOn-CD80/86). Then, NCC incubation with rOn-CD80/86 resulted in a significant production of NCC effector cytokines, including tumor necrosis factor-alpha, cellular apoptosis susceptibility and NK-lysin. Furthermore, NCC treatment with rOn-CD80/86 could significantly improve the ability to kill kidney cells of Grass carp (CIK) and up-regulate the activities of caspase-1 and caspase-3 in CIKs. The yeast, two-hybrid assay showed that On-CD80/86 cannot directly interact with non-specific cytotoxic cell receptor protein-1 of O. niloticus (On-NCCRP-1). The single-cell RNA-Seq data of Nile tilapia head kidney lymphocytes analysis found On-CD28 did not exhibit expression on NCCs subsets. The above results suggest that costimulatory molecules On-CD80/86 is independent of On-NCCRP-1 and On-CD28 receptor in modulating NCC killing activity in vitro of Nile tilapia. The results also provide more insights into the mechanism of NCC activity regulation.

Expression changes of non-specific cytotoxic cell receptor (NCCRP1) and proliferation and migration of NCCs post-Nocardia seriolae infection in Northern Snakehead

Non-specific cytotoxic cells (NCCs) are essential to the cytotoxic cell-mediated immune response in teleost. The fish non-specific cytotoxic cell receptor protein 1 (NCCRP1) plays an important role as a membrane protein in the recognition of target cells and the activation of NCC. However, the roles of fish NCCs during pathogen infection require comprehensive studies. In this study, the coding sequence of northern snakehead (Channa argus) nccrp1 (Canccrp1) was cloned. Canccrp1 contains an open reading frame of 690 bp, encoding a peptide of 229 amino acids with a conserved F-box-associated domain (FBA) and proline-rich motifs (PRMs). Transcriptional expression analysis revealed that the constitutive expression of Canccrp1 was higher in the immune-related organs, such as liver, kidneys, and spleen. Moreover, mRNA and protein expression of Canccrp1 gradually increased in the spleen at 1–6 days post infection (dpi) with Nocardia seriolae, in addition to reaching peak expression in both the kidneys and liver at 2 dpi. A polyclonal antibody prepared against recombinant CaNCCRP1 effectively labeled NCCs in peripheral blood and different tissues. Then, immunofluorescence (IF) staining showed that the number of NCCs was significantly increased and showed a scattered distribution in the early stages of N. seriolae infection (2 and 4 dpi) before the forming of granulomas. At the late stages of N. seriolae infection (6 dpi), more NCCs migrated to preexisting granulomas, showing significant coaccumulation with N. seriolae. All these results clearly indicate the expression changes of CaNCCRP1, and the number and localization changes of NCCs post-N. seriolae infection, implying potential roles for fish NCCs in the antimicrobial infection process in fish.

Transferrin Mediated NCC Killing Activity through NCCRP-1 in Nile Tilapia (Oreochromis niloticus)

Non-specific cytotoxic cell l (NCC) is a kind of important lymphocyte participating in the non-specific immune response in teleost. Non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is a receptor molecule on the surface of NCC and plays an important role in mediating the activity of NCC. However, there are few reports on which signal molecule could transmit signals through NCCRP-1. In this study, yeast two-hybrid library of tilapia liver and head kidney was constructed, and a transferrin from Oreochromis niloticus (On-TF) with interaction protein sequence was obtained by screening the library with bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1). Then, the open reading frame (ORF) of On-TF was cloned, which had 2088 bp in length, encoding polypeptides of 695 amino acids. The deduced amino acid sequence was highly homologous to teleost and similar to mammalian TF, containing two TR_FER domains (25-343th aa and 344-686th aa) for binding iron ions. Furthermore, a point-to-point yeast two-hybrid method was used to further verify whether On-NCCRP-1 could bind to On-TF. The recombinant On-TF (rOn-TF) protein was purified by prokaryotic expression system. In vitro experiments showed that rOn-TF could up-regulate the expression of killing effector molecule of NCC by On-NCCRP-1, and rOn-TF-activated NCCs showed a significantly improved ability to kill FHM cells, indicating that rOn-TF could regulate the NCC signaling pathway through NCC receptor molecule On-NCCRP-1. The results provide a more theoretical basis for understanding the regulation mechanism of NCC activity.

Biological characterisation, expression and functional analysis of non-specific cytotoxic cell receptor protein 1 in Nile tilapia (Oreochromis niloticus)

Non-specific cytotoxic cell receptor protein 1 (NCCRP-1) plays a role in recognition of target cell and activation of non-specific cytotoxic cell (NCC). In this study, the full length of Nile tilapia NCCRP-1 (On-NCCRP-1) was cloned. cDNA is composed of 1045 bp with a 90 bp of 5′-Untranslated Regions (UTR), 702 bp open reading frame (ORF) and 253 bp 3′-UTR, encoding 233 amino acids (GenBank accession no: MF162296). The On-NCCRP-1 genomic sequence is 4471 bp in length and contains six exons and five introns. On-NCCRP-1 possesses some inherent conservative domains, such as proline-rich motifs, antigen recognition site, and F-box-related domain. Subcellular localisation and Western blot analysis indicated that On-NCCRP-1 is located in the cell membrane. The transcript of On-NCCRP-1 was detected in all the examined tissues of healthy Nile tilapia by using qRT-PCR, with the highest expression levels in the liver. Following Streptococcus agalactiae challenged in vivo, the On-NCCRP-1 expression was up-regulated significantly in brain, intestines, head kidney and spleen. In the in vitro analysis, the On-NCCRP-1 expression in NCCs was up-regulated significantly from 8 h to 12 h after LPS challenge, and up-regulated significantly at 12 h after challenged with polyI:C. After NCCs were challenged with inactivated S. agalactiae, the On-NCCRP-1 expression was down-regulated significantly after 24 h. NF-кB pathway was strongly activated by the over-expression of On-NCCRP-1 in HEK-293T cells. These results indicate that On-NCCRP-1, as a membrane surface receptor of NCCs, may play an important role in immune response to pathogenic infection in Nile tilapia.

Salmon louse rhabdoviruses: Impact on louse development and transcription of selected Atlantic salmon immune genes

Recently, it has been shown that the salmon louse (Lepeophtheirus salmonis) is commonly infected by one or two vertically transmitted Lepeophtheirus salmonis rhabdoviruses (LsRVs). As shown in the present study, the viruses have limited effect on louse survival, developmental rate and fecundity. Since the LsRVs were confirmed to be present in the louse salivary glands, the salmon cutaneous immune response towards LsRV positive and negative lice was analyzed. In general, L. salmonis increased the expression of IL1β, IL8 and IL4/13A at the attachment site, in addition to the non-specific cytotoxic cell receptor protein 1 (NCCRP-1). Interestingly, LsRV free lice induced a higher skin expression of IL1β, IL8, and NCCRP-1 than the LsRV infected lice. The inflammatory response is important for louse clearance, and the present results suggest that the LsRVs can be beneficial for the louse by dampening inflammation. Further research is, however; needed to ascertain whether this is a direct modulatory effect of secreted virions, or if virus replication is altering the level of louse salivary gland proteins.

Identification of NCCRP1 as an epigenetically regulated tumor suppressor and biomarker for malignant phenotypes of squamous cell carcinoma of the esophagus.

The poor prognosis and increasing incidence of esophageal squamous cell carcinoma (ESCC) highlight the need for identification of novel ESCC-associated molecular events to improve the diagnosis, and treatment of this disease. Non-specific cytotoxic cell receptor protein 1 (NCCRP1) was reported to be abundantly expressed in human squamous epithelium and to be involved in cell proliferation; however, the role of NCCRP1 in ESCC remains unclear. To elucidate the oncological roles of NCCRP1 in ESCC, NCCRP1 expression, DNA methylation, and copy numbers were analyzed in ESCC cell lines. Nine ESCC cell lines demonstrated different NCCRP1 mRNA expression levels and all exhibited hypermethylation of the NCCRP1 promoter, but no copy number loss. Additionally, NCCRP1 expression was determined in 213 surgically resected esophageal tissue samples. NCCRP1 mRNA expression levels were reduced in ESCC tissues compared with corresponding non-cancerous adjacent tissues in 204 (95.8%) patients. Patients in the low NCCRP1 expression group tended to have a higher recurrence rate and a shorter overall survival time compared with those in the high NCCRP1 expression group. Additionally, multivariate analysis revealed that low NCCRP1 expression was an independent prognostic factor (hazard ratio, 1.75; 95% confidence interval, 1.08-2.87; P=0.022). The findings of the current study indicate that NCCRP1 acts as a putative tumor suppressor that is inactivated through promoter hypermethylation, and serves as a promising biomarker to predict postoperative prognosis in ESCC.

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.

Effects of Fish Meal Replacement by a Soybean Protein on Growth, Histology, Selected Immune and Oxidative Status Markers of Gilthead Sea Bream, Sparus aurata

AbstractThe purpose of the trial was to study the impact of a 6‐mo dietary administration of soybean protein on growth, liver and intestine morphology, immune response, and oxidative stress in gilthead sea bream. The immune response was evaluated by performing immunological assays in blood, head kidney (HK), or serum (respiratory burst activity [RBA], myeloperoxidase content and bacteriolytic activity) and gene expression analysis of immune‐associated genes (MHCIIα [major histocompatibility complex IIα], β2m [β‐2‐microglobulin], CSF‐1R [colony‐stimulating factor‐1 receptor], NCCRP‐1 [nonspecific cytotoxic cell receptor protein 1], TGF‐β1 [transforming growth factor beta‐1], and HSP70 [heat‐shock protein 70]) in HK and intestine. Oxidative stress was evaluated by measuring the activity of liver enzymes associated with antioxidant system. The soybean protein was administrated in the diets at 20, 40, and 60% levels and its effects were evaluated compared with a fish meal diet. Growth and feed efficiency were affected negatively from 40% level. Increased RBA and expression levels of TGF‐β1 and β2m were found in HK only at 40% level. In intestine, at 60% level CSF‐1R expression was upregulated and some signs of inflammation were evident. In liver, at 60% level lipid accumulation in hepatocytes was observed and enzyme activity was increased. Dietary administration of soybean protein indicated changes from 40% in growth and immune response, and exerted an antioxidative effect at 60% level.

Molecular cloning of NCCRP-1 gene from orange-spotted grouper (Epinephelus coioides) and characterization of NCCRP-1+ cells post Cryptocaryon irritans infection

Nonspecific cytotoxic cells (NCCs) are an important cytotoxic cell population in the innate teleost immune system. The receptor designated “NCC receptor protein 1” (NCCRP-1) has been reported to be involved in the recognition and activation of NCCs. In this study, the full-length cDNA of Epinephelus coioides NCCRP-1 (ecnccrp-1) was cloned. The open reading frame (ORF) of ecnccrp-1 is 699bp, encoding a 232 amino acid protein that includes proline-rich motifs at the N-terminus and is related to the F-box associated family. Although a bioinformatics analysis showed that EcNCCRP-1 had no signal peptide or transmembrane helices, a polyclonal antibody directed against recombinant EcNCCRP-1 efficiently labeled a membrane protein in the head kidney, detected with Western blot analysis, which indicated that the protein localized to the cell surface. RT-PCR showed that the constitutive expression of ecnccrp-1 was higher in the lymphoid organs, such as the trunk kidney, spleen, head kidney, and thymus, and lower in brain, heart, fat, liver, muscle, and skin. After infection with Cryptocaryon irritans, the transcription of ecnccrp-1 was analyzed at the infected sites (skin and gills) and in the systemic immune organs (head kidney and spleen). At the infected sites, especially the skin, ecnccrp-1 expression was upregulated at 6h post infection, reaching peak expression on day 3 post the primary infection. However, the expression patterns differed in the systemic immune organs. In the spleen, ecnccrp-1 was gradually increased in the early infection period and decreased sharply on day 3 post the primary infection, whereas in the head kidney, the transcription of ecnccrp-1 was depressed during almost the whole course of infection. An immunohistochemical analysis showed that EcNCCRP-1+ cells accumulated at the sites of infection with C. irritans. These results suggested that NCCs were involved in the process of C. irritans infection in E. coioides.

Cloning and expression analysis of nonspecific cytotoxic cell receptor 1 (Ls-NCCRP1) from red snapper (Lutjanus sanguineus)

It is well known that nonspecific cytotoxic cells (NCCs) are kinds of natural killer cell mediated innate immune responses in teleosts. The nonspecific cytotoxic cell receptor protein 1 (NCCRP-1) is an important cell surface protein on NCC, which serves crucial functions in target cell recognition and cytotoxicity activation. In the present study, a nonspecific cytotoxic cell receptor protein NCCRP-1 (Ls-NCCRP1) was cloned from red snapper, Lutjanus sanguineus. The Ls-NCCRP1 cDNA is composed of 986bp with a 43bp of 5′-UTR, 702bp open reading frame (ORF) and 241bp 3′-UTR, encoding a polypeptide of 233 amino acids (GenBank accession no: ADK32635). Phylogenetic analysis revealed that Ls-NCCRP1 showed highest similarity to sea bream NCCRP-1. Quantitative real-time PCR (qRT-PCR) analysis showed that Ls-NCCRP1 had relatively high expression level in the head kidney, spleen and liver. After Vibrio alginolyticus infection, transcripts of Ls-NCCRP1 increased and reached its peak at 4h p.i. These results indicated that Ls-NCCRP1 may play an important role in innate immune response to bacteria.

Expression of genes involved in the early immune response at the distal segment of the gut in Atlantic cod, Gadus morhua L. after vaccination with a bacterial antigen

The gastro-intestinal tract serves as the route of infection of most bacterial pathogens in fish, and the immune response genes in the gut produce substances that provide an initial defense during pathogen invasion. The expression of some genes involved in antibacterial defense, cell-mediated immunity and inflammation in the distal intestine of Atlantic cod, Gadus morhua L., was investigated following intraperitoneal vaccination with heat-killed Vibrio anguillarum. Tissue samples of the distal intestine were collected at 0, 1, 3, 7 and 10 days post-vaccination (dpv) and processed for semi-quantitative RT-PCR analyses. Expression of the bactericidal/permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme, non-specific cytotoxic cell receptor protein-1 (NCCRP-1) and interleukin-8 (IL-8) significantly increased during the early stages post-vaccination, that is, 1–3 dpv, followed by a decrease in their expression returning to their initial levels. Transferrin and apolipoprotein A-I, although weakly expressed in the distal intestine, also increased in the vaccinated fish. Interleukin-1β (IL-1β) was significantly decreased in its expression level relative to the PBS-injected fish at 3 and 7 dpv. The expression of these immune-related genes in the distal intestine as a result of vaccination provides valuable information on the mechanisms of gut immunity in the fish against pathogenic bacteria, particularly during the early stages of infection.

Characterization of Non-Specific Cytotoxic Cell Receptor Protein 1: A New Member of the Lectin-Type Subfamily of F-Box Proteins

Our previous microarray study showed that the non-specific cytotoxic cell receptor protein 1 (Nccrp1) transcript is significantly upregulated in the gastric mucosa of carbonic anhydrase IX (CA IX)-deficient (Car9−/−) mice. In this paper, we aimed to characterize human NCCRP1 and to elucidate its relationship to CA IX. Recombinant NCCRP1 protein was expressed in Escherichia coli, and a novel polyclonal antiserum was raised against the purified full-length protein. Immunocytochemistry showed that NCCRP1 is expressed intracellularly, even though it has previously been described as a transmembrane protein. Using bioinformatic analyses, we identified orthologs of NCCRP1 in 35 vertebrate genomes, and up to five paralogs per genome. These paralogs are FBXO genes whose protein products are components of the E3 ubiquitin ligase complexes. NCCRP1 proteins have no signal peptides or transmembrane domains. NCCRP1 has mainly been studied in fish and was thought to be responsible for the cytolytic function of nonspecific cytotoxic cells (NCCs). Our analyses showed that in humans, NCCRP1 mRNA is expressed in tissues containing squamous epithelium, whereas it shows a more ubiquitous tissue expression pattern in mice. Neither human nor mouse NCCRP1 expression is specific to immune tissues. Silencing CA9 using siRNAs did not affect NCCRP1 levels, indicating that its expression is not directly regulated by CA9. Interestingly, silencing NCCRP1 caused a statistically significant decrease in the growth of HeLa cells. These studies provide ample evidence that the current name, “non-specific cytotoxic cell receptor protein 1,” is not appropriate. We therefore propose that the gene name be changed to FBXO50.

Molecular Correlates of Experimental Cerebral Malaria Detectable in Whole Blood

Cerebral malaria (CM) is a primary cause of deaths caused by Plasmodium falciparum in young children in sub-Saharan Africa. Laboratory tests based on early detection of host biomarkers in patient blood would help in the prognosis and differential diagnosis of CM. Using the Plasmodium berghei ANKA murine model of experimental cerebral malaria (ECM), we have identified over 300 putative diagnostic biomarkers of ECM in the circulation by comparing the whole-blood transcriptional profiles of resistant mice (BALB/c) to those of two susceptible strains (C57BL/6 and CBA/CaJ). Our results suggest that the transcriptional profile of whole blood captures the molecular and immunological events associated with the pathogenesis of disease. We find that during ECM, erythropoiesis is dysfunctional, thrombocytopenia is evident, and glycosylation of cell surface components may be modified. Furthermore, analysis of immunity-related genes suggests that slightly distinct mechanisms of immunopathogenesis may operate in susceptible C57BL/6 and CBA/CaJ mice. Furthermore, our data set has allowed us to create a molecular signature of ECM composed of a subset of circulatory markers. Complement component C1q, β-chain, nonspecific cytotoxic cell receptor protein 1, prostate stem cell antigen, DnaJC, member 15, glutathione S-transferase omega-1, and thymidine kinase 1 were overexpressed in blood during the symptomatic phase of ECM, as measured by quantitative real-time PCR analysis. These studies provide the first host transcriptome database that is uniquely altered during the pathogenesis of ECM in blood. A subset of these mediators of ECM warrant validation in P. falciparum-infected young African children as diagnostic markers of CM.

Expression profiles of genes associated with immune response and oxidative stress in Atlantic cod, Gadus morhua head kidney leukocytes modulated by live and heat-inactivated intestinal bacteria

The molecular mechanisms of immune response and antioxidant defense in Atlantic cod Gadus morhua head kidney (HK) leukocytes to live and heat-inactivated intestinal bacteria were investigated by transcriptome analyses. The HK leukocytes were incubated with Pseudomonas sp. (GP21) and Psychrobacter sp. (GP12), which are intestinal bacteria of Atlantic cod. The responses of the defense-associated genes at 3 and 24h post-incubation (hpi) were assayed by semi-quantitative RT-PCR. Live and heat-inactivated GP21 caused a significant increase in the transcript levels of bacterial defense genes in the HK leukocytes: BPI/LBP and g-type lysozyme were highest at 24hpi. The levels of BPI/LBP were significantly upregulated at 24hpi by live GP12 but not by the heat-inactivated type. The expression of g-type lysozyme was significantly elevated regardless of the type of GP12. IL-1beta was significantly upregulated by live GP21 and GP12, with maximum expression observed at 3hpi. In contrast, the expression levels of IL-8 in the HK leukocytes were not augmented by both types of GP21 and GP12. A significant upregulation of the non-specific cytotoxic cell receptor protein-1 (NCCRP-1) was observed with live GP12 at 3hpi, whereas in the case of GP21 such a change was noted only with the heat-inactivated type at 24hpi. A definite pattern of granzyme expression was not observed with both the live and heat-inactivated GP21 and GP12. The levels of antioxidant genes (catalase and GSH-Px) remained unchanged except in cells incubated with heat-inactivated GP21, where a significant elevation of GSH-Px was seen at 24hpi. Thus, this in vitro study has revealed that the defense mechanisms in the HK leukocytes can be modulated by the commensal intestinal bacteria of Atlantic cod. The extent of this activation is dependent on the bacterial species and its viability.

Profiling gene expression in the spleen of Atlantic cod, Gadus morhua upon vaccination with Vibrio anguillarum antigen

The mechanisms of physiological responses in Atlantic cod, Gadus morhua following vaccination with a heat-killed Vibrio anguillarum were investigated by transcriptome analysis of spleen tissues. Semi-quantitative RT-PCR of several genes involved in immune defense, inflammation, antioxidant defense and glucose transport were determined in vaccinated fish at 1, 3, 7 and 10 days after vaccination (dpv)and compared with sham-injected fish. Transcript levels of the selected genes involved in bacterial defense such as the bactericidal permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme and transferrin, were significantly upregulated (P < 0.05) throughout the duration of sampling (1-10 dpv). There was differential expression of the genes involved in antiviral activity, cellular immunity, antioxidant defense and glucose transport, while the pro-inflammatory cytokines remained relatively unchanged in both the vaccinated and sham-vaccinated fish. The expressions of interferon stimulated gene-15 (ISG-15) and interferon regulatory factor-1 (IRF-1), which are involved in viral defense, were significantly enhanced (P < 0.05) after vaccination. Likewise, the transcript levels of the non-specific cytotoxic cell receptor protein-1 (NCCRP-1) and granzyme A/K, which are components of the cell-mediated immunity were upregulated. Among the antioxidants, the transcript levels of catalase and phospholipid hydroperoxide glutathione peroxidase (GSH-Px) significantly increased (P < 0.05) following vaccination, while glucose transporter-4 (GLUT-4) was enhanced among the genes involved in glucose transport. Our results indicate that the spleen of Atlantic cod is able to mount a potent physiological response through enhanced transcription of at least the mentioned genes, upon exposure to a bacterial antigen. These genes work synergistically to protect the fish during subsequent infection.

Characterization of rag1 mutant zebrafish leukocytes

BackgroundZebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants).ResultsDifferential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes.Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte-like cells and comprised 7% of the gated cells in the rag1 mutants and 26% in the wild-types.Reverse transcriptase polymerase chain reaction (RT-PCR) assays demonstrated rag1 mutant kidney hematopoietic tissue expressed mRNA encoding Non-specific Cytotoxic cell receptor protein-1 (NCCRP-1) and Natural Killer (NK) cell lysin but lacked T cell receptor (TCR) and immunoglobulin (Ig) transcript expression, while wild-type kidney hematopoietic tissue expressed NCCRP-1, NK lysin, TCR and Ig transcript expression.ConclusionOur study demonstrates that in comparison to wild-type zebrafish, rag1 mutants have a significantly reduced lymphocyte-like cell population that likely includes Non-specific cytotoxic cells (NCC) and NK cells (and lacks functional T and B lymphocytes), a similar macrophage/monocyte population, and a significantly increased neutrophil population. These zebrafish have comparable leukocyte populations to SCID and rag 1 and/or 2 mutant mice, that possess macrophages, natural killer cells and neutrophils, but lack T and B lymphocytes. Rag1 mutant zebrafish will provide the platform for remarkable investigations in fish and innate immunology, as rag 1 and 2 mutant mice did for mammalian immunology.

Role of nonspecific cytotoxic cells in bacterial resistance: Expression of a novel pattern recognition receptor with antimicrobial activity

Pattern recognition receptors (PRR) recognize invariant bacterial, viral, protozoan and certain synthetic ligands. PRR may be expressed as outer membrane (or endosomal) or cytosolic proteins and function to signal cell activation processes during inflammation responses. In the present study, a novel membrane receptor, NCC cationic antimicrobial protein-1 (NCAMP-1), is described that is expressed on nonspecific cytotoxic cell (NCC) membranes and is found in granule extracts from these cells. In recombinant form, full-length (amino acids 1–203) and truncated N (NT; amino acids 1–60) and C (CT; amino acids 116–203) terminal forms of NCAMP-1 had antibacterial activity against bovine, avian and lab strain Escherichia coli. Recombinant NCAMP-1-NT also killed the gram-negative fish pathogen Edwardsiella ictaluri. Maximal bacterial killing of a representative avian E. coli, APEC 3721, occurred at 60 min post-treatment with 2 μg/ml of rNCAMP-1-NT. Killing occurred by NCAMP-1-NT-induced alterations in the permeability of the bacterial cell wall. Polyclonal antibody anti-NCAMP-1 specifically neutralized the antimicrobial activity of recombinant NCAMP-1-NT against E. coli APEC 3751. Expression of NCAMP-1 as a NCC membrane protein was analyzed by flow cytometry using anti-NCAMP-1 monoclonal antibody 9C9. Merged images from immunofluorescence microscopy showed that NCAMP-1 and the NCC receptor protein (NCCRP-1) are co-expressed on NCC membranes. NCAMP-1 was identified in acetic acid granule extracts of NCC by Western blot analysis using polyclonal anti-NCAMP-1 and killing of E. coli by these extracts was specifically inhibited by this polyclonal. These data suggested that NCAMP-1 is a membrane protein and may participate in antibacterial innate immunity by granule exocytosis during inflammatory responses in teleosts.