Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge. (c) 2005 Elsevier Ltd. All rights reserved.

STARVATION-INDUCED CHANGES OF HEMOCYTE PARAMETERS IN THE ZHIKONG SCALLOP CHLAMYS FARRERI

Substantial nutritional and energetic demands m-e associated with immune activation and the maintenance of an efficient immune system. One-year-old Chlamys farreri (Jones and Preston) scallops were maintained ill lantern nets ill different nutritional conditions (satiation and starvation) for 40 days. After the 40-day treatments, the condition index and the total hemocyte count (THC) decreased significantly in the starved group compared with the satiated and initial control groups. The percentage of phagocytic hemocytes also was significantly reduced with starvation. In contrast. no significant effect of starvation was observed oil reactive oxygen species (ROS) production. The acid phosphatase (ACP) activities in cell-free hemolymph increased significantly in scallops in starved and satiated treatments compared with the initial control. whereas ACP activity in hemocyte lysate was significantly lower ill the starved group. These results indicate that starvation stress compromises immunological activities of scallops.

Cloning, characterization, and expression analysis of extracellular copper/zinc superoxide dismutase gene from bay scallop Argopecten irradians

Extracellular superoxide dismutase (ECSOD) is a major extracellular antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned a novel ECSOD from the bay scallop Argopecten irradians (AiECSOD) by 3' and 5' RACE. The full-length cDNA of AiECSOD was 893 bp with a 657 bp open reading frame encoding 218 amino acids. The deduced amino acid sequence contained a putative signal peptide of 20 amino acids, and sequence comparison showed that AiECSOD had low degree of homology to ECSODs of other organisms. The genomic length of the AiECSOD gene was about 5276 bp containing five exons and six introns. The promoter region contained many putative transcription factor binding sites such as c-Myb, Oct-1, Sp1, Kruppel-like, c-ETS, NF kappa B, GATA-1, AP-1, and Ubx binding sites. Furthermore, tissue-specific expressions of AiECSOD and temporal expressions of AiECSOD in haemocytes of bay scallops challenged with bacteria Vibrio anguillarum were quantified using qRT-PCR. High levels of expression were detected in haemocytes, but not in gonad and mantle. The expression of AiECSOD reached the highest level at 12 h post-injection with V. anguillarum and then returned to normal between 24 h and 48 h post-injection. These results indicated that AiECSOD was an inducible protein and that it may play an important role in the immune responses against V anguillarum. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.

Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians: Its gene structure, mRNA expression and recombinant protein

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Two unique and highly compartmentalized bay scallop Argopecten irradians superoxide dismutases: MnSOD and ecCuZnSOD, have been molecularly characterized in our previous study. To complete characterize the SOD family in A. irradians, a novel intracellular copper/zinc SOD from the A. irradians (Ai-icCuZnSOD) was obtained and characterized. The full-length cDNA of Ai-icCuZnSOD was 1047 bp with a 459 bp open reading frame encoding 152 amino acids. The genomic length of the Ai-icCuZnSOD gene was about 4279 bp containing 4 exons and 3 introns. The promoter region containing many putative transcription factor binding sites were analyzed. Furthermore, quantitative reverse transcriptase real-time PCR (qRT-PCR) analysis indicated that the highest expression of the Ai-icCuZnSOD was detected in gill and the expression profiles in hemocytes of bay scallops challenged with bacteria Vibrio anguillarum and lipopolysaccharide (LPS) were different. The result presented an increased expression after injection with LPS whereas no significant changes were observed after V. anguillarum injection. A fusion protein containing Ai-icCuZnSOD was produced in vitro. The rAi-icCuZnSOD is a stable enzyme, retaining more than 80% of its activity between 10 and 60 degrees C and keeping above 88% of its activity at pH values between 5.8 and 9. Ai-icCuZnSOD is more stable under alkaline than acidic conditions. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

The manganese superoxide dismutase gene in bay scallop Argopecten irradians: Cloning, 3D modelling and mRNA expression

A novel manganese superoxide dismutase (MnSOD) was cloned from bay scallop Argopecten irradians by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of MnSOD was of 1207 bp with a 678 bp open reading frame encoding 226 amino acids. The deduced amino acid sequence contained a putative signal peptide of 26 amino acids. Sequence comparison showed that the MnSOD of A. irradians shared high identity with MnSOD in invertebrates and vertebrates, such as MnSOD from abalone Haliotis discus discus (ABG88843) and frog Xenopus laevis (AAQ63483). Furthermore, the 3D structure of bay scallop MnSOD was predicted by SWISS-MODEL Protein Modelling Server and compared with those of other MnSODs. The overall structure of bay scallop MnSOD was similar to those of zebrafish Danio rerio, fruit fly Drosophila melanogaster, Chinese shrimp Fenneropenaeus chinensis, human Homo sapiens, and had the highest similarity to scallop Mizuhopecten yessoensis and abalone H. discus discus. A quantitative real-time PCR (qRT-PCR) assay was developed to detect the mRNA expression of MnSOD in different tissues and the temporal expression in haemocytes following challenge with the bacterium Vibrio anguillarum. A higher-level of mRNA expression of MnSOD was detected in gill and mantle. The expression of MnSOD reached the highest level at 3 h post-injection with V. anguillarum and then slightly recovered from 6 to 48 h. The results indicated that bay scallop MnSOD was a constitutive and inducible protein and thus could play an important role in the immune responses against V anguillarum infection. (c) 2008 Elsevier Ltd. All rights reserved.

Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631 bp, consisting of a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 573 by with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968 bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96 h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12 h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response. (c) 2006 Elsevier Ltd. All rights reserved.

Purification and characterisation of a natural lectin from the serum of the shrimp Litopenaeus vannamei

A natural lectin from the serum of the shrimp Litopenaeus vannamei was purified to homogeneity by a single-step affinity chromatography using fetuin-coupled agarose. The purified serum lectin (named LVL) showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC, chicken RBC and its haemagglutinating (HA) activity was specifically dependent on Ca2+ and reversibly sensitive to EDTA. LVL inactive form had a molecular mass estimate of 172 kDa and was composed of two non-identical subunits (32 and 38 kDa) cross-linked by interchain disulphide bonds. Significant LVL activity was observed between pH 7 and 11. In HA-inhibition assays performed with several carbohydrates and glycoproteins, LVL showed a distinct and unique specificity for GalNAc/GluNAc/NeuAc which had an acetyl group, while glycoproteins fetuin and bovine submaxillary mucin (BSM) had sialic acid. Moreover, this agglutinin appeared to recognise the terminal N- and O-acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of L. vannamei lectin was also susceptible to inhibition by lipopolysaccharides from diverse Gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections. (C) 2006 Elsevier Ltd. All rights reserved.

Purification and characterization of a natural lectin from the plasma of the shrimp Fenneropenaeus chinensis

A natural lectin from the plasma of the shrimp Fenneropenaeus chinensis was purified by singlestep affinity chromatography using fetuin-coupled agarose. The purified plasma lectin showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC and chicken RBC. The hemagglutinating (HA) activity of the lectin was dependent on Ca2+ and reversibly sensitive to EDTA. This lectin was named FC-L and its inactive form had a molecular mass estimate of 168 kDa. Fifteen N-terminal amino acid sequences of this protein were determined. We performed HA-inhibition assays with several carbohydrates and glycoproteins. FC-L showed a distinct and unique specificity to N-acetylated sugars, particularly sialic acid and sialoproteins. The FC-L also has binding activity to some Gram-negative bacteria which caused disease in shrimp and fish. The activity of FC-L was inhibited at temperatures greater than 75 degrees C and at a pH less than 7 or greater than 11. These results suggest that FC-L may play a role as pattern recognition proteins in the reorganization and clearance of invaders in shrimp F. chinensis. Crown Copyright (c) 2008 Published by Elsevier Ltd. All rights reserved.

Exercise training can induce cardiac autophagy at end-stage chronic conditions: Insights from a graft-versus-host-disease mouse model

Chronic graft-versus-host disease (cGVHD) is a frequent cause of morbimortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and severely compromises patients' physical capacity. Despite the aggressive nature of the disease, aerobic exercise training can positively impact survival as well as clinical and functional parameters. We analyzed potential mechanisms underlying the recently reported cardiac function improvement in an exercise-trained cGVHD murine model receiving lethal total body irradiation and immunosuppressant treatment (Fiuza-Luces et al., 2013. Med Sci Sports Exerc 45, 1703-1711). We hypothesized that a cellular quality-control mechanism that is receiving growing attention in biomedicine, autophagy, was involved in such improvement. Our results suggest that exercise training elicits a positive autophagic adaptation in the myocardium that may help preserve cardiac function even at the end-stage of a devastating disease like cGVHD. These preliminary findings might provide new insights into the cardiac exercise benefits in chronic/debilitating conditions.

An artificial immune system for continuous analysis of time-varying data

M. Neal, An Artificial Immune System for Continuous Analysis of Time-Varying Data, in Proceedings of the 1st International Conference on Artificial Immune Systems (ICARIS), 2002, eds J Timmis and P J Bentley, volume 1, pages 76-85,

Meta-stable memory in an artificial immune network

Neal, M., Meta-stable memory in an artificial immune network, Proceedings of the 2nd International Conference on Artificial Immune Systems {ICARIS}, Springer, 168-180, 2003,LNCS 2787/2003

Optimisation of immune effector function within the tumour microenvironment

Cancer represents a leading of cause of death in the developed world, inflicting tremendous suffering and plundering billions from health budgets. The traditional treatment approaches of surgery, radiotherapy and chemotherapy have achieved little in terms of cure for this deadly disease. Instead, life is prolonged for many, with dubious quality of life, only for disease to reappear with the inevitable fatal outcome. “Blue sky” thinking is required to tackle this disease and improve outcomes. The realisation and acceptance of the intrinsic role of the immune system in cancer pathogenesis, pathophysiology and treatment represented such a “blue sky” thought. Moreover, the embracement of immunotherapy, the concept of targeting immune cells rather than the tumour cells themselves, represents a paradigm shift in the approach to cancer therapy. The harnessing of immunotherapy demands radical and innovative therapeutic endeavours – endeavours such as gene and cell therapies and RNA interference, which two decades ago existed as mere concepts. This thesis straddles the frontiers of fundamental tumour immunobiology and novel therapeutic discovery, design and delivery. The work undertaken focused on two distinct immune cell populations known to undermine the immune response to cancer – suppressive T cells and macrophages. Novel RNAi mediators were designed, validated and incorporated into clinically relevant gene therapy vectors – involving a traditional lentiviral vector approach, and a novel bacterial vector strategy. Chapter 2 deals with the design of novel RNAi mediators against FOXP3 – a crucial regulator of the immunosuppressive regulatory T cell population. Two mediators were tested and validated. The superior mediator was taken forward as part of work in chapter 3. Chapter 3 deals with transposing the RNA sequence from chapter 2 into a DNA-based construct and subsequent incorporation into a lentiviral-based vector system. The lentiviral vector was shown to mediate gene delivery in vitro and functional RNAi was achieved against FOXP3. Proof of gene delivery was further confirmed in vivo in tumour-bearing animals. Chapter 4 focuses on a different immune cell population – tumour-associated macrophages. Non-invasive bacteria were explored as a specific means of delivering gene therapy to this phagocytic cell type. Proof of delivery was shown in vitro and in vivo. Moreover, in vivo delivery of a gene by this method achieved the desired immune response in terms of cytokine profile. Overall, the data presented here advance exploration within the field of cancer immunotherapy, introduce novel delivery and therapeutic strategies, and demonstrate pre-clinically the potential for such novel anti-cancer therapies.

Peanut Allergen Threshold Study (PATS): validation of eliciting doses using a novel single-dose challenge protocol

Background: The eliciting dose (ED) for a peanut allergic reaction in 5% of the peanut allergic population, the ED05, is 1.5 mg of peanut protein. This ED05 was derived from oral food challenges (OFC) that use graded, incremental doses administered at fixed time intervals. Individual patients’ threshold doses were used to generate population dose-distribution curves using probability distributions from which the ED05 was then determined. It is important to clinically validate that this dose is predictive of the allergenic response in a further unselected group of peanut-allergic individuals. Methods/Aims: This is a multi-centre study involving three national level referral and teaching centres. (Cork University Hospital, Ireland, Royal Children’s Hospital Melbourne, Australia and Massachusetts General Hospital, Boston, U.S.A.) The study is now in process and will continue to run until all centres have recruited 125 participates in each respective centre. A total of 375 participants, aged 1–18 years will be recruited during routine Allergy appointments in the centres. The aim is to assess the precision of the predicted ED05 using a single dose (6 mg peanut = 1.5 mg of peanut protein) in the form of a cookie. Validated Food Allergy related Quality of Life Questionnaires-(FAQLQ) will be self-administered prior to OFC and 1 month after challenge to assess the impact of a single dose OFC on FAQL. Serological and cell based in vitro studies will be performed. Conclusion: The validation of the ED05 threshold for allergic reactions in peanut allergic subjects has potential value for public health measures. The single dose OFC, based upon the statistical dose-distribution analysis of past challenge trials, promises an efficient approach to identify the most highly sensitive patients within any given food-allergic population.

CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo.

Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II-restricted interferon gamma-producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.

Critical role of interleukin 5 and eosinophils in concanavalin A-induced hepatitis in mice.

BACKGROUND & AIMS: Eosinophils are observed in several liver diseases, but their contribution in the pathogenesis of these disorders remains poorly investigated. Concanavalin A (Con A)-induced hepatitis is an experimental model of immune-mediated liver injury in which natural killer T (NKT) cells play a critical role through the production of interleukin (IL)-4 and the expression of Fas ligand (FasL). Because activated NKT cells also produce IL-5, a critical cytokine for eosinophil maturation and function, the role of IL-5 was investigated in this model. METHODS: IL-5-deficient mice, eosinophil depletion in wild-type (WT) mice, and NKT cell transfer from WT- or IL-5-deficient mice into NKT cell-deficient mice were used to assess the role of IL-5 and eosinophils. RESULTS: Liver eosinophil infiltrate and IL-5 production were observed after Con A challenge. Liver injury was dramatically reduced in IL-5-deficient or eosinophil-depleted mice. In addition, residual hepatitis observed in Fas-deficient mice was abolished after IL-5 neutralization. Finally, we showed that NKT cells constituted a critical source of IL-5. Indeed, transfer of WT NKT cells to mice lacking NKT cells restored liver injury, whereas transfer of IL-5-deficient NKT cells did not. CONCLUSIONS: These observations highlight the pathologic role of IL-5 and eosinophils in experimental immune-mediated hepatitis.