Showing papers in "Developmental and Comparative Immunology in 2002"

Identification and analysis of an interleukin 8-like molecule in rainbow trout Oncorhynchus mykiss

Abstract: An interleukin 8 (IL-8) homologue has been identified in the rainbow trout Oncorhynchus mykiss. The transcript contains an open reading frame of 294 nucleotides that translates into a 97 amino acid putative peptide, with 5' and 3' untranslated regions (UTR) of 171 and 453 nucleotides, respectively. As with previously sequenced lamprey and flounder genes, the trout amino acid sequence lacks the typical ELR motif upstream of the first pair of cysteines, where DLR is present. The trout IL-8 gene contains four exons divided by three short introns of 341, 247 and 292bp, and occupies 1824bp of genomic DNA. RT-PCR reveals a low level constitutive expression of the IL-8 homologue in many tissues, including spleen, heart, liver, head kidney and gill. Expression was not detectable in the brain. Whilst no apparent affect of lipopolysaccharide (LPS) on IL-8 expression was observed in vivo, stimulation of a trout macrophage cell line (RTS-11) with either LPS or poly I:C did result in clear up-regulation of IL-8 expression, detectable by RT-PCR and Northern blot analysis.

Activity of antimicrobial skin peptides from ranid frogs against Batrachochytrium dendrobatidis, the chytrid fungus associated with global amphibian declines

Abstract: Accumulating evidence suggests that a chytrid fungus, Batrachochytrium dendrobatidis, is responsible for recent declines in amphibian populations in Australia, Central America, Europe, and North America. Because the chytrid infects the keratinized epithelium of the skin, we investigated the possible role of antimicrobial peptides produced in the skin as inhibitors of infection and growth. We show here that 10 peptides representing eight families of peptides derived from North American ranid frogs can effectively inhibit growth of this chytrid. The peptides are members of the ranatuerin-1, ranatuerin-2, esculentin-1, esculentin-2, brevinin-2, temporin, palustrin-3, and ranalexin families. All the tested peptides inhibit growth of mature fungal cells at concentrations above 25 microM, and some of them inhibit at concentrations as low as 2 microM. A comparison of the sensitivity of infectious zoospores with that of mature cells showed that the zoospores are inhibited at significantly lower concentrations of peptides. To determine whether cold temperature interferes with the inhibitory effects of these peptides, we tested their effectiveness at both 22 and 10 degrees C. Although the peptides inhibit at both temperatures, they appear to be more effective against zoospores at the lower temperature. These results suggest that the ranid frogs have, within their repertoire of antimicrobial substances, a number of skin peptides that should be a deterrent to chytrid infection. This may provide some natural resistance to infection, but if environmental factors inhibit the synthesis and release of the skin peptides, the pathogen could gain the advantage.

Antimicrobial peptide defenses against pathogens associated with global amphibian declines.

Abstract: Global declines of amphibian populations are a source of great concern. Several pathogens that can infect the skin have been implicated in the declines. The pathogen most frequently associated with recent die-offs is a chytrid fungus, Batrachochytrium dendrobatidis. A second fungus, Basidiobolus ranarum, was isolated from declining populations of Wyoming toads. A third pathogen, Aeromonas hydrophila, is an opportunistic bacterium found in healthy frogs, but capable of inducing disease. Among the immune defense mechanisms used by amphibians is the production of antimicrobial peptides in granular glands in the skin. These packets of natural antibiotics can be emptied onto the skin when the amphibian is injured. To determine whether antimicrobial skin peptides defend against these amphibian pathogens, six peptides (magainin I, magainin II, PGLa, CPF, ranalexin, and dermaseptin), from three species, and representing three structurally different families of peptides, were tested in growth inhibition assays. We show here that the peptides can kill or inhibit growth of both fungi but not Aeromonas. Although each peptide varied in its effectiveness, at least one from each species was effective against both fungi at a concentration of about 10-20 microM. This is the first direct evidence that antimicrobial peptides in the skin can operate as a first line of defense against the organisms associated with global amphibian declines. It suggests that this innate defense mechanism may play a role in preventing or limiting infection by these organisms.

Stress-induced immune changes in the oyster Crassostrea gigas

Abstract: Information concerning the effect of stress on invertebrate immune functions are scarce. The present study investigated the consequences of a 15-min mechanical disturbance on immune parameters in oysters Crassostrea gigas. As indicated by noradrenaline and dopamine measurements, the mechanical disturbance caused a transient state of stress in oysters. The number of circulating hemocytes, the migratory and phagocytic activities and reactive oxygen species production of hemocytes were measured before, during and after application of the stressor. Results show that all immune functions were significantly downregulated during stress and a transient period of immunostimulation was observed 30-240 min after the end of the disturbance. Taken together, these results suggest that stress can exert a profound influence on oyster immune functions and they may explain why stress and the outbreak of disease are often linked in shellfish culture. Furthermore, the present study strongly suggests that checking the stress status of animals may be necessary to avoid biases when studying oyster immune responses in vivo.

Differential expression of two tumor necrosis factor genes in rainbow trout, Oncorhynchus mykiss

Abstract: A second TNF gene (TNF2) has been cloned and sequenced in rainbow trout. In common with the first TNF gene isolated (TNF1), this gene is more TNF alpha-like than TNF beta-like. The full length cDNA is 1519bp, containing a 765bp open reading frame. The gene has four exons, of 380, 49, 60 and 1030bp, respectively. Analysis of the 5' flanking regions of TNF1 and TNF2 reveals several interesting differences in identified transcriptional regulatory elements, with a CATAAA box present 26bp upstream of the transcription start in both genes. Expression analysis in LPS stimulated macrophages has shown a much stronger expression of TNF2 relative to TNF1, with expression being detected earlier and lasting longer.

Channel catfish cytotoxic cells: a mini-review.

Abstract: The use of allogeneic and autologous lymphoid cell lines has facilitated studies of cytotoxic T lymphocytes (CTL) and natural killer (NK)-like cells in channel catfish. Naive catfish leukocytes were shown to spontaneously kill allogeneic cells and virally-infected autologous cells without the need for prior sensitization, and allogeneic cytotoxic responses were greatly enhanced by in vitro alloantigen stimulation. Both catfish CTL and NK-like cells have been successfully cloned from these alloantigen-stimulated cultures, and represent the first cytotoxic cell lines derived from any ectothermic vertebrate. These cloned cytotoxic cells contain granules and likely induce apoptosis in sensitive targets via a putative perforin/granzyme mechanism. In addition, some catfish CTL clones may also kill targets by an additional mechanism, possibly by Fas/FasL-like interactions. Importantly, these cytotoxic cells do not express the marker for catfish nonspecific cytotoxic cells (NCCs), and thus represent cell types distinct from NCCs. The use of monoclonal antibodies against the catfish F and G immunoglobulin light chain isotypes revealed the presence of a putative Fc receptor for IgM (Fc mu R) on some catfish NK-like cells that appears to 'arm' these cells with surface IgM. In addition, a potentially important monoclonal antibody (CC41) developed against catfish NK-like cells was found to recognize an approximately 150kDa molecule on the surface of catfish cytotoxic cells. These studies clearly demonstrate that catfish possess an array of different cytotoxic cells. The availability of various cloned cytotoxic cell lines should enable unambiguous functional studies to be performed in ways not currently possible with any other fish species.

Cytotoxic T cell function in fish.

Abstract: Fish possess immunoglobulins, major histocompatibility complex (MHC), T-cell receptors, and lymphocyte populations analogous to B and T cells and can evoke specific immune responses against a variety of antigens. However, T-cell subsets have yet to be demonstrated and the information on cell-mediated immunity is limited. Here we briefly review our recent studies on specific cell-mediated immunity, particularly on cytotoxic T-cell function employing isogeneic fish and cell lines. Analyses of the graft-versus host reaction (GVHR) and cell-mediated cytotoxicity (CMC) against allogeneic erythrocytes or cell lines show alloantigen-specific cytotoxicity in clonal ginbuna crucian carp. We also describe specific cytotoxicity against virus-infected cells using clonal ginbuna and a syngeneic cell line. Lastly, we report MHC-restriction in CMC against virus-infected cells using homozygous clonal rainbow trout and trout cell line which share the same MHC class I allele. These studies on CMC strongly suggest the presence of antigen specific cytotoxic T cells in teleosts and functional similarities between the immune systems of fish and higher vertebrates. Experimental model systems established in these studies can be applied to the investigation of protective antigens to induce cell-mediated immunity for the development of fish vaccines.

Immunity induced shortly after DNA vaccination of rainbow trout against rhabdoviruses protects against heterologous virus but not against bacterial pathogens

Abstract: It was recently reported that DNA vaccination of rainbow trout fingerlings against viral hemorrhagic septicaemia virus (VHSV) induced protection within 8 days after intramuscular injection of plasmid DNA. In order to analyse the specificity of this early immunity, fish were vaccinated with plasmid DNA encoding the VHSV or the infectious haematopoietic necrosis virus (IHNV) glycoprotein genes and later challenged with homologous or heterologous pathogens. Challenge experiments revealed that immunity established shortly after vaccination was cross-protective between the two viral pathogens whereas no increased survival was found upon challenge with bacterial pathogens. Within two months after vaccination, the cross-protection disappeared while the specific immunity to homologous virus remained high. The early immunity induced by the DNA vaccines thus appeared to involve short-lived non-specific anti-viral defence mechanisms.

Dynamics of immune cell infiltration in the caecal lamina propria of chickens after neonatal infection with a Salmonella enteritidis strain.

Abstract: Dynamics of leucocyte infiltration and bacterial invasion in the caecal wall were studied after oral infection of 2-day-old chicks with Salmonella enterica ser. Enteritidis. Bacteria invaded the lamina propria of the caecal wall from 12 h post-challenge onwards. Bacteriological examination of internal organs (liver, spleen) showed a peak in Salmonella bacteria at 3 days post-infection, after which the number of bacteria decreased. Immunohistochemistry revealed macrophages and T-lymphocytes invading the caecal propria mucosae from 24 h after challenge onwards, while B-lymphocytes came somewhat later, subsequently organising into follicular aggregates. An early increase in granulocytes was partly masked by the response to natural flora. While the B-lymphocyte and granulocyte populations were maintained, T-lymphocyte and macrophage populations were already reducing by 10 days post-challenge. The infiltration of macrophages and T-lymphocytes in the caecal wall, followed by B-lymphocytes, is the result of an inflammatory response, caused by invading bacteria at this site. The structural maturation of the mucosa-associated lymphoid tissues is antigen driven, since B-cells organise in a follicular pattern.

Evolution of the acute phase response: iron release by echinoderm (Asterias forbesi) coelomocytes, and cloning of an echinoderm ferritin molecule.

Abstract: That the plasma concentration of certain divalent cations change during an inflammatory insult provides a major host defense response in vertebrate animals This study was designed to investigate the involvement of iron sequestration in invertebrate immune responses A ferritin molecule was cloned from an echinoderm coelomocyte cDNA library The amino acid sequence showed sequence homology with vertebrate ferritin The cDNA contained a conserved iron responsive element sequence Studies showed that stimulated coelomocytes released iron into in vitro culture supernatants The amount of iron in the supernatants decreased over time when the amebocytes were stimulated with LPS or PMA Coelomocytes increased expression of ferritin mRNA after stimulation In vertebrates, cytokines can cause changes in iron levels in macrophages Similarly, echinoderm macrokines produced decreases in iron levels in coelomocyte supernatant fluids These results suggest that echinoderm ferritin is an acute phase protein and suggest that sequestration of iron is an ancient host defense response in animals

A β-1,3-glucan binding protein from the black tiger shrimp, Penaeus monodon

Abstract: A β-1,3-glucan binding protein (GBP) has been isolated from a shrimp hemocyte cDNA library. Its open reading frame consists of 1314 nucleotides with a polyadenylated sequence and a poly A tail. It encodes a polypeptide of 370 amino acids including a 17 amino acid-signal peptide. The mature protein has an estimated molecular mass of 39.5 kDa and a predicted pI of 5.5. Sequence comparison shows a high degree of similarity to invertebrate recognition proteins with glucanase-like domains for example, the lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) from the freshwater crayfish, Pacifastacus leniusculus , coelomic cytolytic factor-1 from the earthworm, Eisenia foetida and the Gram negative bacteria binding protein from the mosquito, Anopheles gambiae as well as to sea urchin β-1,3-glucanases and bacterial β-1,3-glucanases or β-1,3-, 1,4-glucanases. Northern blot analysis showed that the shrimp protein is constitutively expressed in hemocytes. Animals injected with curdlan or heat-killed bacterial cell of Vibrio harveyi , a shrimp pathogen, showed no significant change in the mRNA expression profile within 12 h post-injection. After incubation of shrimp hemocyte lysate supernatant (HLS) with curdlan or zymosan, a protein with a molecular mass of 31 kDa was eluted from the incubated curdlan or zymosan, and, by immunoblotting, this 31-kDa band could be detected by an affinity-purified anti-crayfish LGBP antibody. In contrast, incubation of shrimp HLS with LPS showed no any reactive band detected on SDS–PAGE or by immunoblotting suggesting that the binding is specific for β-1,3-glucan.

Affinity maturation in trout: clonal dominance of high affinity antibodies late in the immune response

Abstract: The ability of an animal to develop a highly specific antibody response through affinity maturation has been considered an integral part of the adaptive immune response. However, much of the literature dealing with teleostean antibody responses suggests that little or no affinity maturation may occur within these taxa. As trout antibodies are similar to multimeric mammalian IgM, it has been reasoned that affinity maturational shifts in intrinsic affinity might be similarly small. Such small increases in affinity can, however, lead to potentially great avidity changes for multimeric antibodies. We therefore employed a partition-based immunoassay that permits the dissection of a single antiserum into discrete, affinity-based antibody subpopulations. Such partitioning assays provide for enhanced sensitivity and resolution of these affinity subpopulations over that which can be obtained by fluorescence quenching or equilibrium dialysis. Through the use of the partition-based immunoassay, we were able to detect a consistent increase in affinity within trout anti-TNP antisera. Furthermore, it was determined that trout are capable of generating new, higher affinity antibodies relatively late in the antibody response, which then come into dominance. Such evidence suggests that either somatic mutation does occur or that a unique form of affinity-based regulation of antibody expression is employed.

Signaling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 MAP kinases.

Abstract: In this work the mechanisms of transduction triggered in Mytilus galloprovincialis hemocytes by bacterial challenge were investigated in an in vitro model of infection of hemocyte monolayers with Escherichia coli. Western blot analyses of hemocyte extracts with phospho-specific anti-MAPK (Mitogen Activated Protein Kinase) antibodies indicate that E. coli induced a time dependent activation of different classes of MAPKs, mainly of the stress-activated p38 MAPK. P38 activation was confirmed by the use of the selective kinase inhibitor SB203580. Moreover, hemocyte pretreatment with SB203580 significantly reduced bacterial killing, whereas PD98059, an inhibitor of extracellularly regulated kinase (ERK) activation, was ineffective. Interestingly, the PI3-kinase (phosphatidylinositol-3-OH-kinase) inhibitor, Wortmannin, reduced both p38 activation and bacterial killing, indicating a critical role also for this lipid kinase in the hemocyte immune response.

Antibody–antigen kinetics following immunization of rainbow trout (Oncorhynchus mykiss) with a T-cell dependent antigen

Abstract: Enhancement of the immune response through affinity maturation of the antibody response is a feature of the mammalian immune system and has important implications with respect to development of vaccination strategies. However, an absence of germinal centres and apparent lack of somatic hypermutation of immunoglobulin V genes suggests that this phenomenon does not occur in fish. We investigated the question of affinity maturation in rainbow trout (Oncorhynchus mykiss) by measuring antibody-antigen binding kinetics using a BIAcore biosensor. Following immunization with a T-cell dependent antigen (FITC-KLH), relative binding affinities of serum and mucosal antibodies were assessed based on their dissociation rate constants (k(diss.)). A detectable serum anti-FITC response developed by 4 weeks post-immunization, and a consistent shift to higher affinity antibody production (i.e. a decrease in k(diss.)) was observed over the ensuing course of the immune response. An average k(diss.) of 3.5 x 10(-4)+/-0.27 x 10(-4)sec(-1) was observed during early stages of the response (4 weeks), while by 6 weeks this decreased significantly (p<0.05). Further reduction in k(diss.) was observed, with a low of 1.2 x 10(-4)+/-0.06 x 10(-4)sec(-1) being observed by week 12. Analysis of the anti-FITC response in skin-derived mucus revealed a similar pattern of decreasing k(diss.) as the immune response progressed. While these data clearly demonstrate a 2-3 fold increase in antibody-antigen binding during the course of the immune response in trout, the magnitude of this increase is much less than that seen in the mammalian immune response. This may reflect differences in the mechanisms underpinning this phenomenon in divergent species.

Comparative study on characteristics of lysozymes from the hemolymph of three lepidopteran larvae, Galleria mellonella, Bombyx mori, Agrius convolvuli.

Abstract: Lysozymes were purified from the hemolymph of three immunized Lepidopteran larvae, Galleria mellonella, Bombyx mori, Agrius convolvuli to compare their physico-chemical properties and antibacterial activities with those of chicken lysozyme. Four lysozymes including the one from chicken had similar molecular masses and chromatographic behavior on reverse phase-high pressure liquid chromatography. Western blotting analysis using an antibody raised against G. mellonella revealed that lysozyme cross-reacted with two other insect lysozymes but not with commercial chicken lysozyme. Antibacterial activities of lysozymes were measured in two types of tests: radial diffusion assay and colony count assay. Our antibacterial tests revealed that all lysozymes have strong activities against Gram-positive bacteria and three insect lysozymes still retain a little potency against Gram-negative bacteria, while chicken lysozyme has no activity against Gram-negative bacteria. Taken together, we conclude three Lepidopteran lysozymes have a common distinct structure and have an antibacterial activity, which is absent in chicken lysozyme, against Gram-negative bacteria.

Effects of ovotransferrin on chicken macrophages and heterophil-granulocytes.

Abstract: Ovotransferrin (OTF) is an acute phase protein in chickens, serum levels of which increase in inflammation and infections. To understand the significance of OTF in inflammation, we studied its in vitro effects on HD11 cells, a macrophage cell line, and heterophils isolated from blood using a panel of variables indicative of cellular activation. These included the production of interleukin-6 (IL-6), nitrite, matrix metalloproteinase (MMP), oxidation of dichlorofluorescein diacetate for respiratory burst and the degranulation of heterophils by the loss of fluorescein isothiocyanate positive cytoplasmic granules. The results show that ovotransferrin stimulates the production of IL-6, nitrite and MMP by HD11 cells and augments phorbol ester-induced respiratory burst. Ovotransferrin stimulated heterophils to produce IL-6, and MMP, but failed to produce nitrite, enhanced respiratory burst activity and degranulation. These results suggest that ovotransferrin can modulate macrophage and heterophil functions in chickens.

Cloning of a Salmo salar interleukin-1 receptor-like cDNA.

Abstract: The interleukin-1 receptor/toll-like receptor (IL-1R/TLR) superfamily, defined by a cytosolic Toll/IL-1R (TIR) signalling domain, participates in host responses to injury and infection. We describe in this study the cloning of a cDNA encoding a Salmo salar interleukin-1 receptor-like protein (SalIL-1RLP). SalIL-1RLP comprises a potential signal peptide, three extracellular immunoglobulin domains, a short transmembrane region and an intracellular region that contains the TIR domain. The predicted amino acid sequence of SalIL-1RLP displays 43-44% similarities and 31% identities to chicken and human IL-1RI sequences. Within the intracellular region, SalIL-1RLP displays highest similarity (59%) and identity (46%) to the chicken IL-1RI sequence. Two different 5' distal UTRs were identified among six salmon IL-1RLP clones. The six clones, however, displayed identical 5' proximal UTRs, coding regions and 3' UTRs. SalIL-1RLP expression is induced in liver, head kidney, spleen and gills upon injection of salmon with bacterial lipopolysaccharide. Sequence comparisons, protein domain structures, expression patterns and phylogenetic analyses indicate that SalIL-1RLP is most closely related to type I interleukin-1 receptors and interleukin-1 receptor related proteins.

Immunohistochemical localization of rhamnose-binding lectins in the steelhead trout (Oncorhynchus mykiss).

Abstract: The localization of three l -rhamnose-binding lectins named STL1, STL2, and STL3 from eggs of steelhead trout ( Oncorhynchus mykiss ) was analyzed by indirect immunohistochemical staining with specific antisera against individual lectins. In early previtellogenic oocyte, STL1 was localized not only in the cortical vesicles, but also in the plasma membrane and germinal vesicle. On the other hand, STL2 and STL3 were localized only in the cortical vesicles. In pre-fertilization mature egg, STLs were localized in a thin layer of cortical granules at the cytoplasmic side of the plasma membrane. STLs were accumulated on the surface of cytoplasm and inner membrane 30 min after fertilization. The strong staining with anti-STL1 antiserum was observed in several tissues and cells of the steelhead trout, such as spleen, thrombocytes, and blood leukocytes, but not erythrocytes. STL1 was also identified in exocrine cells, such as goblet cells of intestine and mucous cells of gill. These results indicate that the multiple lectins found in eggs of the steelhead trout play physiological roles not only in eggs, but also in various cells related to the innate immunity.

The immune system of sea bass, Dicentrarchus labrax, reared in aquaculture

Abstract: The sea bass Dicentrarchus labrax is one the most important seawater fish species of south Europe and Mediterranean aquaculture, and studies on its immune system are important for both scientific and applied purposes. In this paper, we summarise the results obtained in studies of the immune system in this species, and present original data on cell-mediated acquired immune response.

Bacteriolytic activity of rainbow trout (Oncorhynchus mykiss) complement

Abstract: The total bacteriolytic activity comprising of the classical, alternative and possible lectine pathways as well as the bacteriolytic activity of the alternative pathway (AP) of rainbow trout (Oncorhynchus mykiss) complement was assessed in temperatures ranging from 0 to 35 °C against a recombinant strain Escherichia coli containing two reporter genes gfp and lucFF. At 35 °C there was no difference between the total (TC) activity and the activity of the AP, but at 10 °C the TC was notably higher than the AP. Total activity peaked at 30 °C and gradually grew smaller towards 0 °C. The activity of the AP was similarly temperature-dependent, but CB50 value was found to be beyond measurable range at temperatures below 10 °C. When compared to human serum complement, the peak human TC activity at 37 °C was four times higher than the TC of rainbow trout at 30 °C. Human TC activity was 10.1-fold lower at 25 °C when compared to the activity at 37 °C. At 37 °C the human AP bacteriolytic activity was 4.5-fold less effective than human TC, but at 25 °C there was no difference between human TC and AP. In contrast to previous reports where AP activity of fish was assayed as hemolytic activity our study showed that the bacteriolytic activity of AP was lower than that of TC and very low at temperatures below 10 °C suggesting that the earlier proposed particular importance of AP in fish should be reconsidered.

Calreticulin enriched as an early-stage encapsulation protein in wax moth Galleria mellonella larvae.

Abstract: To investigate the molecular mechanism of the early-stage encapsulation reaction in insects, we purified a 47kDa protein from injected beads into Galleria mellonella larvae. When a cDNA clone was isolated, the 47kDa protein showed high homology with Drosophila and human calreticulin. Western blotting analysis showed that the 47kDa protein was present in the hemocytes, but not in the plasma. When the early-stage encapsulated beads were coated with 47kDa protein antibody and reinjected into G. mellonella larvae, any further encapsulation reaction was inhibited. These results suggest that calreticulin is involved in non-self recognition in invertebrate cellular defense reactions.

Characterization and partial purification of a lectin from the hemolymph of the white shrimp Litopenaeus schmitti.

Abstract: The agglutinating activity of the hemolymph of Litopenaeus schmitti is insensitive to calcium and specific for acetylated sugars, particularly sialic acid (Neu5Ac) and O -sialoglycoconjugates (bovine submaxillary mucin) and has varying specificity for different LPS, which may suggest a putative role in microorganism recognition. Affinity chromatography on fetuin–agarose of the agglutinin resulted in a 220 kDa band (lectin), and a 82.5 kDa band, which probably is hemocyanin. The 220 kDa protein consists of 31 and 34 kDa subunits, suggesting that this lectin is multimeric. The lectin molecular mass was estimated by gel filtration to be 153±10 kDa. The hemolymph of L. schmitti comprises at least another soluble lectin, with distinct chemical and carbohydrate specificity than the 220 kDa lectin.

Differential effects of age on chicken heterophil functional activation by recombinant chicken interleukin-2.

Abstract: Interleukin-2 (IL-2) exercises an array of biological effects on many cells including the functional activation of cells of the innate immune response. Heterophils, the avian equivalent of the neutrophil, function as professional phagocytes to aid in regulation of innate host defenses. The objective of the present studies was to examine the effects of recombinant chicken IL-2 (rChIL-2) on functional activities of heterophils from chickens during the first 3 weeks after hatch. Peripheral blood heterophils were isolated and incubated with either COS cell-derived rChIL-2 or supernatants from mock-transfected COS cells. rChIL-2 had no effect on the functional activities of heterophils from day-of-hatch chickens, but significantly increased the phagocytosis and bactericidal activity of heterophils from 7- and 14-day-old chickens. rChIL-2 induced no direct stimulation of the respiratory burst by heterophils, but primed heterophils from 7- and 14-day-old birds for an enhanced respiratory burst in response to phorbol ester stimulation. Lastly, rChIL-2 had neither direct nor priming effects on heterophil degranulation. The enhancing effects on heterophil functional activity by rChIL-2 were abated by a neutralizing anti-chicken IL-2 mAb and were therefore specific for this cytokine. These results show that rChIL-2 can directly activate chicken heterophils to exert effector functions, and that heterophil activation by rChIL-2 is also an age-dependent event.

The effect of aging on T cell responses in the horse.

Abstract: Horses greater than 20 years of age exhibit alterations in their immune responses similar to those observed in other aged individuals The purpose of this study was to characterize immunosenescence in a population of aged ponies The peripheral blood mononuclear cells (PBMC) from aged ponies exhibited a decreased proliferative response to various mitogens that was not overcome by the addition of interleukin 2 (IL-2) to the cultures No difference in overall expression of the IL-2 receptor was seen between young and aged ponies, though CD8(+) cells from aged ponies exhibited increased levels of IL-2 receptor expression The kinetics of the response to both mitogen and IL-2 did not appear to be affected in the aged PBMCs These results indicate that the age-related decrease in the proliferative response to mitogens is not due to a failure to produce or respond to IL-2 but probably involves some other process

Activation of nonspecific cytotoxic cells (NCC) with synthetic oligodeoxynucleotides and bacterial genomic DNA: binding, specificity and identification of unique immunostimulatory motifs.

Abstract: We have analyzed the effects of synthetic oligodeoxynucleotides (sODNs) and bacterial DNA (bDNA) on the in vitro activation of NCC. Teleost NCC recognition of DNA appeared to differ from that which occurs in higher vertebrates. NCC contain at least two different receptor specificities for DNA. Both oligodeoxyguanosine 20-mers (dG20) and 5'-TGCTGCTTGTGCTTGTGCTT-3' (4GC-2T) bound specifically to NCC. The existence of different receptor specificities was indicated by reciprocal cold target inhibition experiments. dG20 competed with 4GC-2T binding but sODNs composed of GpC or CpG nests did not compete with recognition by NCC of the dG20. ODN binding by NCC primarily depended on the presence of GpC or CpG nests with a preference for -G- serving as the anchor nucleotide. Secondarily, and similar to models of ODN activation in mammals, palindrome sequences of pu-pu-CpG-py-py activated NCC cytotoxicity. Additional analysis of the requirements for ODN activation indicated that guanosine could not substitute for adenosine as a purine spacer and that CpG motifs containing flanking thymidine (i.e.-GTCpGTT-) augmented the activity of the sODN containing this flanking base. Other evidence for the participation of both G and C in the recognition of specific nucleotides by NCC was that poly-dC20, dA20 or dT20 had no activating properties. Methylation of all cytosine nucleotides within an ODN abrogated activation. A canonical ODN motif of 5'-C/AT/AGCTT-3' can now be suggested for teleosts. Additional studies were done to examine the effects of in vitro treatment of NCC with bDNA. bDNA from three different disease isolates of Streptococcus iniae activated NCC cytotoxicity. Treatment of the bDNA with DNase abrogated the enhancement of cytotoxicity. Also, treatment of NCC with eukaryotic DNA had no effects on cytotoxicity. These studies suggested that NCC recognize bacterial nonmethylated DNA. The consequences of these interactions may be increased innate and acquired anti-bacterial immunity.

Diversity of complement factor B/C2 in the common carp (Cyprinus carpio): three isotypes of B/C2-A expressed in different tissues.

Abstract: Complement factor B and C2 are two critical proteases for complement activation. Some bony fish have been reported to possess duplicated genes for B/C2, but there is no direct evidence regarding possible functional divergence. Here, we report the isolation of the second and third isotypes of carp B/C2-A, a close relative of other bony fish B reported to date, and designated B/C2-A2 and B/C2-A3. B/C2-A1 (previously reported B/C2-A) and B/C2-A2 share 78% amino acid identity and are synthesized mainly in hepatopancreas. On the other hand, B/C2-A3 showed less (∼60%) sequence identity with the other two isotypes. It was expressed mainly in kidney and spleen, and was up-regulated after injection of carp with scleroglucan or sodium alginate, known immunostimulants for fish. Phylogenetic analysis suggests that B/C2-A3 diverged before separation of carp and zebrafish. B/C2-A3 represent a novel B/C2-lineage functioning as an acute phase reactant in cyprinid fish.

The mysterious immunoglobulin light chain.

Abstract: The immunoglobulin light chain is mysterious from different points of view. In an evolutionary perspective there seems to be at least three major pathways but it is today impossible to say which of them is the most ancient one and which isotype belongs to which branch. It is also difficult to assess the contribution of the light chain to the binding capacity of the antibodies. It is possible that it differs from antibody to antibody but the fact that there are antibodies in camels and sharks without any light chain suggests that it is perhaps not necessary for good antigen binding.

Isolation and characterization of channel catfish natural resistance associated macrophage protein gene.

Abstract: Natural resistance associated macrophage protein 1 (Nramp1) affects the ability of macrophages to kill pathogens. We cloned Nramp cDNA of channel catfish to identify potential molecular markers for disease resistance. Three different Nramp transcripts were identified: NrampCa-2912 nucleotides (nt), NrampCb-3245 nt, and NrampCc-3721 nt. At the 5′ end, the transcripts have a common 2263 nt sequence containing the open reading frame. The differences are in the 3′ untranslated region resulting from alternative splicing and polyadenylation. NrampCc is the predominant form expressed. The deduced 550 amino acid sequence of the channel catfish Nramp (NrampC) has high homology to Nramp from other vertebrates and a predicted conserved structure. The NrampC contains the 12 transmembrane domains, and the consensus transport motif. Post-transcriptional processing is also conserved. Phylogenetic analysis grouped NrampC with other fish Nramps and closer to Nramp2 than to Nramp1 of mammals. However, the catfish transcript does not contain an iron-responsive regulatory-protein binding site, a characteristic of Nramp2, and, like Nramp1, NrampC expression is induced in macrophage-rich tissues after exposure to lipopolysaccharide and in a macrophage cell line when stimulated. Thus NrampC is structurally closer to mammalian Nramp2 but may function similar to Nramp1.

Characterisation of echidna IgM provides insights into the time of divergence of extant mammals.

Abstract: The immunobiology of monotremes is poorly understood. In this paper, we describe the characterisation of the heavy chain of IgM from Tachyglossus aculeatus, the short-beaked echidna. The echidna heavy chain constant region of IgM (Cμ) was isolated from a spleen cDNA library using a Trichosurus vulpecula probe. It has approximately 46.5% amino acid identity to marsupial and eutherian Cμs, and approximately 30% amino acid identity with Cμs from birds and reptiles. Phylogenetic analysis of mammalian Cμ provides strong support for the Theria hypothesis, with a sister grouping of the eutherians and marsupials to the exclusion of the monotremes. Cμ sequences suggest that monotremes and therians separated approximately 170 million years ago (mya), marsupials and eutherians separated approximately 130 mya, and Australian and American marsupials separated approximately 65 mya.

Trypanosoma cruzi is lysed by coelomic cytolytic factor-1, an invertebrate analogue of tumor necrosis factor, and induces phenoloxidase activity in the coelomic fluid of Eisenia foetida foetida.

Abstract: A cytolytic protein named Coelomic Cytolytic Factor-1 (CCF-1) was isolated from the coelomic fluid of the earthworm Eisenia foetida foetida. Despite the absence of any gene homology, CCF-1 showed functional analogy with the mammalian cytokine tumour necrosis factor (TNF), particularly based on similar lectin-like activity. Indeed, both CCF-1 and TNF recognise N,N′-diacetylchitobiose and exert lytic activity on African Trypanosoma brucei brucei. In this report, we show that South-American Trypanosoma cruzi trypomastigotes, but not epimastigotes, were lysed by earthworm coelomic fluid or purified CCF-1. However, T. cruzi was less susceptible to lysis than T. brucei brucei. This lytic effect of coelomic fluid and CCF-1 on T. cruzi trypomastigotes was partially inhibited in the presence of anti-CCF-1 monoclonal antibody, antibody neutralising the lectin-like activity of TNF or N,N′-diacetylchitobiose. In contrast, this lytic effect was completely inhibited when using T. b. brucei. In addition, T. cruzi components, upon recognition by CCF-1 in E. f. foetida coelomic fluid, triggered the prophenoloxidase cascade, an invertebrate defence mechanism. These results further extend the functional analogies of CCF-1 and TNF, suggesting that both molecules share a similar lectin-like activity that has been conserved as an innate recognition mechanism in invertebrates and vertebrates. They also establish a link between stercorarian (T. cruzi) and salivarian (T. brucei) trypanosomatids having divergent phylogenetic origins and patterns of evolution, but possessing closely related cell surface sugar moieties.