Interspecific exchange of avian influenza virus genes in Alaska: the influence of trans-hemispheric migratory tendency and breeding ground sympatry

The movement and transmission of avian influenza viral strains via wild migratory birds may vary by host species as a result of migratory tendency and sympatry with other infected individuals. To examine the roles of host migratory tendency and species sympatry on the movement of Eurasian low-pathogenic avian influenza (LPAI) genes into North America, we characterized migratory patterns and LPAI viral genomic variation in mallards (Anas platyrhynchos) of Alaska in comparison with LPAI diversity of northern pintails (Anas acuta). A 50-year band-recovery data set suggests that unlike northern pintails, mallards rarely make trans-hemispheric migrations between Alaska and Eurasia. Concordantly, fewer (14.5%) of 62 LPAI isolates from mallards contained Eurasian gene segments compared to those from 97 northern pintails (35%), a species with greater inter-continental migratory tendency. Aerial survey and banding data suggest that mallards and northern pintails are largely sympatric throughout Alaska during the breeding season, promoting opportunities for interspecific transmission. Comparisons of full-genome isolates confirmed near-complete genetic homology (>99.5%) of seven viruses between mallards and northern pintails. This study found viral segments of Eurasian lineage at a higher frequency in mallards than previous studies, suggesting transmission from other avian species migrating inter-hemispherically or the common occurrence of endemic Alaskan viruses containing segments of Eurasian origin. We conclude that mallards are unlikely to transfer Asian-origin viruses directly to North America via Alaska but that they are likely infected with Asian-origin viruses via interspecific transfer from species with regular migrations to the Eastern Hemisphere.     

Influence of body condition on influenza A virus infection in mallard ducks: experimental infection data

Migrating waterfowl are implicated in the global spread of influenza A viruses (IAVs), and mallards (Anas platyrhynchos) are considered a particularly important IAV reservoir. Prevalence of IAV infection in waterfowl peaks during autumn pre-migration staging and then declines as birds reach wintering areas. Migration is energetically costly and birds often experience declines in body condition that may suppress immune function. We assessed how body condition affects susceptibility to infection, viral shedding and antibody production in wild-caught and captive-bred juvenile mallards challenged with low pathogenic avian influenza virus (LPAIV) H5N9. Wild mallards (n = 30) were separated into three experimental groups; each manipulated through food availability to a different condition level (−20%, −10%, and normal ±5% original body condition), and captive-bred mallards (n = 10) were maintained at normal condition. We found that wild mallards in normal condition were more susceptible to LPAIV infection, shed higher peak viral loads and shed viral RNA more frequently compared to birds in poor condition. Antibody production did not differ according to condition. We found that wild mallards did not differ from captive-bred mallards in viral intensity and duration of infection, but they did exhibit lower antibody titers and greater variation in viral load. Our findings suggest that reduced body condition negatively influences waterfowl host competence to LPAIV infection. This observation is contradictory to the recently proposed condition-dependent hypothesis, according to which birds in reduced condition would be more susceptible to IAV infection. The mechanisms responsible for reducing host competency among birds in poor condition remain unknown. Our research indicates body condition may influence the maintenance and spread of LPAIV by migrating waterfowl.     

NO modulation of myocardial performance in fish hearts

In the mammalian heart, intracardiac nitric oxide (NO) regulates in an autocrine-paracrine manner cardiac function in the beat-to-beat response (Starling's law of the heart), short-term response (phasic control, e.g. excitation-contraction coupling, responses to neurotransmitters and endocrines) and long-term response (tonic control by altering gene expression). This trio of NO temporal-dependent actions has a long evolutionary history, as we have documented in the prototypic vertebrate heart, the teleost heart. This heart shares a common structural and functional scenario with higher vertebrate hearts exhibiting, at the same time, differences in myoarchitecture (trabecular vs. compact type), blood supply (lacunary vs. vascular) and pumping performance (sensitivity to filling pressure), thus providing challenging opportunities for revealing aspects of unity and diversity of cardiac NO in vertebrates. Using in vitro working teleost heart preparations we have shown that, under basal conditions, NO through a cGMP-mediated mechanism modulates ventricular performance (negative inotropism) and remarkably increases the sensitivity to filling pressure (i.e. the Frank-Starling response). NO-cGMP mechanism also influences the short-term response elicited by inotropic agents such as acetylcholine and angiotensin II. A role of NO in long-term cardiac adaptation is illustrated by morphologic evidence (e.g. NOS immuno-localization in phylogenetically distant species) which emphasizes the importance of NO in reshaping the angio-myoarchitecture of the fish heart ventricle (i.e. compensation for regional heterogeneity). Finally, by studying the avascular hearts of teleosts and amphibians that lack vascular endothelium, a relevant role of endocardial endothelium-NO signalling in intracavitary regulation of myocardial performance has been firmly established, thus revealing its early evolutionary role in non-mammalian vertebrates.     

Effects of intra-peritoneal injection with NH4Cl, urea, or NH4Cl+urea on nitrogen excretion and metabolism in the African lungfish Protopterus dolloi

This study aimed to (1) determine if ammonia (as NH(4)Cl) injected intra-peritoneally into the ureogenic slender African lungfish, Protopterus dolloi, was excreted directly rather than being converted to urea; (2) examine if injected urea was retained in this lungfish, leading to decreases in liver arginine and brain tryptophan levels, as observed during aestivation on land; and (3) elucidate if increase in internal ammonia level would affect urea excretion, when ammonia and urea are injected simultaneously into the fish. Despite being ureogenic, P. dolloi rapidly excreted the excess ammonia as ammonia within the subsequent 12 h after NH(4)Cl was injected into its peritoneal cavity. Injected ammonia was not detoxified into urea through the ornithine-urea cycle, probably because it is energetically intensive to synthesize urea and because food was withheld before and during the experiment. In addition, injected ammonia was likely to stay in extracellular compartments available for direct excretion. At hour 24, only a small amount of ammonia accumulated in the muscle of these fish. In contrast, when urea was injected intra-peritoneally into P. dolloi, only a small percentage (34%) of it was excreted during the subsequent 24-h period. A significant increase in the rate of urea excretion was observed only after 16 h. At hour 24, significant quantities of urea were retained in various tissues of P. dolloi. Injection with urea led to an apparent reduction in endogenous ammonia production, a significant decrease in the hepatic arginine content, and a significantly lower level of brain tryptophan in this lungfish. All three phenomena had been observed previously in aestivating P. dolloi. Hence, it is logical to deduce that urea synthesis and accumulation could be one of the essential factors in initiating and perpetuating aestivation in this lungfish. Through the injection of NH(4)Cl + urea, it was demonstrated that an increase in urea excretion occurred in P. dolloi within the first 12 h post-injection, which was much earlier than that of fish injected with urea alone. These results suggest that urea excretion in P. dolloi is likely to be regulated by the level of internal ammonia in its body.     

Distinct maturation of, but not migration between, human monocyte-derived dendritic cells upon ingestion of apoptotic cells of early or late phases

Cell death via apoptosis is a normal physiological process. Rapid, but silent, removal of apoptotic cells (ACs) plays an essential role in maintaining homeostasis in the immune system. Defective clearance of ACs allows ACs to accumulate and undergo late phase apoptosis, also known as secondary necrosis, which may generate danger signals, leading to inflammation or autoimmunity. In this study we investigate the outcome of dendritic cells (DCs), which are potent APCs, on the interaction with ACs of early or late phase. Immature DCs internalized ACs of both early and late phases with similar efficiency. However, DCs that had taken up ACs of early phase acquired a non-fully mature DC phenotype, expressing low MHC class II complex, costimulatory molecule CD40, and mature DC-restricted marker CD83, and had a low capacity to stimulate allogeneic CD4+ T cell proliferation, whereas DCs that had taken up ACs of late phase acquired a mature DC phenotype with enhanced T cell stimulatory capacity. Ingestion of either early or late ACs induced minimal production of IL-12 and modulated CC chemokine and CCR expression in DCs. In particular, there was down-regulation of CCR5 and up-regulation of CCR7, resulting in switches in responsiveness from inflammatory to lymphoid chemokines. We conclude from these data that after taking up ACs of either early or late phases, DCs acquire the capability of homing to draining lymph nodes, and the distinct maturation between DCs taking up early or late ACs may contribute to DC function in the induction of T cell tolerance or Ag-specific T cell response, respectively.     

Functional analysis of Toll-related genes in Drosophila

The Drosophila genome encodes a total of nine Toll and related proteins. The immune and developmental functions of Toll and 18Wheeler (18W) have been analyzed extensively, while the in vivo functions of the other Toll-related proteins require further investigation. We performed transgenic experiments and found that overexpression of Toll-related genes caused different extents of lethality and developmental defects. Moreover, 18w, Toll-6, Toll-7 and Toll-8 often caused related phenotypic changes, consistent with the idea that these four genes have more conserved molecular structure and thus may regulate similar processes in vivo. Deletion alleles of Toll-6, Toll-7 and Toll-8 were generated by targeted homologous recombination or P element excision. These mutant alleles were viable, fertile, and had no detectable defect in the inducible expression of antimicrobial peptide genes except for the Toll-8 mutant had some defects in leg development. The expression of 18w, Toll-7 and Toll-8 mRNA showed wide and overlapping patterns in imaginal discs and the 18w, Toll-8 double and Toll-7, Toll-8 double mutants showed substantially increased lethality. Overall our results suggest that some of the Toll-related proteins, such as 18W, Toll-7 and Toll-8, may have redundant functions in regulating developmental processes.     

Deficiency of mannose-binding lectin greatly increases susceptibility to postburn infection with Pseudomonas aeruginosa

Burn injury disrupts the mechanical and biological barrier that the skin presents against infection by symbionts like the Pseudomonas aeruginosa, a Gram-negative bacteria. A combination of local factors, antimicrobial peptides, and resident effector cells form the initial response to mechanical injury of the skin. This activity is followed by an inflammatory response that includes influx of phagocytes and serum factors, such as complement and mannose-binding lectin (MBL), which is a broad-spectrum pattern recognition molecule that plays a key role in innate immunity. A growing consensus from studies in humans and mice suggests that lack of MBL together with other comorbid factors predisposes the host to infection. In this study we examined whether MBL deficiency increases the risk of P. aeruginosa infection in a burned host. We found that both wild-type and MBL null mice were resistant to a 5% total body surface area burn alone or s.c. infection with P. aeruginosa alone. However, when mice were burned then inoculated s.c. with P. aeruginosa at the burn site, all MBL null mice died by 42 h from septicemia, whereas only one-third of wild-type mice succumbed (p = 0.0005). This result indicates that MBL plays a key role in containing and preventing a systemic spread of P. aeruginosa infection following burn injury and suggests that MBL deficiency in humans maybe a premorbid variable in the predisposition to infection in burn victims.     

Pctaire1 phosphorylates N-ethylmaleimide-sensitive fusion protein: implications in the regulation of its hexamerization and exocytosis

Pctaire1, a member of the cyclin-dependent kinase (Cdk)-related family, has recently been shown to be phosphorylated and regulated by Cdk5/p35. Although Pctaire1 is expressed in both neuronal and non-neuronal cells, its precise functions remain elusive. We performed a yeast two-hybrid screen to identify proteins that interact with Pctaire1. N-Ethylmaleimide-sensitive fusion protein (NSF), a crucial factor in vesicular transport and membrane fusion, was identified as one of the Pctaire1 interacting proteins. We demonstrate that the D2 domain of NSF, which is required for the oligomerization of NSF subunits, binds directly to and is phosphorylated by Pctaire1 on serine 569. Mutation of this phosphorylation site on NSF (S569A) augments its ability to oligomerize. Moreover, inhibition of Pctaire1 activity by transfecting its kinase-dead (KD) mutant into COS-7 cells enhances the self-association of NSF. Interestingly, Pctaire1 associates with NSF and synaptic vesicle-associated proteins in adult rat brain. To investigate whether Pctaire1 phosphorylation of NSF is involved in regulation of Ca(2+)-dependent exocytosis, we examined the effect of expressing Pctaire1 or NSF phosphorylation mutants on the regulated secretion of growth hormone from PC12 cells. Interestingly, expression of either Pctaire1-KD or NSF-S569A in PC12 cells significantly increases high K(+)-stimulated growth hormone release. Taken together, our findings provide the first demonstration that Pctaire1 phosphorylation of NSF regulates the ability of NSF to oligomerize, implicating an unexpected role of this kinase in modulating exocytosis. These findings open a new avenue of research in studying the functional roles of Pctaire1 in the nervous system.