Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function in situ remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4+ and CD8+ T-cells during skin infection with HSV-1. IFN-?-producing CD4+ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-?-producing CD8+ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-? production by CD4+ T cells, CD8+ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-? production by CD8+ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4+ and CD8+ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC).
To elucidate the effects of a controlled exposure to ethanol on gene expression, we studied lymphoblastoid cell lines (LCLs) from 21 alcoholics and 21 controls. We cultured each cell line for 24 h with and without 75 mM ethanol and measured gene expression using microarrays. Differences in expression between LCLs from alcoholics and controls included 13 genes previously identified as associated with alcoholism or related traits, including KCNA3, DICER1, ZNF415, CAT, SLC9A9, and PPARGC1B. The paired design allowed us to detect very small changes due to ethanol treatment: ethanol altered the expression of 37% of the probe sets (51% of the unique named genes) expressed in these LCLs, most by modest amounts. Ninety-nine percent of the named genes expressed in the LCLs were also expressed in brain. Key pathways affected by ethanol include cytokine, TNF, and NF?B signaling. Among the genes affected by ethanol were ANK3, EPHB1, SLC1A1, SLC9A9, NRD1, and SH3BP5, which were reported to be associated with alcoholism or related phenotypes in 2 genome-wide association studies. Genes that either differed in expression between alcoholics and controls or were affected by ethanol exposure are candidates for further study.
Trophic island biogeography theory predicts that the effects of predators on prey diversity are context dependent in heterogeneous landscapes. Specifically, models predict that the positive effect of habitat area on prey diversity should decline in the presence of predators, and that predators should modify the partitioning of alpha and beta diversity across patchy landscapes. However, experimental tests of the predicted context dependency in top-down control remain limited. Using a factorial field experiment we quantify the effects of a focal predatory fish species (grouper) and habitat characteristics (patch size, fragmentation) on the partitioning of diversity and assembly of coral reef fish communities. We found independent effects of groupers and patch characteristics on prey communities. Groupers reduced prey abundance by 50% and gamma diversity by 45%, with a disproportionate removal of rare species relative to common species (64% and 36% reduction, respectively; an oddity effect). Further, there was a 77% reduction in beta diversity. Null model analysis demonstrated that groupers increased the importance of stochastic community assembly relative to patches without groupers. With regard to patch size, larger patches contained more fishes, but a doubling of patch size led to a modest (36%) increase in prey abundance. Patch size had no effect on prey diversity; however, fragmented patches had 50% higher species richness and modified species composition relative to unfragmented patches. Our findings suggest two different pathways (i.e., habitat or predator shifts) by which natural and/or anthropogenic processes can drive variation in fish biodiversity and community assembly.
The ability of CMVs to evade the immune system of the host is dependent on the expression of a wide array of glycoproteins, many of which interfere with natural killer cell function. In murine CMV, two large protein families mediate this immune-evasive function. Although it is established that the m145 family members mimic the structure of MHC-I molecules, the structure of the m02 family remains unknown. The most extensively studied m02 family member is m04, a glycoprotein that escorts newly assembled MHC-I molecules to the cell surface, presumably to avoid "missing self" recognition. Here we report the crystal structure of the m04 ectodomain, thereby providing insight into this large immunoevasin family. m04 adopted a ?-sandwich immunoglobulin variable (Ig-V)-like fold, despite sharing very little sequence identity with the Ig-V superfamily. In addition to the Ig-V core, m04 possesses several unique structural features that included an unusual ?-strand topology, a number of extended loops and a prominent ?-helix. The m04 interior was packed by a myriad of hydrophobic residues that form distinct clusters around two conserved tryptophan residues. This hydrophobic core was well conserved throughout the m02 family, thereby indicating that murine CMV encodes a number of Ig-V-like molecules. We show that m04 binds a range of MHC-I molecules with low affinity in a peptide-independent manner. Accordingly, the structure of m04, which represents the first example of an murine CMV encoded Ig-V fold, provides a basis for understanding the structure and function of this enigmatic and large family of immunoevasins.
Global climate change is rapidly altering disturbance regimes in many ecosystems including coral reefs, yet the long-term impacts of these changes on ecosystem structure and function are difficult to predict. A major ecosystem service provided by coral reefs is the provisioning of physical habitat for other organisms, and consequently, many of the effects of climate change on coral reefs will be mediated by their impacts on habitat structure. Therefore, there is an urgent need to understand the independent and combined effects of coral mortality and loss of physical habitat on reef-associated biota. Here, we use a unique series of events affecting the coral reefs around the Pacific island of Moorea, French Polynesia to differentiate between the impacts of coral mortality and the degradation of physical habitat on the structure of reef fish communities. We found that, by removing large amounts of physical habitat, a tropical cyclone had larger impacts on reef fish communities than an outbreak of coral-eating sea stars that caused widespread coral mortality but left the physical structure intact. In addition, the impacts of declining structural complexity on reef fish assemblages accelerated as structure became increasingly rare. Structure provided by dead coral colonies can take up to decades to erode following coral mortality, and, consequently, our results suggest that predictions based on short-term studies are likely to grossly underestimate the long-term impacts of coral decline on reef fish communities.
A 2-year-old boxer dog from southern Ontario was evaluated because of acute onset lethargy. Exploratory laparotomy revealed a hemorrhagic, destructive, liver mass. Histology, immunohistochemistry, and polymerase chain reaction confirmed Echinococcus multilocularis as the cause of the hepatic mass. This constitutes the first description of endemic E. multilocularis in Ontario.
Signaling from JAK (Janus kinase) protein kinases to STAT (signal transducers and activators of transcription) transcription factors is key to many aspects of biology and medicine, yet the mechanism by which cytokine receptors initiate signaling is enigmatic. We present a complete mechanistic model for activation of receptor-bound JAK2, based on an archetypal cytokine receptor, the growth hormone receptor. For this, we used fluorescence resonance energy transfer to monitor positioning of the JAK2 binding motif in the receptor dimer, substitution of the receptor extracellular domains with Jun zippers to control the position of its transmembrane (TM) helices, atomistic modeling of TM helix movements, and docking of the crystal structures of the JAK2 kinase and its inhibitory pseudokinase domain with an opposing kinase-pseudokinase domain pair. Activation of the receptor dimer induced a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel TM helix pair to a left-handed crossover arrangement. This separation leads to removal of the pseudokinase domain from the kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans-activation. This model may well generalize to other class I cytokine receptors.
Alcohol and drug use disorders are individually heritable (50%). Twin studies indicate that alcohol and substance use disorders share common genetic influences, and therefore may represent a more heritable form of addiction and thus be more powerful for genetic studies. This study utilized data from 2322 subjects from 118 European-American families in the Collaborative Study on the Genetics of Alcoholism sample to conduct genome-wide association analysis of a binary and a continuous index of general substance dependence liability. The binary phenotype (ANYDEP) was based on meeting lifetime criteria for any DSM-IV dependence on alcohol, cannabis, cocaine or opioids. The quantitative trait (QUANTDEP) was constructed from factor analysis based on endorsement across the seven DSM-IV criteria for each of the four substances. Heritability was estimated to be 54% for ANYDEP and 86% for QUANTDEP. One single-nucleotide polymorphism (SNP), rs2952621 in the uncharacterized gene LOC151121 on chromosome 2, was associated with ANYDEP (P?=?1.8?×?10(-8) ), with support from surrounding imputed SNPs and replication in an independent sample [Study of Addiction: Genetics and Environment (SAGE); P?=?0.02]. One SNP, rs2567261 in ARHGAP28 (Rho GTPase-activating protein 28), was associated with QUANTDEP (P?=?3.8?×?10(-8) ), and supported by imputed SNPs in the region, but did not replicate in an independent sample (SAGE; P?=?0.29). The results of this study provide evidence that there are common variants that contribute to the risk for a general liability to substance dependence.
Clinical practice guidelines have been widely developed and disseminated with the aim of improving healthcare processes and patient outcomes but the uptake of evidence-based practice remains haphazard. There is a need to develop effective implementation methods to achieve large-scale adoption of proven innovations and recommended care. Clinical networks are increasingly being viewed as a vehicle through which evidence-based care can be embedded into healthcare systems using a collegial approach to agree on and implement a range of strategies within hospitals. In Australia, the provision of evidence-based care for men with prostate cancer has been identified as a high priority. Clinical audits have shown that fewer than 10% of patients in New South Wales (NSW) Australia at high risk of recurrence after radical prostatectomy receive guideline recommended radiation treatment following surgery. This trial will test a clinical network-based intervention to improve uptake of guideline recommended care for men with high-risk prostate cancer.
Whole-exome sequencing (WES) studies have demonstrated the contribution of de novo loss-of-function single-nucleotide variants (SNVs) to autism spectrum disorder (ASD). However, challenges in the reliable detection of de novo insertions and deletions (indels) have limited inclusion of these variants in prior analyses. By applying a robust indel detection method to WES data from 787 ASD families (2,963 individuals), we demonstrate that de novo frameshift indels contribute to ASD risk (OR = 1.6; 95% CI = 1.0-2.7; p = 0.03), are more common in female probands (p = 0.02), are enriched among genes encoding FMRP targets (p = 6 × 10(-9)), and arise predominantly on the paternal chromosome (p < 0.001). On the basis of mutation rates in probands versus unaffected siblings, we conclude that de novo frameshift indels contribute to risk in approximately 3% of individuals with ASD. Finally, by observing clustering of mutations in unrelated probands, we uncover two ASD-associated genes: KMT2E (MLL5), a chromatin regulator, and RIMS1, a regulator of synaptic vesicle release.
We assessed gene expression profiles in 2,752 twins, using a classic twin design to quantify expression heritability and quantitative trait loci (eQTLs) in peripheral blood. The most highly heritable genes (?777) were grouped into distinct expression clusters, enriched in gene-poor regions, associated with specific gene function or ontology classes, and strongly associated with disease designation. The design enabled a comparison of twin-based heritability to estimates based on dizygotic identity-by-descent sharing and distant genetic relatedness. Consideration of sampling variation suggests that previous heritability estimates have been upwardly biased. Genotyping of 2,494 twins enabled powerful identification of eQTLs, which we further examined in a replication set of 1,895 unrelated subjects. A large number of non-redundant local eQTLs (6,756) met replication criteria, whereas a relatively small number of distant eQTLs (165) met quality control and replication standards. Our results provide a new resource toward understanding the genetic control of transcription.
Killer Ig-like receptors (KIRs) control the activation of human NK cells via interactions with peptide-laden HLAs. KIR3DL1 is a highly polymorphic inhibitory receptor that recognizes a diverse array of HLA molecules expressing the Bw4 epitope, a group with multiple polymorphisms incorporating variants within the Bw4 motif. Genetic studies suggest that KIR3DL1 variation has functional significance in several disease states, including HIV infection. However, owing to differences across KIR3DL1 allotypes, HLA-Bw4, and associated peptides, the mechanistic link with biological outcome remains unclear. In this study, we elucidated the impact of KIR3DL1 polymorphism on peptide-laden HLA recognition. Mutational analysis revealed that KIR residues involved in water-mediated contacts with the HLA-presented peptide influence peptide binding specificity. In particular, residue 282 (glutamate) in the D2 domain underpins the lack of tolerance of negatively charged C-terminal peptide residues. Allotypic KIR3DL1 variants, defined by neighboring residue 283, displayed differential sensitivities to HLA-bound peptide, including the variable HLA-B*57:01-restricted HIV-1 Gag-derived epitope TW10. Residue 283, which has undergone positive selection during the evolution of human KIRs, also played a central role in Bw4 subtype recognition by KIR3DL1. Collectively, our findings uncover a common molecular regulator that controls HLA and peptide discrimination without participating directly in peptide-laden HLA interactions. Furthermore, they provide insight into the mechanics of interaction and generate simple, easily assessed criteria for the definition of KIR3DL1 functional groupings that will be relevant in many clinical applications, including bone marrow transplantation.
Studies have shown association between common variants in the ?6-?3 nicotinic receptor subunit gene cluster and nicotine dependence in European ancestry populations. We investigate whether this generalizes to African Americans, whether the association is specific to nicotine dependence and whether this region contains additional genetic contributors to nicotine dependence.
The rotational spectra of cyclopentanone and its van der Waals complexes with argon and neon have been observed with a Balle-Flygare type pulsed jet Fourier transform microwave spectrometer in the 6 to 20 GHz region. This work improves the rotational constants and quartic centrifugal distortion constants for cyclopentanone and its five (13)C and the (18)O isotopologues. The argon-(12)C5H8(16)O van der Waals complex has rotational constants of A = 2611.6688, B = 1112.30298, and C = 971.31969 MHz. The (20)Ne-(12)C5H8(16)O complex has rotational constants of A = 2728.8120, B = 1736.5882, and C = 1440.4681 MHz. In addition, the five unique, singly substituted (13)C and (18)O isotopologues of the argon complex are reported. The five single-substituted (13)C of the (20)Ne complex and the (22)Ne-(12)C5H8(16)O complex are reported. The rare gases are in van der Waals contact with the carbonyl ? carbon and nearly in contact with the hydrogen on ? and ? carbons toward the back of the ring.
Genomes of men and women differ in only a limited number of genes located on the sex chromosomes, whereas the transcriptome is far more sex-specific. Identification of sex-biased gene expression will contribute to understanding the molecular basis of sex-differences in complex traits and common diseases.
The activation of murine and human natural killer (NK) cells is regulated by families of receptors including the Ly49 and Killer immunoglobulin-like receptors, respectively, both of which contain activating and inhibitory members. The archetypal role of inhibitory Ly49 receptors is to attenuate NK cell responses to normal cells that express major histocompatibility complex (MHC) class-I molecules, in essence allowing for more robust responses to infected or cancerous cells that lack MHC-I on their cell surface. However, it is now evident that Ly49 receptors have an appreciably more sophisticated array of functions. In particular, some activating Ly49 receptors can bind directly to MHC-I-like viral gene products such as m157, whereas others recognize self-MHC-I but only in the presence of viral chaperones. Although Ly49 receptor recognition is centred on the MHC-I-like fold, these NK cell receptors can also engage related ligands in unexpected ways. Herein we review the varied strategies employed by Ly49 receptors to recognize both self and viral ligands, with particular emphasis on the recently determined mode of Ly49-m157 ligation, and highlight the versatile nature of this family in the control of viral infections.
Understanding dogs' perceptual experience of both conspecifics and humans is important to understand how dogs evolved and the nature of their relationships with humans and other dogs. Olfaction is believed to be dogs' most powerful and perhaps important sense and an obvious place to begin for the study of social cognition of conspecifics and humans. We used fMRI in a cohort of dogs (N=12) that had been trained to remain motionless while unsedated and unrestrained in the MRI. By presenting scents from humans and conspecifics, we aimed to identify the dimensions of dogs' responses to salient biological odors - whether they are based on species (dog or human), familiarity, or a specific combination of factors. We focused our analysis on the dog's caudate nucleus because of its well-known association with positive expectations and because of its clearly defined anatomical location. We hypothesized that if dogs' primary association to reward, whether it is based on food or social bonds, is to humans, then the human scents would activate the caudate more than the conspecific scents. Conversely, if the smell of conspecifics activated the caudate more than the smell of humans, dogs' association to reward would be stronger to their fellow canines. Five scents were presented (self, familiar human, strange human, familiar dog, strange dog). While the olfactory bulb/peduncle was activated to a similar degree by all the scents, the caudate was activated maximally to the familiar human. Importantly, the scent of the familiar human was not the handler, meaning that the caudate response differentiated the scent in the absence of the person being present. The caudate activation suggested that not only did the dogs discriminate that scent from the others, they had a positive association with it. This speaks to the power of the dog's sense of smell, and it provides important clues about the importance of humans in dogs' lives. This article is part of a Special Issue entitled: Canine Behavior.
The growth hormone receptor was the first cytokine receptor to be cloned and crystallized, and provides a valuable exemplar for activation of its cognate kinase, JAK2. We review progress in understanding its activation mechanism, in particular the molecular movements made by this constitutively dimerized receptor in response to ligand binding, and how these lead to a separation of JAK-binding Box1 motifs. Such a separation leads to removal of the pseudokinase inhibitory domain from the kinase domain of a partner JAK2 bound to the receptor, and vice versa, leading to apposition of the kinase domains and transactivation. This may be a general mechanism for class I cytokine receptor action.
There is much interest in the potential of Ab-dependent cellular cytotoxicity (ADCC) to slow disease progression following HIV infection. Despite several studies demonstrating a positive association between ADCC and slower disease progression, it is possible that continued stimulation of NK cells by ADCC during chronic HIV infection could render these cells dysfunctional. Indeed, activation of NK cells by ADCC results in matrix metalloproteinase-induced reductions in CD16 expression and activation refractory periods. In addition, ex vivo analyses of NK cells from HIV-infected individuals revealed other alterations in phenotype, such as decreased expression of the activating NKp46 receptor that is essential for NK-mediated antitumor responses and immunity from infection. Because NKp46 shares a signaling pathway with CD16, we hypothesized that activation-induced downregulation of both receptors could be controlled by a common mechanism. We found that activation of NK cells by anti-HIV or anti-CD16 Abs resulted in NKp46 downregulation. The addition of a matrix metalloproteinase inhibitor attenuated NKp46 downregulation following NK cell activation by anti-HIV Abs. Consequently, these results suggest that continued stimulation through CD16 has the potential to impair natural cytotoxicity via attenuation of NKp46-dependent signals.
The long pentraxin, pentraxin 3 (PTX3), can play beneficial or detrimental roles during infection and disease by modulating various aspects of the immune system. There is growing evidence to suggest that PTX3 can mediate antiviral activity in vitro and in vivo. Previous studies demonstrated that PTX3 and the short pentraxin serum amyloid P express sialic acids that are recognized by the hemagglutinin (HA) glycoprotein of certain influenza A viruses (IAV), resulting in virus neutralization and anti-IAV activity. In this study, we demonstrate that specificity of both HA and the viral neuraminidase for particular sialic acid linkages determines the susceptibility of H1N1, H3N2, and H7N9 strains to the antiviral activities of PTX3 and serum amyloid P. Selection of H3N2 virus mutants resistant to PTX3 allowed for identification of amino acid residues in the vicinity of the receptor-binding pocket of HA that are critical determinants of sensitivity to PTX3; this was supported by sequence analysis of a range of H3N2 strains that were sensitive or resistant to PTX3. In a mouse model of infection, the enhanced virulence of PTX3-resistant mutants was associated with increased virus replication and elevated levels of proinflammatory cytokines in the airways, leading to pulmonary inflammation and lung injury. Together, these studies identify determinants in the viral HA that can be associated with sensitivity to the antiviral activities of PTX3 and highlight its importance in the control of IAV infection.
Specific protein receptors that mediate internalization and entry of influenza A virus (IAV) have not been identified for any cell type. Sialic acid (SIA), the primary attachment factor for IAV hemagglutinin, is expressed by numerous cell-surface glycoproteins and glycolipids, confounding efforts to identify specific receptors involved in virus infection. Lec1 Chinese hamster ovary (CHO) epithelial cells express cell-surface SIA and bind IAV, yet are largely resistant to infection. Herein, we demonstrate that expression of the murine macrophage galactose-type lectin (MGL)-1 by Lec1 cells enhanced Ca(2+)-dependent IAV binding and restored permissivity to infection. Lec1 cells expressing MGL1 were infected in the presence or absence of cell-surface SIA, indicating that MGL1 can act as a primary receptor or as a co-receptor with SIA. Lec1 cells expressing endocytosis-deficient MGL1 mediated Ca(2+)-dependent IAV binding, but were less sensitive to IAV infection, indicating that direct internalization via MGL1 can result in cellular infection. Together, these studies identify MGL1 as a cell-surface glycoprotein that can act as an authentic receptor for both attachment and infectious entry of IAV.
During the 2009 pandemic of influenza A virus subtype H1N1 (A[H1N1]pdm09) infection, older individuals were partially protected from severe disease. It is not known whether preexisting antibodies with effector functions such as antibody-dependent cellular cytotoxicity (ADCC) contributed to the immunity observed.
Natural killer (NK) cells possess effector and immunoregulatory functions that are controlled by a myriad of receptor-ligand pairs, including human killer inhibitory receptor (KIR) and mouse Ly49-MHC class I interactions. We have recently shown that the NK cell inhibitory molecule Ly49A binds the non-classical MHC molecule H2-M3, thus regulating host innate immune responses to tumor initiation and metastasis.
The effects of inbreeding on the health of offspring can be studied by measuring genome-wide autozygosity as the proportion of the genome in runs of homozygosity (F roh) and relate F roh to outcomes such as psychiatric phenotypes. To successfully conduct these studies, the main patterns of variation for genome-wide autozygosity between and within populations should be well understood and accounted for. Within population variation was investigated in the Dutch population by comparing autozygosity between religious and non-religious groups. The Netherlands have a history of societal segregation and assortment based on religious affiliation, which may have increased parental relatedness within religious groups. Religion has been associated with several psychiatric phenotypes, such as major depressive disorder (MDD). We investigated whether there is an association between autozygosity and MDD, and the extent to which this association can be explained by religious affiliation. All F roh analyses included adjustment for ancestry-informative principal components (PCs) and geographic factors. Religious affiliation was significantly associated with autozygosity, showing that F roh has the ability to capture within population differences that are not captured by ancestry-informative PCs or geographic factors. The non-religious group had significantly lower F roh values and significantly more MDD cases, leading to a nominally significant negative association between autozygosity and depression. After accounting for religious affiliation, MDD was not associated with F roh, indicating that the relation between MDD and inbreeding was due to stratification. This study shows how past religious assortment and recent secularization can have genetic consequences in a relatively small country. This warrants accounting for the historical social context and its effects on genetic variation in association studies on psychiatric and other related traits.
Class I HLAs generally present peptides of 8-10 aa in length, although it is unclear whether peptide length preferences are affected by HLA polymorphism. In this study, we investigated the CD8(+) T cell response to the BZLF1 Ag of EBV, which includes overlapping sequences of different size that nevertheless conform to the binding motif of the large and abundant HLA-B*44 supertype. Whereas HLA-B*18:01(+) individuals responded strongly and exclusively to the octamer peptide (173)SELEIKRY(180), HLA-B*44:03(+) individuals responded to the atypically large dodecamer peptide (169)EECDSELEIKRY(180), which encompasses the octamer peptide. Moreover, the octamer peptide bound more stably to HLA-B*18:01 than did the dodecamer peptide, whereas, conversely, HLA-B*44:03 bound only the longer peptide. Furthermore, crystal structures of these viral peptide-HLA complexes showed that the Ag-binding cleft of HLA-B*18:01 was more ideally suited to bind shorter peptides, whereas HLA-B*44:03 exhibited characteristics that favored the presentation of longer peptides. Mass spectrometric identification of > 1000 naturally presented ligands revealed that HLA-B*18:01 was more biased toward presenting shorter peptides than was HLA-B*44:03. Collectively, these data highlight a mechanism through which polymorphism within an HLA class I supertype can diversify determinant selection and immune responses by varying peptide length preferences.
The mucosal-associated invariant T-cell antigen receptor (MAIT TCR) recognizes MR1 presenting vitamin B metabolites. Here we describe the structures of a human MAIT TCR in complex with human MR1 presenting a non-stimulatory ligand derived from folic acid and an agonist ligand derived from a riboflavin metabolite. For both vitamin B antigens, the MAIT TCR docks in a conserved manner above MR1, thus acting as an innate-like pattern recognition receptor. The invariant MAIT TCR ?-chain usage is attributable to MR1-mediated interactions that prise open the MR1 cleft to allow contact with the vitamin B metabolite. Although the non-stimulatory antigen does not contact the MAIT TCR, the stimulatory antigen does. This results in a higher affinity of the MAIT TCR for a stimulatory antigen in comparison with a non-stimulatory antigen. We formally demonstrate a structural basis for MAIT TCR recognition of vitamin B metabolites, while illuminating how TCRs recognize microbial metabolic signatures.
The Laura-Normanby River (catchment area: 24,350 km(2)), which drains into Princess Charlotte Bay, has been identified in previous studies as the third largest contributor of sediment to the Great Barrier Reef World Heritage Area. These catchment scale modelling studies also identified surface soil erosion as supplying >80% of the sediment. Here we use activity concentrations of the fallout radionuclides (137)Cs and (210)Pbex to test the hypothesis that surface soil erosion dominates the supply of fine (<10 ?m) sediment in the river systems draining into Princess Charlotte Bay. Our results contradict these previous studies, and are consistent with channel and gully erosion being the dominant source of fine sediment in this catchment. The hypothesis that surface soil erosion dominates the supply of fine sediment to Princess Charlotte Bay is rejected. River sediment samples were collected using both time-integrated samplers and sediment drape deposits. We show that there is no detectable difference in (137)Cs and (210)Pbex activity concentrations between samples collected using these two methods. Two methods were also used to collect samples to characterise (137)Cs and (210)Pbex concentrations in sediment derived from surface soil erosion; sampling of surface-wash deposits and deployment of surface runoff traps that collected samples during rain events. While there was no difference in the (137)Cs activity concentrations for samples collected using these two methods, (210)Pbex activity concentrations were significantly higher in the samples collected using the runoff traps. The higher (210)Pbex concentrations are shown to be correlated with loss-on-ignition (r(2) = 0.79) and therefore are likely to be related to higher organic concentrations in the runoff trap samples. As a result of these differences we use a three end member mixing model (channel/gully, hillslope surface-wash and hillslope runoff traps) to determine the relative contribution from surface soil erosion. Probability distributions for (137)Cs and (210)Pbex concentrations were determined for each of the end members, with these distributions then used to estimate the surface soil contribution to each of the collected river sediment samples. The mean estimate of contribution of surface derived sediment for all river samples (n = 70) is 16 ± 2%. This study reinforces the importance of testing model predictions before they are used to target investment in remedial action and adds to the body of evidence that the primary source of sediment delivered to tropical river systems is derived from subsoil erosion.
The Fellowship Examination is the final summative assessment before the Surgical Education and Training trainees are awarded Fellowship of the Royal Australasian College of Surgeons. Conducted in nine specialties, it is aligned with the curriculum of each specialty training programme. The Fellowship Examination focuses on specific surgical competencies; in particular, the clinical application of knowledge, operative decision making and professional judgement. As a true exit examination, it has to be conducted at the correct cognitive level for surgeons about to enter practice without direct supervision. This requires examiners to have specific skills and expertise for which training is required. This paper outlines the process of training undertaken by newly appointed examiners, and describes some of the areas of knowledge that they have to master before examining at the consistently high level that is now expected.
There is mounting evidence that the mesolimbic dopamine system carries valuation signals not only for appetitive or gain-related stimuli, with which it is traditionally associated, but also for aversive and loss-related stimuli. Cellular-level studies demonstrate that the neuronal architecture to support aversive stimuli encoding in this system does exist. Both cellular-level and human neuroimaging research suggest the co-existence of appetitive and aversive prediction-error signals within the mesocorticolimbic system. These findings shift the view of the mesocorticolimbic system as a singular pathway for reward to a system with multiple signals across a wide range of domains that drive value-based decision making.
The novel radical cation salt (BEDT-TTF)3(sulfamate)2·2H2O (BEDT-TTF = bis(ethylenedithio)tetrathiafulvalene) is semiconducting with donor stacks comprised of pairs of partially oxidized molecules and a single more highly oxidized molecule which is twisted out of the stack by ca. 30°. Hydrogen bonded pairs of sulfamate ions are linked into parallel ribbons by further hydrogen bonding between sulfamates and bridging water molecules. In contrast, the BEDT-TTF salt with pentaborate contains infinite layers formed of a network of hydrogen bonded pentaborate anions. Two new bromide salts of BEDT-TTF are reported, one is a semiconducting 1 : 1 salt in which the bromide is integrated among the BEDT-TTF donors, while the other contain a square of four bromide ions linked together by hydrogen bonding to a centrally located H5O2(+) cation for every five BEDT-TTF molecules.
Activating and inhibitory receptors on natural killer (NK) cells have a crucial role in innate immunity, although the basis of the engagement of activating NK cell receptors is unclear. The activating receptor Ly49H confers resistance to infection with murine cytomegalovirus by binding to the immunoevasin m157. We found that m157 bound to the helical stalk of Ly49H, whereby two m157 monomers engaged the Ly49H dimer. The helical stalks of Ly49H lay centrally across the m157 platform, whereas its lectin domain was not required for recognition. Instead, m157 targeted an aromatic peg motif present in stalks of both activating and inhibitory receptors of the Ly49 family, and substitution of this motif abrogated binding. Furthermore, ligation of m157 to Ly49H or Ly49C resulted in intracellular signaling. Accordingly, m157 has evolved to tackle the legs of a family of NK cell receptors.
Caveolae and caveolin-1 (CAV1) have been linked to several cellular functions. However, a model explaining their roles in mammalian tissues in vivo is lacking. Unbiased expression profiling in several tissues and cell types identified lipid metabolism as the main target affected by CAV1 deficiency. CAV1-/- mice exhibited impaired hepatic peroxisome proliferator-activated receptor ? (PPAR?)-dependent oxidative fatty acid metabolism and ketogenesis. Similar results were recapitulated in CAV1-deficient AML12 hepatocytes, suggesting at least a partial cell-autonomous role of hepatocyte CAV1 in metabolic adaptation to fasting. Finally, our experiments suggest that the hepatic phenotypes observed in CAV1-/- mice involve impaired PPAR? ligand signaling and attenuated bile acid and FXR? signaling. These results demonstrate the significance of CAV1 in (1) hepatic lipid homeostasis and (2) nuclear hormone receptor (PPAR?, FXR?, and SHP) and bile acid signaling.
Members of the pentraxin family, including PTX3 and serum amyloid P component (SAP), have been reported to play a role in innate host defence against a range of microbial pathogens, yet little is known regarding their antiviral activities. In this study, we demonstrate that human SAP binds to human influenza A virus (IAV) strains and mediates a range of antiviral activities, including inhibition of IAV-induced hemagglutination (HA), neutralization of virus infectivity and inhibition of the enzymatic activity of the viral neuraminidase (NA). Characterization of the anti-IAV activity of SAP after periodate or bacterial sialidase treatment demonstrated that ?(2,6)-linked sialic acid residues on the glycosidic moiety of SAP are critical for recognition by the HA of susceptible IAV strains. Other proteins of the innate immune system, namely human surfactant protein A and porcine surfactant protein D, have been reported to express sialylated glycans which facilitate inhibition of particular IAV strains, yet the specific viral determinants for recognition of these inhibitors have not been defined. Herein, we have selected virus mutants in the presence of human SAP and identified specific residues in the receptor-binding pocket of the viral HA which are critical for recognition and therefore susceptibility to the antiviral activities of SAP. Given the widespread expression of ?(2,6)-linked sialic acid in the human respiratory tract, we propose that SAP may act as an effective receptor mimic to limit IAV infection of airway epithelial cells.
Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the viral hemagglutinin (HA). Glycans on the head of HA promote virus survival by shielding antigenic sites, but highly glycosylated seasonal IAV are inactivated by soluble lectins of the innate immune system. In 2009, human strains of pandemic H1N1 [A(H1N1)pdm] expressed a single glycosylation site (Asn(104)) on the head of HA. Since then, variants with additional glycosylation sites have been detected, and the location of these sites has been distinct to those of recent seasonal H1N1 strains. We have compared wild-type and reverse-engineered A(H1N1)pdm IAV with differing potential glycosylation sites on HA for sensitivity to collectins and to neutralizing Abs. Addition of a glycan (Asn(136)) to A(H1N1)pdm HA was associated with resistance to neutralizing Abs but did not increase sensitivity to collectins. Moreover, variants expressing Asn(136) showed enhanced growth in A(H1N1)pdm-vaccinated mice, consistent with evasion of Ab-mediated immunity in vivo. Thus, a fine balance exists regarding the optimal pattern of HA glycosylation to facilitate evasion of Ab-mediated immunity while maintaining resistance to lectin-mediated defenses of the innate immune system.
IFN-? is critical for immunity against infections with intracellular pathogens, such as Salmonella enterica. However, which of the many cell types capable of producing IFN-? controls Salmonella infections remains unclear. Using a mouse model of systemic Salmonella infection, we observed that only a lack of all lymphocytes or CD90 (Thy1)(+) cells, but not the absence of T cells, Retinoic acid-related orphan receptor (ROR)-?t-dependent lymphocytes, (NK)1.1(+) cells, natural killer T (NKT), and/or B cells alone, replicated the highly susceptible phenotype of IFN-?-deficient mice to Salmonella infection. A combination of antibody depletions and adoptive transfer experiments revealed that early protective IFN-? was provided by Thy1-expressing natural killer (NK) cells and that these cells improved antibacterial immunity through the provision of IFN-?. Further analysis of NK cells producing IFN-? in response to Salmonella indicated that less mature NK cells were more efficient at mediating antibacterial effector function than terminally differentiated NK cells. Inspired by recent reports of Thy1(+) NK cells contributing to immune memory, we analyzed their role in secondary protection against otherwise lethal WT Salmonella infections. Notably, we observed that a newly generated Salmonella vaccine strain not only conferred superior protection compared with conventional regimens but that this enhanced efficiency of recall immunity was afforded by incorporating CD4(-)CD8(-)Thy1(+) cells into the secondary response. Taken together, these findings demonstrate that Thy1-expressing NK cells play an important role in antibacterial immunity.
Natural killer (NK) cell recognition of the nonclassical human leukocyte antigen (HLA) molecule HLA-E is dependent on the presentation of a nonamer peptide derived from the leader sequence of other HLA molecules to CD94-NKG2 receptors. However, human cytomegalovirus can manipulate this central innate interaction through the provision of a "mimic" of the HLA-encoded peptide derived from the immunomodulatory glycoprotein UL40. Here, we analyzed UL40 sequences isolated from 32 hematopoietic stem cell transplantation recipients experiencing cytomegalovirus reactivation. The UL40 protein showed a "polymorphic hot spot" within the region that encodes the HLA leader sequence mimic. Although all sequences that were identical to those encoded within HLA-I genes permitted the interaction between HLA-E and CD94-NKG2 receptors, other UL40 polymorphisms reduced the affinity of the interaction between HLA-E and CD94-NKG2 receptors. Furthermore, functional studies using NK cell clones expressing either the inhibitory receptor CD94-NKG2A or the activating receptor CD94-NKG2C identified UL40-encoded peptides that were capable of inhibiting target cell lysis via interaction with CD94-NKG2A, yet had little capacity to activate NK cells through CD94-NKG2C. The data suggest that UL40 polymorphisms may aid evasion of NK cell immunosurveillance by modulating the affinity of the interaction with CD94-NKG2 receptors.
A better understanding of immunity to influenza virus is needed to generate cross-protective vaccines. Engagement of Ab-dependent cellular cytotoxicity (ADCC) Abs by NK cells leads to killing of virus-infected cells and secretion of antiviral cytokines and chemokines. ADCC Abs may target more conserved influenza virus Ags compared with neutralizing Abs. There has been minimal interest in influenza-specific ADCC in recent decades. In this study, we developed novel assays to assess the specificity and function of influenza-specific ADCC Abs. We found that healthy influenza-seropositive young adults without detectable neutralizing Abs to the hemagglutinin of the 1968 H3N2 influenza strain (A/Aichi/2/1968) almost always had ADCC Abs that triggered NK cell activation and in vitro elimination of influenza-infected human blood and respiratory epithelial cells. Furthermore, we detected ADCC in the absence of neutralization to both the recent H1N1 pandemic strain (A/California/04/2009) as well as the avian H5N1 influenza hemagglutinin (A/Anhui/01/2005). We conclude that there is a remarkable degree of cross-reactivity of influenza-specific ADCC Abs in seropositive humans. Targeting cross-reactive influenza-specific ADCC epitopes by vaccination could lead to improved influenza vaccines.
Strong evidence exists to support preoperative pelvic floor muscle training (PFMT) to reduce the severity and duration of urinary incontinence after radical prostatectomy. Receipt of preoperative PFMT amongst men having radical prostatectomy in Western Sydney, however, is suboptimal. This study was undertaken to investigate barriers and enablers to provision/receipt of preoperative PFMT from the perspectives of potential referrers to and providers of PFMT, and of men having radical prostatectomy.
Genetic variation in a population can be summarized through principal component analysis (PCA) on genome-wide data. PCs derived from such analyses are valuable for genetic association studies, where they can correct for population stratification. We investigated how to capture the genetic population structure in a well-characterized sample from the Netherlands and in a worldwide data set and examined whether (1) removing long-range linkage disequilibrium (LD) regions and LD-based SNP pruning significantly improves correlations between PCs and geography and (2) whether genetic differentiation may have been influenced by migration and/or selection. In the Netherlands, three PCs showed significant correlations with geography, distinguishing between: (1) North and South; (2) East and West; and (3) the middle-band and the rest of the country. The third PC only emerged with minimized LD, which also significantly increased correlations with geography for the other two PCs. In addition to geography, the Dutch North-South PC showed correlations with genome-wide homozygosity (r=0.245), which may reflect a serial-founder effect due to northwards migration, and also with height (?: r=0.142, ?: r=0.153). The divergence between subpopulations identified by PCs is partly driven by selection pressures. The first three PCs showed significant signals for diversifying selection (545 SNPs - the majority within 184 genes). The strongest signal was observed between North and South for the functional SNP in HERC2 that determines human blue/brown eye color. Thus, this study demonstrates how to increase ancestry signals in a relatively homogeneous population and how those signals can reveal evolutionary history.
Previously, we demonstrated the possibility of fMRI in two awake and unrestrained dogs. Here, we determined the replicability and heterogeneity of these results in an additional 11 dogs for a total of 13 subjects. Based on an anatomically placed region-of-interest, we compared the caudate response to a hand signal indicating the imminent availability of a food reward to a hand signal indicating no reward. 8 of 13 dogs had a positive differential caudate response to the signal indicating reward. The mean differential caudate response was 0.09%, which was similar to a comparable human study. These results show that canine fMRI is reliable and can be done with minimal stress to the dogs.
Variants within the gene cluster encoding ?3, ?5, and ?4 nicotinic receptor subunits are major risk factors for substance dependence. The strongest impact on risk is associated with variation in the CHRNA5 gene, where at least two mechanisms are at work: amino acid variation and altered mRNA expression levels. The risk allele of the non-synonymous variant (rs16969968; D398N) primarily occurs on the haplotype containing the low mRNA expression allele. In populations of European ancestry, there are approximately 50 highly correlated variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and the adjacent PSMA4 gene region that are associated with CHRNA5 mRNA levels. It is not clear which of these variants contribute to the changes in CHRNA5 transcript level. Because populations of African ancestry have reduced linkage disequilibrium among variants spanning this gene cluster, eQTL mapping in subjects of African ancestry could potentially aid in defining the functional variants that affect CHRNA5 mRNA levels. We performed quantitative allele specific gene expression using frontal cortices derived from 49 subjects of African ancestry and 111 subjects of European ancestry. This method measures allele-specific transcript levels in the same individual, which eliminates other biological variation that occurs when comparing expression levels between different samples. This analysis confirmed that substance dependence associated variants have a direct cis-regulatory effect on CHRNA5 transcript levels in human frontal cortices of African and European ancestry and identified 10 highly correlated variants, located in a 9 kb region, that are potential functional variants modifying CHRNA5 mRNA expression levels.
Increased habitat diversity is often predicted to promote the diversity of animal communities because a greater variety of habitats increases the opportunities for species to specialize on different resources and coexist. Although positive correlations between the diversities of habitat and associated animals are often observed, the underlying mechanisms are only now starting to emerge, and none have been tested specifically in the marine environment. Scleractinian corals constitute the primary habitat-forming organisms on coral reefs and, as such, play an important role in structuring associated reef fish communities. Using the same field experimental design in two geographic localities differing in regional fish species composition, we tested the effects of coral species richness and composition on the diversity, abundance, and structure of the local fish community. Richness of coral species overall had a positive effect on fish species richness but had no effect on total fish abundance or evenness. At both localities, certain individual coral species supported similar levels of fish diversity and abundance as the high coral richness treatments, suggesting that particular coral species are disproportionately important in promoting high local fish diversity. Furthermore, in both localities, different microhabitats (coral species) supported very different fish communities, indicating that most reef fish species distinguish habitat at the level of coral species. Fish communities colonizing treatments of higher coral species richness represented a combination of those inhabiting the constituent coral species. These findings suggest that mechanisms underlying habitat-animal interaction in the terrestrial environment also apply to marine systems and highlight the importance of coral diversity to local fish diversity. The loss of particular key coral species is likely to have a disproportionate impact on the biodiversity of associated fish communities.
Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10-deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.
Minimotif Miner (MnM available at http://minimotifminer.org or http://mnm.engr.uconn.edu) is an online database for identifying new minimotifs in protein queries. Minimotifs are short contiguous peptide sequences that have a known function in at least one protein. Here we report the third release of the MnM database which has now grown 60-fold to approximately 300,000 minimotifs. Since short minimotifs are by their nature not very complex we also summarize a new set of false-positive filters and linear regression scoring that vastly enhance minimotif prediction accuracy on a test data set. This online database can be used to predict new functions in proteins and causes of disease.
The primary function of the monomorphic MHC class Ib molecule Qa-1(b) is to present peptides derived from the leader sequences of other MHC class I molecules for recognition by the CD94-NKG2 receptors expressed by NK and T cells. Whereas the mode of peptide presentation by its ortholog HLA-E, and subsequent recognition by CD94-NKG2A, is known, the molecular basis of Qa-1(b) function is unclear. We have assessed the interaction between Qa-1(b) and CD94-NKG2A and shown that they interact with an affinity of 17 ?M. Furthermore, we have determined the structure of Qa-1(b) bound to the leader sequence peptide, Qdm (AMAPRTLLL), to a resolution of 1.9 Å and compared it with that of HLA-E. The crystal structure provided a basis for understanding the restricted peptide repertoire of Qa-1(b). Whereas the Qa-1(b-AMAPRTLLL) complex was similar to that of HLA-E, significant sequence and structural differences were observed between the respective Ag-binding clefts. However, the conformation of the Qdm peptide bound by Qa-1(b) was very similar to that of peptide bound to HLA-E. Although a number of conserved innate receptors can recognize heterologous ligands from other species, the structural differences between Qa-1(b) and HLA-E manifested in CD94-NKG2A ligand recognition being species specific despite similarities in peptide sequence and conformation. Collectively, our data illustrate the structural homology between Qa-1(b) and HLA-E and provide a structural basis for understanding peptide repertoire selection and the specificity of the interaction of Qa-1(b) with CD94-NKG2 receptors.
Airway M? and DCs are important components of innate host defense and can play a critical role in limiting the severity of influenza virus infection. Although it has been well established that cell-surface SA acts as a primary attachment receptor for IAV, the particular receptor(s) or coreceptor(s) that mediate IAV entry into any cell, including M? and DC, have not been clearly defined. Identifying which receptors are involved in attachment and entry of IAV into immune cells may have important implications in regard to understanding IAV tropism and pathogenesis. Recent evidence suggests that specialized receptors on M? and DCs, namely CLRs, can act as capture and/or entry receptors for many viral pathogens, including IAV. Herein, we review the early stages of infection of M? and DC by IAV. Specifically, we examine the potential role of CLRs expressed on M? and DC to act as attachment and/or entry receptors for IAV.
Whats known on the subject? and What does the study add? The Australian private health sector is increasingly being recognised as an opportunity for advanced surgical training. Little is known about what the patients think of urology training in the private sector. Patients perceptions on the place of urology registrars in the Australian private sector are assessed for the first time. We can confirm that there appears to be acceptance of urology training in the private sector.
The species composition of coral communities has shifted in many areas worldwide through the relative loss of important ecosystem engineers such as highly branched corals, which are integral in maintaining reef biodiversity. We assessed the degree to which the performance of recently recruited branching corals was influenced by corallivory, competition, sedimentation, and the interactions between these factors. We also explored whether the species-specific influence of these biotic and abiotic constraints helps to explain recent shifts in the coral community in lagoons of Moorea, French Polynesia. Population surveys revealed evidence of a community shift away from a historically acroporid-dominated community to a pocilloporid- and poritid-dominated community, but also showed that the distribution and abundance of coral taxa varied predictably with location in the lagoon. At the microhabitat scale, branching corals grew mainly on dead or partially dead massive Porites ("bommies"), promontories with enhanced current velocities and reduced sedimentation. A demographic study revealed that growth and survival of juvenile Pocillopora verrucosa and Acropora retusa, the two most common branching species of each taxon, were affected by predation and competition with vermetid gastropods. By 24 months of age, 20-60% of juvenile corals suffered partial predation by corallivorous fishes, and injured corals experienced reduced growth and survival. A field experiment confirmed that partial predation by corallivorous fishes is an important, but habitat-modulated, constraint for branching corals. Competition with vermetid gastropods reduced growth of both branching species but unexpectedly also provided an associational defense against corallivory. Overall, the impact of abiotic constraints was habitat-specific and similar for Acropora and Pocillopora, but biotic interactions, especially corallivory, had a greater negative effect on Acropora than Pocillopora, which may explain the local shift in coral community composition.
Natural killer T cell antigen receptors (NKT TCRs) recognize lipid-based antigens (Ags) presented by CD1d. Although the TCR ?-chain is invariant, NKT TCR V? exhibits greater diversity, with one (V?11) and three (V?8, V?7, and V?2) V? chains in humans and mice, respectively. With the exception of the V?2 NKT TCR, NKT TCRs possess canonical tyrosine residues within complementarity determining region (CDR) 2? that are critical for CD1d binding. Thus, how V?2 NKT TCR docks with CD1d-Ag was unclear. Despite the absence of the CDR2?-encoded tyrosine residues, we show that the V?2 NKT TCR engaged CD1d-Ag in a similar manner and with a comparable affinity and energetic footprint to the manner observed for the V?8.2 and V?7 NKT TCRs. Accordingly, the germline-encoded regions of the TCR ?-chain do not exclusively dictate the innate NKT TCR-CD1d-Ag docking mode. Nevertheless, clear fine specificity differences for the CD1d-Ag existed between the V?2 NKT TCR and the V?8.2 and V?7 NKT TCRs, with the V?2 NKT TCR exhibiting greater sensitivity to modifications to the glycolipid Ag. Furthermore, within the V?2 NKT TCR-CD1d-?GalCer complex, the CDR2? loop mediated fewer contacts with CD1d, whereas the CDR1? and CDR3? loops contacted CD1d to a much greater extent compared with most V?11, V?8.2, and V?7 NKT TCRs. Accordingly, there is a greater interplay between the germline- and nongermline-encoded loops within the TCR ?-chain of the V?2 NKT TCR that enables CD1d-Ag ligation.
Murine cerebral malaria is a complex disease caused by Plasmodium berghei ANKA infection. Several cell types, including CD8(+) T cells, are essential effectors of disease. Although the use of transgenic parasites expressing model antigens has revealed the induction of cytotoxic T lymphocytes (CTL) specific for these model antigens, there is no direct evidence for a response to authentic blood-stage parasite antigens, nor any knowledge of its magnitude. Our studies show that there is a dramatic primary parasite-specific CTL response, akin to viral immunity, reaching approximately 30% of splenic CD8(+) T cells, with many producing interferon-? and tumor necrosis factor-?. These cells express granzyme B and other markers of specific responders, are cytolytic, and respond to a broad array of major histocompatibility complex (MHC) I-restricted epitopes, 5 of which are identified here. Our studies indicate that vigorous CTL responses can be induced to pathogens even when they largely reside in red blood cells, which lack MHC I processing machinery.
Neutrophil activation, whilst a key component of host defence, must be tightly regulated in order to avoid an inappropriate cellular response. Annexin-1, which is present in large amounts in neutrophils, and its N-terminal peptides, reduce neutrophil accumulation but annexin peptides have also been shown to exhibit neutrophil activating properties. We have recently shown annexin-1 to be present in equine neutrophils and demonstrated that the annexin-1-derived peptide, Ac2-26, can both reduce superoxide production by these cells in response to other stimuli and directly induce free radical production at a higher concentration. In the present study, we have further characterised the effects of Ac2-26 on equine neutrophil function. In addition, as anti-inflammatory glucocorticoids are known to up-regulate annexin-1, we have examined the effects of dexamethasone on annexin-1 expression in equine leukocytes. The effects of Ac2-26 alone and on agonist (CXCL8, leukotriene (LT)B(4) and PAF)-induced adherence and migration were examined by measuring adhesion of neutrophils to serum-coated plastic and by use of a ChemoTx migration assay. The role of formyl peptide receptors (FPRs) in mediating the effects of Ac2-26 was examined using the pan-FPR antagonist, BOC-2. Flow cytometry was used to measure the effects of dexamethasone on annexin-1 expression. Pre-incubation with Ac2-26 (10(-5)M) significantly inhibited neutrophil adhesion and migration in response to other agonists but when used alone could also induce these responses. The stimulatory and inhibitory effects of Ac2-26 were reduced by BOC-2, indicating a dependency on FPR activation. Dexamethasone increased the percentage of annexin-1 positive neutrophils and mononuclear cells by 1h post treatment (from 45±5% to 93±1% and 62±14% to 87±9% for neutrophils and monocytes, respectively) but by 4h there was no difference from control cells. No difference was seen between the percentages of annexin-1 positive cells pre- and post-treatment in animals that had undergone a dexamethasone suppression test. The attenuation of agonist-induced adherence and migration by Ac2-26 may play a part in regulating recruitment of equine neutrophils in inflammatory conditions of the horse. However, if high concentrations are produced in vivo following release of annexin-1 from activated cells, direct stimulatory effects may occur which could be either beneficial or detrimental. The therapeutic efficacy of anti-inflammatory steroids in the horse may be mediated in part by increasing annexin-1 expression although this effect appears to be short-lived.
The growth hormone receptor (GHR) was the first class 1 cytokine receptor to be cloned, and it has been studied intensively. The crystal structures of the bound and unbound forms have been solved and the energetic contributions of residues involved in the binding interaction have been quantified. Two receptor subunits bind to opposite sides of the hormone through site 1 and site 2, and a third interaction occurs between receptors in the lower ?-sandwich module at site 3. All three interactions are required for receptor activation, which was thought to be a consequence of hormone-induced receptor dimerization. However, substantial data support the existence of a constitutive receptor dimer that interacts via the transmembrane domain (TMD), with receptor activation triggered by a hormone-induced conformational change. Mutagenesis studies and crystal structure data indicate that receptor activation involves a relative rotation and scissor movement of subunits to activate the associated tyrosine kinase, Janus kinase 2 (JAK2). We have recently reported that a second tyrosine kinase, an Src-family kinase, also associates constitutively with the receptor and activates the Ras-extracellular-signal-regulated kinase pathway. Activation of this kinase requires a conformational change in a loop of the lower sandwich module, and a different orientation of the TMDs than needed for JAK2 activation.
Oligosaccharides on the hemagglutinin (HA) and neuraminidase of influenza A virus (IAV) are a target for recognition by lectins of the innate immune system, including soluble surfactant protein-D and the macrophage mannose receptor on airway macrophages. Glycans attached to the head of H1 subtype of IAV differ markedly in number and location. A reverse genetic approach was used to define the importance of particular N-glycosylation sites on H1 in determining sensitivity to innate immune defenses and virulence in mice. The HA of A/PR/8/34 (PR8, H1N1) and A/Brazil/11/78 (Brazil, H1N1) express zero and four glycosylation sites on the head of HA, respectively. Site-directed mutagenesis was used to add (PR8) or delete (Brazil) glycosylation sites, and IAV expressing wild-type or mutant HA were generated on a PR8 backbone. Addition or removal of particular glycans modulated sensitivity to mouse lung fluids but was not a major factor determining susceptibility of airway macrophages to infection. PR8 is a mouse-adapted virus, and mutations in multiple IAV genes have been shown to contribute to virulence, yet addition of glycosylation to PR8 HA was sufficient to attenuate disease. In contrast, removal of glycans from Brazil HA resulted in severe disease and death. These studies provide insight regarding the mechanisms by which IAV can induce disease in mice. Moreover, reduced glycosylation of HA is likely to be an important factor associated with adaptation of human IAV to growth in mouse lung.
Infections localized to peripheral tissues such as the skin result in the priming of T-cell responses that act to control pathogens. Activated T cells undergo migrational imprinting within the draining lymph nodes, resulting in memory T cells that provide local and systemic protection. Combinations of migrating and resident memory T cells have been implicated in long-term peripheral immunity, especially at the surfaces that form pathogen entry points into the body. However, T-cell immunity consists of separate CD4(+) helper T cells and CD8(+) killer T cells, with distinct effector and memory programming requirements. Whether these subsets also differ in their ability to form a migrating pool involved in peripheral immunosurveillance or a separate resident population responsible for local infection control has not been explored. Here, using mice, we show key differences in the migration and tissue localization of memory CD4(+) and CD8(+) T cells following infection of the skin by herpes simplex virus. On resolution of infection, the skin contained two distinct virus-specific memory subsets; a slow-moving population of sequestered CD8(+) T cells that were resident in the epidermis and confined largely to the original site of infection, and a dynamic population of CD4(+) T cells that trafficked rapidly through the dermis as part of a wider recirculation pattern. Unique homing-molecule expression by recirculating CD4(+) T effector-memory cells mirrored their preferential skin-migratory capacity. Overall, these results identify a complexity in memory T-cell migration, illuminating previously unappreciated differences between the CD4(+) and CD8(+) subsets.
Coral reefs world-wide are threatened by escalating local and global impacts, and some impacted reefs have shifted from coral dominance to a state dominated by macroalgae. Therefore, there is a growing need to understand the processes that affect the capacity of these ecosystems to return to coral dominance following disturbances, including those that prevent the establishment of persistent stands of macroalgae. Unlike many reefs in the Caribbean, over the last several decades, reefs around the Indo-Pacific island of Moorea, French Polynesia have consistently returned to coral dominance following major perturbations without shifting to a macroalgae-dominated state. Here, we present evidence of a rapid increase in populations of herbivorous fishes following the most recent perturbation, and show that grazing by these herbivores has prevented the establishment of macroalgae following near complete loss of coral on offshore reefs. Importantly, we found the positive response of herbivorous fishes to increased benthic primary productivity associated with coral loss was driven largely by parrotfishes that initially recruit to stable nursery habitat within the lagoons before moving to offshore reefs later in life. These results underscore the importance of connectivity between the lagoon and offshore reefs for preventing the establishment of macroalgae following disturbances, and indicate that protecting nearshore nursery habitat of herbivorous fishes is critical for maintaining reef resilience.
Members of the killer cell immunoglobulin-like receptor (KIR) family, a large group of polymorphic receptors expressed on natural killer (NK) cells, recognize particular peptide-laden human leukocyte antigen (pHLA) class I molecules and have a pivotal role in innate immune responses. Allelic variation and extensive polymorphism within the three-domain KIR family (KIR3D, domains D0-D1-D2) affects pHLA binding specificity and is linked to the control of viral replication and the treatment outcome of certain haematological malignancies. Here we describe the structure of a human KIR3DL1 receptor bound to HLA-B*5701 complexed with a self-peptide. KIR3DL1 clamped around the carboxy-terminal end of the HLA-B*5701 antigen-binding cleft, resulting in two discontinuous footprints on the pHLA. First, the D0 domain, a distinguishing feature of the KIR3D family, extended towards ?2-microglobulin and abutted a region of the HLA molecule with limited polymorphism, thereby acting as an innate HLA sensor domain. Second, whereas the D2-HLA-B*5701 interface exhibited a high degree of complementarity, the D1-pHLA-B*5701 contacts were suboptimal and accommodated a degree of sequence variation both within the peptide and the polymorphic region of the HLA molecule. Although the two-domain KIR (KIR2D) and KIR3DL1 docked similarly onto HLA-C and HLA-B respectively, the corresponding D1-mediated interactions differed markedly, thereby providing insight into the specificity of KIR3DL1 for discrete HLA-A and HLA-B allotypes. Collectively, in association with extensive mutagenesis studies at the KIR3DL1-pHLA-B*5701 interface, we provide a framework for understanding the intricate interplay between peptide variability, KIR3D and HLA polymorphism in determining the specificity requirements of this essential innate interaction that is conserved across primate species.
The disposition effect is a phenomenon in which investors hold onto losing assets longer than they hold onto gaining assets. In this study, we used functional magnetic resonance imaging (fMRI) to measure the response of valuation regions in the brain during the decision to keep or to sell an asset that followed a random walk in price. The most common explanation for the disposition effect is preference-based: namely, that people are risk-averse over gains and risk-seeking over losses. This explanation would predict correlations between individuals risk-preferences, the magnitude of their disposition effect, and activation in valuation structures of the brain. We did not observe these correlations. Nor did we find evidence for a realization utility explanation, which would predict differential responses in valuation regions during the decision to sell versus keep an asset that correlated with the magnitude of the disposition effect. Instead, we found an attenuated ventral striatum response to upticks in value below the purchase price in some individuals with a large disposition effect. Given the role of the striatum in signaling prediction error, the blunted striatal response is consistent with the expectation that an asset will rise when it is below the purchase price, thus spurring loss-holding behavior. This suggests that for some individuals, the disposition effect is likely driven by a belief that the asset will eventually return to the purchase price, also known as mean reversion.
Lead (Pb) exposure has been associated with a host of pathological conditions in humans. In rodents Pb exposure has been shown to alter the hypothalamic-pituitary-adrenal (HPA) axis function.Objective: We investigated the effects of lead on responses of the HPA axis to a psychosocial laboratory stressor administered to Pb-exposed workers.
We have undertaken a genome-wide analysis of rare copy-number variation (CNV) in 1124 autism spectrum disorder (ASD) families, each comprised of a single proband, unaffected parents, and, in most kindreds, an unaffected sibling. We find significant association of ASD with de novo duplications of 7q11.23, where the reciprocal deletion causes Williams-Beuren syndrome, characterized by a highly social personality. We identify rare recurrent de novo CNVs at five additional regions, including 16p13.2 (encompassing genes USP7 and C16orf72) and Cadherin 13, and implement a rigorous approach to evaluating the statistical significance of these observations. Overall, large de novo CNVs, particularly those encompassing multiple genes, confer substantial risks (OR = 5.6; CI = 2.6-12.0, p = 2.4 × 10(-7)). We estimate there are 130-234 ASD-related CNV regions in the human genome and present compelling evidence, based on cumulative data, for association of rare de novo events at 7q11.23, 15q11.2-13.1, 16p11.2, and Neurexin 1.
Neutrophils have an important role in early host protection during influenza A virus infection. Their ability to modulate the virus-specific adaptive immune response is less clear. Here, we have used a mouse model to examine the impact of neutrophils on CD8(+) T-cell responses during influenza virus infection. CD8(+) T-cell priming, expansion, migration, cytokine secretion and cytotoxic capacity were investigated in the virus-infected airways and secondary lymphoid organs. To do this, we utilised a Ly6G-specific monoclonal antibody (mAb; 1A8) that specifically depletes neutrophils in vivo. Neutrophil depletion early after infection with influenza virus strain HKx31 (H3N2) did not alter influenza virus-derived antigen presentation or naïve CD8(+) T-cell expansion in the secondary lymphoid organs. Trafficking of virus-specific CD8(+) T cells into the infected pulmonary airways was also unaltered. Instead, early neutropenia reduced both the overall magnitude of influenza virus-specific CD8(+) T cells, together with impaired cytokine production and cytotoxic effector function. Therefore, neutrophils are important participants in anti-viral mechanisms that sustain effective CD8(+) T-cell responses in the respiratory tract of influenza virus-infected mice.
Influenza A virus (IAV) infection is associated with outcomes ranging from subclinical infection to severe pneumonia. In this study, we compared IAV strains BJx109 (H3N2), HKx31 (H3N2), and PR8 (H1N1), for their ability to elicit innate immune responses from mouse airway cells in vitro and their virulence in mice. The viruses differed markedly in their ability to induce disease in mice (PR8 > HKx31 > BJx109). In particular, PR8 infection was associated with high levels of virus replication and pulmonary inflammation. We next compared the ability of each virus strain to infect and induce inflammatory mediators from mouse airway cells. First, major differences were observed in the ability of viruses to infect and induce chemokines and cytokines from mouse alveolar macrophages (BJx109 > HKx31 > PR8), but not from airway epithelial cells (AEC) in vitro. Second, C-type lectins of the innate immune system in mouse lung fluids blocked the ability of BJx109, but not PR8, to infect mouse macrophages and AEC. The failure of the virulent PR8 virus to elicit responses from airway macrophages, combined with resistance to antiviral proteins in mouse airway fluids, likely contribute to virulence in mice. These findings provide insight into the mechanisms underlying disease severity in the mouse model of influenza infection.
Antiretroviral therapy (ART) has reduced morbidity and mortality in HIV-1 infection; however HIV-1-associated neurocognitive disorders (HAND) persist despite treatment. The reasons for the limited efficacy of ART in the brain are unknown. Here we used functional genomics to determine ART effectiveness in the brain and to identify molecular signatures of HAND under ART. We performed genome-wide microarray analysis using Affymetrix U133 Plus 2.0 Arrays, real-time PCR, and immunohistochemistry in brain tissues from seven treated and eight untreated HAND patients and six uninfected controls. We also determined brain virus burdens by real-time PCR. Treated and untreated HAND brains had distinct gene expression profiles with ART transcriptomes clustering with HIV-1-negative controls. The molecular disease profile of untreated HAND showed dysregulated expression of 1470 genes at p<0.05, with activation of antiviral and immune responses and suppression of synaptic transmission and neurogenesis. The overall brain transcriptome changes in these patients were independent of histological manifestation of HIV-1 encephalitis and brain virus burdens. Depending on treatment compliance, brain transcriptomes from patients on ART had 83% to 93% fewer dysregulated genes and significantly lower dysregulation of biological pathways compared to untreated patients, with particular improvement indicated for nervous system functions. However a core of about 100 genes remained similarly dysregulated in both treated and untreated patient brain tissues. These genes participate in adaptive immune responses, and in interferon, cell cycle, and myelin pathways. Fluctuations of cellular gene expression in the brain correlated in Pearsons formula analysis with plasma but not brain virus burden. Our results define for the first time an aberrant genome-wide brain transcriptome of untreated HAND and they suggest that antiretroviral treatment can be broadly effective in reducing pathophysiological changes in the brain associated with HAND. Aberrantly expressed transcripts common to untreated and treated HAND may contribute to neurocognitive changes defying ART.
Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V(?)14-J(?)18) T cell antigen receptor (TCR) ?-chain and recognition of the glycolipid ?-galactosylceramide (?-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of ?-GalCer-reactive NKT cells that expressed a canonical V(?)10-J(?)50 TCR ?-chain, which showed a preference for ?-glucosylceramide (?-GlcCer) and bacterial ?-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCR? sequence identity, the V(?)10 TCR-CD1d-?-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-?-GalCer complexes, although differences at the antigen-binding interface accounted for the altered antigen specificity. Our findings provide new insight into the structural basis and evolution of glycolipid antigen recognition and have notable implications for the scope and immunological role of glycolipid-specific T cell responses.
Difficulties in scaling up theoretical and experimental results have raised controversy over the consequences of biodiversity loss for the functioning of natural ecosystems. Using a global survey of reef fish assemblages, we show that in contrast to previous theoretical and experimental studies, ecosystem functioning (as measured by standing biomass) scales in a non-saturating manner with biodiversity (as measured by species and functional richness) in this ecosystem. Our field study also shows a significant and negative interaction between human population density and biodiversity on ecosystem functioning (i.e., for the same human density there were larger reductions in standing biomass at more diverse reefs). Human effects were found to be related to fishing, coastal development, and land use stressors, and currently affect over 75% of the worlds coral reefs. Our results indicate that the consequences of biodiversity loss in coral reefs have been considerably underestimated based on existing knowledge and that reef fish assemblages, particularly the most diverse, are greatly vulnerable to the expansion and intensity of anthropogenic stressors in coastal areas.
Neutrophils have been implicated in both protective and pathological responses following influenza virus infections. We have used mAb 1A8 (anti-Ly6G) to specifically deplete LyG6(high) neutrophils and induce neutropenia in mice infected with virus strains known to differ in virulence. Mice were also treated with mAb RB6-8C5 (anti-Ly6C/G or anti-Gr-1), a mAb widely used to investigate the role of neutrophils in mice that has been shown to bind and deplete additional leukocyte subsets. Using mAb 1A8, we confirm the beneficial role of neutrophils in mice infected with virus strains of intermediate (HKx31; H3N2) or high (PR8; H1N1) virulence whereas treatment of mice infected with an avirulent strain (BJx109; H3N2) did not affect disease or virus replication. Treatment of BJx109-infected mice with mAb RB6-8C5 was, however, associated with significant weight loss and enhanced virus replication indicating that other Gr-1(+) cells, not neutrophils, limit disease severity during mild influenza infections.
Natural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t(1/2) life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.
Collectins in airway fluids and membrane-associated lectins such as the macrophage mannose receptor (MMR) recognize mannose-rich glycans on the envelope glycoproteins of influenza A viruses. In this study, we used a reverse genetic approach to examine the role of particular N-linked glycosylation sites on the hemagglutinin (HA) of A/Beijing/353/89 (Beij/89, H3N2) in determining sensitivity to lectin-mediated immune defenses and virulence in mice. We generated 7:1 reassortant viruses on an A/PR/8/34 backbone with Beij/89 HA or HA lacking one or more glycosylation sites. Asn(165) was an important determinant of sensitivity to mouse collectins and virulence but did not alter susceptibility of airway macrophages to infection. Removal of both Asn(165) and Asn(246) led to a further increase in virulence, characterized by enhanced virus replication, pulmonary inflammation and vascular leak. These studies define the importance of particular glycans on H3 HA in determining sensitivity to airway collectins and virulence in mice.
Copy number variations (CNVs) are a major source of alterations among individuals and are a potential risk factor in many diseases. Numerous diseases have been linked to deletions and duplications of these chromosomal segments. Data from genome-wide association studies and other microarrays may be used to identify CNVs by several different computer programs, but the reliability of the results has been questioned.
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