Evaluation of kurtosis-corrected sound exposure level as a metric for predicting onset of hearing threshold shifts in harbor porpoises (Phocoena phocoena).

Application of a kurtosis correction to frequency-weighted sound exposure level (SEL) improved predictions of risk of hearing damage in humans and terrestrial mammals for sound exposures with different degrees of impulsiveness. To assess whether kurtosis corrections may lead to improved predictions for marine mammals, corrections were applied to temporary threshold shift (TTS) growth measurements for harbor porpoises (Phocoena phocoena) exposed to different sounds. Kurtosis-corrected frequency-weighted SEL predicted accurately the growth of low levels of TTS (TTS1-4 < 10 dB) for intermittent sounds with short (1-13 s) silence intervals but was not consistent with frequency-weighted SEL data for continuous sound exposures.

Impulsive sounds change European seabass swimming patterns: Influence of pulse repetition interval.

Seismic shootings and offshore pile-driving are regularly performed, emitting significant amounts of noise that may negatively affect fish behaviour. The pulse repetition interval (PRI) of these impulsive sounds may vary considerably and influence the behavioural impact and recovery. Here, we tested the effect of four PRIs (0.5-4.0s) on European seabass swimming patterns in an outdoor basin. At the onset of the sound exposures, the fish swam faster and dived deeper in tighter shoals. PRI affected the immediate and delayed behavioural changes but not the recovery time. Our study highlights that (1) the behavioural changes of captive European seabass were consistent with previous indoor and outdoor studies; (2) PRI could influence behavioural impact differentially, which may have management implications; (3) some acoustic metrics, e.g. SELcum, may have limited predictive power to assess the strength of behavioural impacts of noise. Noise impact assessments need to consider the contribution of sound temporal structure.

Interleukin-23 is sufficient to induce rapid de novo gut tumorigenesis, independent of carcinogens, through activation of innate lymphoid cells.

Chronic inflammation has been associated with increased risk for developing gastrointestinal cancer. Interleukin-23 (IL-23) receptor signaling has been correlated with inflammatory bowel disease pathogenesis, as well as promotion of tumor growth. However, little is known about the relative potential for IL-23-directed causality in gut tumorigenesis. We report that IL-23 transgene expression was sufficient to induce rapid (3-4 weeks) de novo development of intestinal adenomas with 100% incidence. Initiation of tumorigenesis was independent of exogenous carcinogens, Helicobacter colonization, or pre-existing tumor-suppressor gene mutations. Tumorigenesis was mediated by Thy1(+)IL-23R(+) innate lymphoid cells (ILC3), in part, through IL-17 responses as tumor development was inhibited in RAG(-/-) × IL-17(-/-) double knockout mice. Remarkably, IL-23 initiation of tumorigenesis by resident ILCs consistently occurred before recruitment of conspicuous inflammatory infiltrates. Our results reveal an explicit role for IL-23-mediated initiation of gut tumorigenesis and implicate a key role for IL-23R(+) ILC3 in the absence of overt cellular infiltrate recruitment.

Expression of interleukin-27 by human trophoblast cells.

Cytokines produced at the fetal-maternal interface play a key role in regulating maternal tolerance to the fetus and successful pregnancy. Previously, we showed that EBV-induced gene 3 (EBI3), an interleukin (IL)-12 p40 homologue, was expressed at very high levels by syncytiotrophoblasts and extravillous trophoblasts throughout human pregnancy. EBI3 was recently shown to associate with a novel ligand, p28, to form a new heterodimeric cytokine with important immunoregulatory functions, IL-27. In this study, we investigated whether EBI3 expression by trophoblast cells is associated with that of p28 to form IL-27. We found that genes encoding IL-27 (EBI3 and p28) and its receptor (IL-27R and gp130) were expressed in the placenta at various stages of pregnancy. Co-immunoprecipitation experiments performed from placental lysates, and ELISA of culture supernatants from placental explants, showed that IL-27 heterodimer was produced and released from placental cells. In situ studies of placentae of first, second and third trimester of pregnancy, and of choriocarcinomas, demonstrated that syncytiotrophoblast cells co-expressed EBI3 and p28. Similarly, extravillous trophoblast cells invading the decidua were found to co-express both subunits of IL-27. These data suggest that IL-27 may be part of the cytokine network regulating local immune responses and angiogenesis during human pregnancy.

Variable expression of Epstein-Barr virus-induced gene 3 during normal B-cell differentiation and among B-cell lymphomas.

Epstein-Barr virus (EBV)-induced gene 3 (EBI3) is expressed by tumour cells in several EBV-associated malignancies. EBI3 was recently found to associate with a novel peptide, p28, to form a new heterodimeric cytokine, called interleukin-27. In this study, we investigated EBI3 and p28 expression in normal human B lymphocytes and in non-EBV-associated B-cell lymphomas. Low levels of EBI3 were detected in purified tonsillar B cells and expression was upregulated upon anti-CD40 or anti-micro stimulation via NF-kappaB activation. In non-neoplastic tissues, EBI3 expression by lymphocytes was largely restricted to a subset of germinal centre (GC) B cells located at the margin of the light zone, in close contact with CD3+ T lymphocytes. Over 50% of EBI3+ GC B cells were engaged in cell proliferation as assessed by Ki67 expression, and 10-30% expressed MUM1, an early marker of plasma cell differentiation expressed by late centrocytes. Many EBI3+ GC B cells had downregulated bcl-6 expression, which further suggests that these cells correspond to late CD40-activated centrocytes. Immunohistochemical analysis of 64 B-cell lymphomas showed that the highest EBI3 levels were detected in follicular lymphomas and in diffuse large B-cell lymphomas of both GC B-cell-like or non-GC B-cell-like types. No or rare p28 expression was detected in normal or tumour B cells. This constitutive expression of EBI3 by neoplastic B cells may be involved in lymphomagenesis, and may be a useful marker for lymphoma diagnosis.

Differences in the response of a striped dolphin (Stenella coeruleoalba) and a harbour porpoise (Phocoena phocoena) to an acoustic alarm.

Small cetacean bycatch in gillnet fisheries may be reduced by deterring odontocetes from nets acoustically. However, different odontocete species may respond differently to acoustic signals from alarms. Therefore, in this study a striped dolphin and a harbour porpoise were subjected simultaneously to sounds produced by the XP-10 experimental acoustic alarm. The alarm produced 0.3s tonal signals randomly selected from a set of 16 with fundamental frequencies between 9 and 15kHz, with a constant pulse interval of 4.0s (duty cycle 8%) and a Source Level range of 133-163dB re 1muPa (rms). The effect of the alarm was judged by comparing the animals' respiration rate and position relative to the alarm during test periods with those during baseline periods. As in a previous study on two porpoises with the same alarm, the porpoise in the present study reacted strongly to the alarm by swimming away from it and increasing his respiration rate. The striped dolphin, however, showed no reaction to the active alarm. Based on harbour porpoise audiograms and the specific audiogram of the striped dolphin in the present study, and the low background noise levels during the experiment, both animals must have heard the alarm signals clearly. This study indicates that cetacean species are not equally sensitive to human-made noise disturbance. Therefore, source levels of acoustic alarms should be adapted to the species they are supposed to deter. In addition, alarms should be tested on each odontocete species for which they are intended to reduce bycatch.

The influence of acoustic emissions for underwater data transmission on the behaviour of harbour porpoises (Phocoena phocoena) in a floating pen.

To prevent grounding of ships and collisions between ships in shallow coastal waters, an underwater data collection and communication network is currently under development: Acoustic Communication network for Monitoring of underwater Environment in coastal areas (ACME). Marine mammals might be affected by ACME sounds since they use sounds of similar frequencies (around 12 kHz) for communication, orientation, and prey location. If marine mammals tend to avoid the vicinity of the transmitters, they may be kept away from ecologically important areas by ACME sounds. One marine mammal species that may be affected in the North Sea is the harbour porpoise. Therefore, as part of an environmental impact assessment program, two captive harbour porpoises were subjected to four sounds, three of which may be used in the underwater acoustic data communication network. The effect of each sound was judged by comparing the animals' positions and respiration rates during a test period with those during a baseline period. Each of the four sounds could be made a deterrent by increasing the amplitude of the sound. The porpoises reacted by swimming away from the sounds and by slightly, but significantly, increasing their respiration rate. From the sound pressure level distribution in the pen, and the distribution of the animals during test sessions, discomfort sound level thresholds were determined for each sound. In combination with information on sound propagation in the areas where the communication system may be deployed, the extent of the 'discomfort zone' can be estimated for several source levels (SLs). The discomfort zone is defined as the area around a sound source that harbour porpoises are expected to avoid. Based on these results, SLs can be selected that have an acceptable effect on harbour porpoises in particular areas. The discomfort zone of a communication sound depends on the selected sound, the selected SL, and the propagation characteristics of the area in which the sound system is operational. In shallow, winding coastal water courses, with sandbanks, etc., the type of habitat in which the ACME sounds will be produced, propagation loss cannot be accurately estimated by using a simple propagation model, but should be measured on site. The SL of the communication system should be adapted to each area (taking into account bounding conditions created by narrow channels, sound propagation variability due to environmental factors, and the importance of an area to the affected species). The discomfort zone should not prevent harbour porpoises from spending sufficient time in ecologically important areas (for instance feeding areas), or routes towards these areas.

Underwater audiogram of a Pacific walrus (Odobenus rosmarus divergens) measured with narrow-band frequency-modulated signals.

The underwater hearing sensitivity of an 18-year-old male Pacific walrus was measured in a pool by using a go/no-go response paradigm and the up-down staircase method. Auditory sensitivity was measured using narrow-band, frequency-modulated signals (1500 ms duration) with center frequencies ranging from 0.125 to 15 kHz. The resulting underwater audiogram (50% detection thresholds) for this individual walrus shows the typical mammalian U-shape. Maximum sensitivity (67 dB re 1 microPa) occurred at 12 kHz. The range of best hearing (10 dB from the maximum sensitivity) was from 1 to 12 kHz. Sensitivity fell gradually below 1 kHz and dropped off sharply above 12 kHz. The animal showed a peculiar insensitivity for 2 kHz signals. His much higher sensitivity for 1.5- and 3-kHz signals indicated that this is a narrow-band phenomenon. Walrus hearing is relatively sensitive to low frequency sound, thus the species is likely to be susceptible to anthropogenic noise. The thresholds found during a small test with four frequencies with signal durations of 300 ms did not differ significantly from those obtained with signal durations of 1500 ms.

Food intake and body measurements of Atlantic bottlenose dolphins (Tursiops truncates) in captivity.

The food consumption (recorded in kg of individual fish species), body length and mass of 11 Atlantic bottlenose dolphins kept first at Windsor Safari Park, UK (1979-1993/1994), and later at Harderwijk Marine Mammal Park, The Netherlands (1993/1994-1995) are reported. This broad-scale, longitudinal study is based on historical data that were originally recorded for short-term husbandry purposes. The chemical composition and caloric value of the diet were variable and were not recorded. The food intake quantities should therefore be viewed as rough weight estimates of what wild conspecifics might eat (depending on their diet). The average annual food consumption of adult males and non-pregnant, non-lactating females was approximately 2000 kg of fish (estimated at 176 x 10(5) kJ). Food consumption showed little increase during gestation, but was 58-97% higher during lactation than during similar periods in non-reproductive years. All six calves began to eat solid food within a year of birth although suckling continued for 14-37 months after birth. The pattern of food intake of mothers and calves varied substantially between suckling periods. No seasonal changes in food consumption were detected, although there were small seasonal changes in water temperature. Births occurred at various times of year, since the timing of mating varied between years. The animals' body length increased rapidly during the first 3 years of life after which the growth rate decreased. Body length reached asymptote at approximately 270 cm. Adults of both sexes weighed around 260 kg. The relationship between standard body length (in cm) and body mass (in kg), although based on a small sample size (n = 16), can be expressed as body mass = 17.261e(0.0156(body length-100)). Animals weighing 155-225 kg consumed between 2 and 4% of their body mass per day.

The influence of three acoustic alarms on the behaviour of harbour porpoises (Phocoena phocoena) in a floating pen.

Harbour porpoise bycatch may be reduced by deterring porpoises from nets acoustically. In this study, two harbour porpoises were subjected to three acoustic alarms. The effect of each alarm was judged by comparing the animals' position and respiration rate during a test period with that during a baseline period. The XP-10 alarm produced 0.3 s tonal signals randomly selected from a set of 16 with fundamental frequencies between 9 and 15 kHz, with a constant pulse interval of 4.8 s (duty cycle 6%). The 2MP alarm produced 0.3 s tonal signals randomly selected from a set of 16 with similar fundamental frequencies but with random pulse intervals of between 2 and 5 s (duty cycle 8%). The frequency spectra and source levels of the 2MP and XP-10 alarms varied depending on the signal selected. The HS20-80 alarm produced a constant, but asymmetrical frequency modulated sinewave between 20 and 80 kHz with total pulse duration of 0.3 s. with random pulse intervals of between 2 and 5 s (duty cycle 4.6%). The porpoises reacted to all three alarms by swimming away from them and by increasing their respiration rate. The XP-10, which on average had the highest source level, had the strongest effect.

Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens.

Toll-like receptors (TLRs) are ancient microbial pattern recognition receptors highly conserved from Drosophila to humans. To investigate if subsets of human dendritic cell precursors (pre-DC), including monocytes (pre-DC1), plasmacytoid DC precursors (pre-DC2), and CD11c(+) immature DCs (imDCs) are developed to recognize different microbes or microbial antigens, we studied their TLR expression and responses to microbial antigens. We demonstrate that whereas monocytes preferentially express TLR 1, 2, 4, 5, and 8, plasmacytoid pre-DC strongly express TLR 7 and 9. In accordance with these TLR expression profiles, monocytes respond to the known microbial ligands for TLR2 (peptidoglycan [PGN], lipoteichoic acid) and TLR4 (lipopolysaccharide), by producing tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. In contrast, plasmacytoid pre-DCs only respond to the microbial TLR9-ligand, CpG-ODNs (oligodeoxynucleotides [ODNs] containing unmethylated CpG motifs), by producing IFN-alpha. CD11c(+) imDCs preferentially express TLR 1, 2, and 3 and respond to TLR 2-ligand PGN by producing large amounts of TNF-alpha, and to viral double-stranded RNA-like molecule poly I:C, by producing IFN-alpha and IL-12. The expression of distinct sets of TLRs and the corresponding difference in reactivity to microbial molecules among subsets of pre-DCs and imDCs support the concept that they have developed through distinct evolutionary pathways to recognize different microbial antigens.

A critical role for interleukin 18 in primary and memory effector responses to Listeria monocytogenes that extends beyond its effects on Interferon gamma production.

The stimulation of interferon (IFN)-gamma by interleukin (IL)-12 has been shown to provide protection from intracellular pathogens such as Listeria monocytogenes. Tumor necrosis factor (TNF) is also a major player in the resolution of Listeria infections and is suggested to have more global effects than can be explained by the induction of IFN-gamma alone. Since IL-18 synergizes with IL-12 to induce IFN-gamma production by natural killer and T helper (Th)1 cells, we determined its role in responses to Listeria. IL-18 appeared to be even more potent than either IL-12 or IFN-gamma for protection against this pathogen and IL-18 enhanced bacterial clearance in the complete absence of IFN-gamma. Indeed IL-18 was comparable to TNF in its ability to resolve the infection and showed a lowered protective capacity in the absence of TNF. Moreover, IL-18 induced macrophages to secrete both TNF and nitric oxide after a Listeria infection. IL-18 was also essential for optimal IFN-gamma production by antigen-specific T cells. Therefore, IL-18 operates via its effects on both the innate immune response, including macrophages, as well as on Th1 cells, to protect against Listeria.

Two novel IL-1 family members, IL-1 delta and IL-1 epsilon, function as an antagonist and agonist of NF-kappa B activation through the orphan IL-1 receptor-related protein 2.

IL-1 is of utmost importance in the host response to immunological challenges. We identified and functionally characterized two novel IL-1 ligands termed IL-1delta and IL-1epsilon. Northern blot analyses show that these IL-1s are highly abundant in embryonic tissue and tissues containing epithelial cells (i.e., skin, lung, and stomach). In extension, quantitative real-time PCR revealed that of human skin-derived cells, only keratinocytes but not fibroblasts, endothelial cells, or melanocytes express IL-1delta and epsilon. Levels of keratinocyte IL-1delta are approximately 10-fold higher than those of IL-1epsilon. In vitro stimulation of keratinocytes with IL-1beta/TNF-alpha significantly up-regulates the expression of IL-1epsilon mRNA, and to a lesser extent of IL-1delta mRNA. In NF-kappaB-luciferase reporter assays, we demonstrated that IL-1delta and epsilon proteins do not initiate a functional response via classical IL-1R pairs, which confer responsiveness to IL-1alpha and beta or IL-18. However, IL-1epsilon activates NF-kappaB through the orphan IL-1R-related protein 2 (IL-1Rrp2), whereas IL-1delta, which shows striking homology to IL-1 receptor antagonist, specifically and potently inhibits this IL-1epsilon response. In lesional psoriasis skin, characterized by chronic cutaneous inflammation, the mRNA expression of both IL-1 ligands as well as IL-1Rrp2 are increased relative to normal healthy skin. In total, IL-1delta and epsilon and IL-1Rrp2 may constitute an independent signaling system, analogous to IL-1alphabeta/receptor agonist and IL-1R1, that is present in epithelial barriers of our body and takes part in local inflammatory responses.

Human thymic stromal lymphopoietin preferentially stimulates myeloid cells.

The sequence of a novel hemopoietic cytokine was discovered in a computational screen of genomic databases, and its homology to mouse thymic stromal lymphopoietin (TSLP) suggests that it is the human orthologue. Human TSLP is proposed to signal through a heterodimeric receptor complex that consists of a new member of the hemopoietin family termed human TSLP receptor and the IL-7R alpha-chain. Cells transfected with both receptor subunits proliferated in response to purified, recombinant human TSLP, with induced phosphorylation of Stat3 and Stat5. Human TSLPR and IL-7Ralpha are principally coexpressed on monocytes and dendritic cell populations and to a much lesser extent on various lymphoid cells. In accord, we find that human TSLP functions mainly on myeloid cells; it induces the release of T cell-attracting chemokines from monocytes and, in particular, enhances the maturation of CD11c(+) dendritic cells, as evidenced by the strong induction of the costimulatory molecules CD40 and CD80 and the enhanced capacity to elicit proliferation of naive T cells.

Ubiquitous transgenic expression of the IL-23 subunit p19 induces multiorgan inflammation, runting, infertility, and premature death.

p19, a molecule structurally related to IL-6, G-CSF, and the p35 subunit of IL-12, is a subunit of the recently discovered cytokine IL-23. Here we show that expression of p19 in multiple tissues of transgenic mice induced a striking phenotype characterized by runting, systemic inflammation, infertility, and death before 3 mo of age. Founder animals had infiltrates of lymphocytes and macrophages in skin, lung, liver, pancreas, and the digestive tract and were anemic. The serum concentrations of the proinflammatory cytokines TNF-alpha and IL-1 were elevated, and the number of circulating neutrophils was increased. In addition, ubiquitous expression of p19 resulted in constitutive expression of acute phase proteins in the liver. Surprisingly, liver-specific expression of p19 failed to reproduce any of these abnormalities, suggesting specific requirements for production of biologically active p19. Bone marrow transfer experiments showed that expression of p19 by hemopoietic cells alone recapitulated the phenotype induced by its widespread expression, pointing to hemopoietic cells as the source of biologically active p19. These findings indicate that p19 shares biological properties with IL-6, IL-12, and G-CSF and that cell-specific expression is required for its biological activity.

Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.

A novel sequence discovered in a computational screen appears distantly related to the p35 subunit of IL-12. This factor, which we term p19, shows no biological activity by itself; instead, it combines with the p40 subunit of IL-12 to form a novel, biologically active, composite cytokine, which we term IL-23. Activated dendritic cells secrete detectable levels of this complex. IL-23 binds to IL-12R beta 1 but fails to engage IL-12R beta 2; nonetheless, IL-23 activates Stat4 in PHA blast T cells. IL-23 induces strong proliferation of mouse memory (CD4(+)CD45Rb(low)) T cells, a unique activity of IL-23 as IL-12 has no effect on this cell population. Similar to IL-12, human IL-23 stimulates IFN-gamma production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.

Computational identification, cloning, and characterization of IL-1R9, a novel interleukin-1 receptor-like gene encoded over an unusually large interval of human chromosome Xq22.2-q22.3.

The Interleukin-1 receptor (IL-1R) and Toll signaling pathways share the evolutionarily conserved Toll homology domain (THD), which is a critical component in the signaling cascade of the host defense responses to infection and inflammation. Our initial genomic database searches uncovered a novel THD signature sequence between DNA markers DXS87 and DXS366. The feasibility of subsequently applying a coordinated computational approach, including various exon-finding programs, homology-based searches, and receptor profile searches, in revealing the exons encoding this novel IL-1R family member is described. IL-1R9 shows restricted expression in fetal brain and is highly homologous to IL1RAPL (A. Carrie et al., 1999 Nat. Genet. 23: 25-31), which is reportedly involved in nonsyndromic X-linked mental retardation. These genes are scattered over separate genomic intervals in excess of 1.0 Mb and encode receptors with extended C-terminal tails. In our functional NF-kappaB reporter assays, IL1RAPL, IL-1R9, or versions lacking the extended C-terminal sequences failed in responding either to IL-1 directly or to IL-18 when various permutations of IL-18R ectodomain chimeras were fused to their cytoplasmic domains. Evolutionary sequence analyses reinforce our conclusion that these novel orphan receptors probably form a functionally distinct subset of the IL-1R superfamily.

IL-18 receptors, their role in ligand binding and function: anti-IL-1RAcPL antibody, a potent antagonist of IL-18.

IL-18 is critical in eliciting IFN-gamma production from Th1 cells both in vitro and in vivo. Th1 cells have been implicated in the pathogenesis of autoimmune disorders, making antagonists of IL-18 promising therapeutics. However, specificity and binding characteristics of IL-18R components have only been superficially explored. In this study, we show that IL-1R related protein 1 (IL-1Rrp1) and IL-1R accessory protein-like (IL-1RAcPL) confer responsiveness to IL-18 in a highly specific (no response to other IL-1 ligands) and unique manner (no functional pairing with other IL-1Rs and IL-1R-like molecules). Cotransfection with both receptor components resulted in expression of both low and high affinity binding sites for IL-18 (K:(d) of 11 and 0.4 nM, respectively). We prepared anti-IL-1RAcPL mAb TC30-28E3, which, in contrast to soluble R proteins, effectively inhibited the IL-18-induced activation of NF-kappaB. Quantitative PCR showed that Th1 but not Th2 cells are unique in that they coexpress IL-1Rrp1 and IL-1RAcPL. mAb TC30-28E3 inhibited IL-18-induced production of IFN-gamma by Th1 cells, being at least 10-fold more potent than anti-IL-18 ligand mAb. This study shows that IL-1RAcPL is highly specific to IL-18, is required for high affinity binding of IL-18, and that the anti-IL-1RAcPL mAb TC30-28E3 potently antagonizes IL-18 responses in vitro, providing a rationale for the use of anti-IL-1RAcPL Abs to inhibit Th1-mediated inflammatory pathologies.