Search

Leukocyte immune-type receptors (LITRs) belong to a large family of teleost immunoregulatory receptors that share phylogenetic and syntenic relationships with mammalian Fc receptor-like molecules (FCRLs). Recently, several putative stimulatory Carassius auratus (Ca)-LITR transcripts, including CaLITR3, have been identified in goldfish. CaLITR3 has four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane domain containing a positively charged histidine residue, and a short cytoplasmic tail region. Additionally, the calitr3 transcript is highly expressed by goldfish primary kidney neutrophils (PKNs) and macrophages (PKMs). To further investigate the immunoregulatory potential of CaLITR3 in goldfish myeloid cells, we developed and characterized a CaLITR3-epitope-specific polyclonal antibody (anti-CaL3.D1 pAb). We show that the anti-CaL3.D1 pAb stains various hematopoietic cell types within the goldfish kidney, as well as in PKNs and PKMs. Moreover, cross-linking of the anti-CaL3.D1-pAb on PKN membranes induces phosphorylation of p38 and ERK1/2, critical components of the MAPK pathway involved in controlling a wide variety of innate immune effector responses such as NETosis, respiratory burst, and cytokine release. These findings support the stimulatory potential of CaLITR3 proteins as activators of fish granulocytes and pave the way for a more in-depth examination of the immunoregulatory functions of CaLITRs in goldfish myeloid cells., (Copyright © 2024. Published by Elsevier Ltd.)

Leukocyte immune-type receptors (LITRs) represent a polymorphic and polygenic family of immunoregulatory proteins originally discovered in channel catfish (Ictalurus punctatus; IpLITRs). Belonging to the immunoglobulin superfamily (IgSF), IpLITRs are generally classified as stimulatory or inhibitory types based on their utilization of various intracellular tyrosine-based signaling motifs. While research has shown that IpLITRs can activate as well as abrogate different immune cell effector responses including phagocytosis, recent identification of LITRs within the zebrafish genome (Danio rerio; DrLITRs) revealed the existence of fish LITR-types uniquely containing counteracting stimulatory and inhibitory cytoplasmic tail (CYT) region motifs (i.e., an immunoreceptor tyrosine-based activation motif; ITAM, and immunoreceptor tyrosine-based inhibitory motif; ITIM) within the same receptor. This arrangement is unusual as these motifs typically exist on separate stimulatory (i.e., ITAM-containing) or inhibitory (i.e., ITIM-containing) immunoregulatory receptors that then co-engage to fine-tune cellular signaling and effector responses. Using a flow cytometric-based phagocytosis assay, we show here that engagement of DrLITR 1.2-expressing cells with antibody coated 4.5 μm beads causes a robust ITAM-dependent phagocytic response and reveal that its tandem ITIM motif surprisingly enhances the DrLITR 1.2-induced phagocytic activity while simultaneously decreasing the receptors ability to bind the beads. Confocal microscopy studies also revealed that the ITIM-associated inhibitory signaling molecule SHP-2 is localized to the phagocytic synapse during the phagocytic response. Overall, these results provide the first functional characterization of teleost immune receptors containing a tandem ITAM and ITIM and allow for the proposal of an intracytoplasmic tail signaling model for ITIM-mediated enhancement of ITAM-dependent cellular activation., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Oil sands process affected water (OSPW) is produced during the surface mining of the oil sands bitumen deposits in Northern Alberta. OSPW contains variable quantities of organic and inorganic components causing toxic effects on living organisms. Advanced Oxidation Processes (AOPs) are widely used to degrade toxic organic components from OSPW including naphthenic acids (NAs). However, there is no established biological procedure to assess the effectiveness of the remediation processes. Our previous study showed that human macrophage cells (THP-1) can be used as a bioindicator system to evaluate the effectiveness of OSPW treatments through examining the proinflammatory gene transcription levels. In the present study, we investigated the immunotoxicological changes in THP-1 cells following exposure to untreated and AOP-treated OSPW. Specifically, using proinflammatory cytokine protein secretion assays we showed that AOP treatment significantly abrogates the ability of OSPW to induce the secretion of IL-1β, IL-6, IL-8, TNF-α, IL-1Ra and MCP-1. By measuring transcriptional activity as well as surface protein expression levels, we also showed that two select immune cell surface markers, CD40 and CD54, were significantly elevated following OSPW exposure. However, AOP treatments abolished the immunostimulatory properties of OSPW to enhance the surface expression of these immune proteins. Finally, a transcriptome-based approach was used to examine the proinflammatory effects of OSPW as well as the abrogation of immunotoxicity following AOP treatments. Overall, this research shows how a human macrophage cell-based biomonitoring system serves as an effective in vitro tool to study the immunotoxicity of OSPW samples before and after targeted remediation strategies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Leukocyte immune-type receptors (LITRs) are a large family of teleost immunoregulatory receptor-types belonging to the immunoglobulin superfamily. These immune genes are phylogenetically and syntenically related to Fc receptor-like protein genes (fcrls) present in other vertebrates, including amphibians, birds, mice, and man. In vitro-based functional analyses of LITRs, using transfection approaches, have shown that LITRs have diverse immunoregulatory potentials including the activation and inhibition of several innate immune effector responses such as cell-mediated killing responses, degranulation, cytokine secretion, and phagocytosis. The purpose of this mini review is to provide an overview of fish LITR-mediated immunoregulatory potentials obtained from various teleost model systems, including channel catfish, zebrafish, and goldfish. We will also describe preliminary characterization of a new goldish LITR-specific polyclonal antibody (pAb) and discuss the significance of this tool for further investigation of the functions of fish LITRs., (Copyright © 2023. Published by Elsevier Ltd.)

In this study, we provide evidence that oil sands process-affected waters (OSPW) contain factors that activate the antimicrobial and proinflammatory responses of immune cells. Specifically, using the murine macrophage RAW 264.7 cell line, we establish the bioactivity of two different OSPW samples and their isolated fractions. Here, we directly compared the bioactivity of two pilot scale demonstration pit lake (DPL) water samples, which included expressed water from treated tailings (termed the before water capping sample; BWC) as well as an after water capping (AWC) sample consisting of a mixture of expressed water, precipitation, upland runoff, coagulated OSPW and added freshwater. Significant inflammatory (i.e. macrophage activating) bioactivity was associated with the AWC sample and its organic fraction (OF), whereas the BWC sample had reduced bioactivity that was primarily associated with its inorganic fraction (IF). Overall, these results indicate that at non-toxic exposure doses, the RAW 264.7 cell line serves as an acute, sensitive and reliable biosensor for the screening of inflammatory constituents within and among discrete OSPW samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2022. Published by Elsevier B.V.)

The oil sands in Northern Alberta are the largest oil sands in the world, providing an important economic resource for the Canadian energy industry. The extraction of petroleum in the oil sands begins with the addition of hot water to the bituminous sediment, generating oil sands process-affected water (OSPW), which is acutely toxic to organisms. Trillions of litres of OSPW are stored on oil sands mining leased sites in man-made reservoirs called tailings ponds. As the volume of OSPW increases, concerns arise regarding the reclamation and eventual release of this water back into the environment. OSPW is composed of a complex and heterogeneous mix of components that vary based on factors such as company extraction techniques, age of the water, location, and bitumen ore quality. Therefore, the effective remediation of OSPW requires the consideration of abiotic and biotic constituents within it to understand short and long term effects of treatments used. This review summarizes selected chemicals and organisms in these waters and their interactions to provide a holistic perspective on the physiochemical and microbial dynamics underpinning OSPW ., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Carassius auratus leukocyte immune-type receptors (CaLITRs) were recently discovered immunoregulatory receptors in goldfish that have diverse immunoglobulin-like (Ig-like) ectodomains and intracellular signaling motifs. Genomic analysis shows that CaLITR-types are also located as distinct gene clusters across multiple goldfish chromosomes. For example, CaLITR1 (unplaced) is a functionally ambiguous receptor having two Ig-like domains, a transmembrane domain (TM), and a short cytoplasmic tail (CYT) devoid of any recognizable signaling motifs. CaLITR2 (Chr47) is a putative inhibitory receptor containing four Ig-like domains, a TM, and a long CYT with an immunoreceptor tyrosine-based inhibition motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). A putative activating receptor-type, CaLITR3 (Chr3), has four Ig-like domains, a TM, and a short CYT containing a positively charged histidine residue and CaLITR4 (ChrLG28B) is a receptor with putative multifunctional signaling potential as well as five Ig-like domains, a TM, and a long tyrosine-motif containing CYT region. The variable genomic locations of the CaLITRs suggest that they are likely under the influence of different cis- and/or trans-regulatory elements. To better understand the transcriptional activities of select CaLITRs from variable genomic regions, we used an RT-qPCR-based approach to examine the expression of CaLITR1, CaLITR2, CaLITR3, and CaLITR4 during goldfish primary kidney macrophage (PKM) development and in mixed leukocyte reaction cultures (MLRs) of the goldfish. Our results showed that the select CaLITRs are differentially expressed during PKM development and in goldfish MLRs exposed to T-cell mitogens/immunosuppressive drugs, supporting that the transcription of these CaLITRs is likely regulated by distinct cis- and/or trans-regulatory elements., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Oil sands process water (OSPW) is an industrial process effluent that contains organic compounds such as naphthenic acids (NAs) and polyaromatic hydrocarbons (PAHs), as well as large quantities of inorganic compounds in its mixture. OSPW requires effective treatment for successful reclamation and water reuse. This study investigated the impact of solar-activated zinc oxide (ZnO) photocatalysis on the degradation and removal of NAs and PAHs in OSPW, as well as the elimination of its acute toxicity. With catalyst particles suspended in the effluent (at 1 g/L) under simulated solar radiation of steady irradiance of ~278 W/m 2 , more than 99% removal of NAs was achieved after 4 h of treatment, while nearly all PAHs were simultaneously oxidized within the same reaction time. The photocatalytic treatment appeared to selectively convert classical NAs faster than oxidized NAs. Additionally, NAs with higher double-bond equivalents (DBEs) and higher carbon numbers seemed more susceptible to photocatalytic destruction than others. An overall pseudo first-order rate constant of 1.14 × 10 -2  min -1 , and a fluence-based rate constant of 6.81 × 10 -1  m 2 /MJ were recorded in apparently hydroxyl radicals (OH) and superoxide (O 2 - ) radicals mediated NAs degradation mechanisms. Assessment of the toxicity levels in raw and treated OSPW samples by using Microtox® bioassay indicated that the photocatalytic treatment resulted in ~50% reduction in acute toxicity. Furthermore, we showed that by monitoring the expression levels of key proinflammatory genes using qPCR that treated OSPW significantly reduced the ability of raw OSPW to activate the inflammatory response of immune cells. This indicates that at acute sub-lethal exposure doses, photocatalytic treatment also reduces immunotoxicity. Overall, our results suggest that the ZnO-based photocatalytic degradation of these NAs and PAHs in OSPW could be a significant treatment process aimed at detoxifying OSPW., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Advances in flow cytometry have allowed for innovative functional investigations of innate immune cell responses. Imaging flow cytometers combine the imaging capabilities of microscopy with rapid, high-throughput data acquisition attributes of standard flow cytometers. Here, we describe a detailed method for co-expressing stimulatory and inhibitory immunoregulatory receptor-types in AD293 cells and then measuring receptor cross-talk during the regulation of the phagocytic response. Information on reagent selection, imaging flow cytometry calibration, and automated template analyses are included., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Development of elastin-like polypeptide (ELP) biomaterials is widespread, but information critical for clinical deployment is limited, with biocompatibility studies focused on a narrow cross-section of ELP sequences. Macrophages can impair biomaterial systems by degrading or isolating the biomaterial and by activating additional immune functions. Their phagocytic response will reveal early immune biocompatibility of ELP nanoparticles (NPs). This study examines that response, induced by the adsorbed protein corona, as a function of ELP guest amino acid, chain length and NP diameter. The breadth of proteins adsorbed to ELP NPs varied, with valine-containing ELP NPs adsorbing fewer types of proteins than leucine-containing constructs. Particle diameter was also a factor, with smaller leucine-containing ELP NPs adsorbing the broadest range of proteins. Macrophage viability was unaffected by the ELP NPs, and their phagocytic capabilities were unimpeded except when incubated with a 500 nm valine-containing 40-mer. This NP significantly decreased the phagocytic capacity of macrophages relative to the control and to a corresponding 500 nm leucine-containing 40-mer. NP size and the proportion of opsonin to dysopsonin proteins likely influenced this outcome. These results suggest that certain combinations of ELP sequence and particle size can result in an adsorbed protein corona, which may hinder macrophage function., (© 2021 Wiley Periodicals LLC.)

Leukocytes offer a critical layer of protection to the host following skin infections. Delineating the kinetics of cutaneous leukocyte recruitment as well as their anti-microbial and regulatory profiles is challenging since it requires the isolation of adequate cell numbers and maintenance of their functional properties. Herein, we took advantage of a modified procedure to gain insights into the contributions of fish phagocytes through induction and resolution phases of acute cutaneous inflammation in goldfish ( Carassius auratus ). Our data shows early upregulation of pro-inflammatory cytokines and chemokines, which was paired with neutrophil-dominant leukocyte migration of neutrophils from circulation to the injury site. Recruited neutrophils were associated with high levels of reactive oxygen species (ROS). Following pathogen elimination, a reduction in ROS levels and pro-inflammatory cytokines expression preceded the resolution of inflammation. These results provide a better understanding of the cutaneous immune responses in fish. Moreover, the increased viability and functionality of isolated skin leukocytes opens the door to better understand a range of additional skin diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Soliman, Yoon, Wang, Stafford and Barreda.)

Background: Stress is an important transdiagnostic risk factor in adolescence and predicts a host of physical and psychological problems in adolescence and adulthood. Adolescence is also a developmental stage in which people may be more sensitive or reactive to stress. Indeed, research has shown that adolescents report high levels of stress, particularly when enrolled in school. However, adolescents report engaging in few, if any, stress management techniques. Consequently, the development of effective programs to help address adolescent stress is particularly important. To date, most stress management programs for adolescents are delivered within schools, and the evidence for such programs is mixed. Furthermore, most of these programs rely on traditional stress management techniques rather than incorporating methods to address the underlying negative cognitive processes, such as rumination, that may contribute to or exacerbate the effects of perceived stress., Objective: The aim of this study is to test the short-term effects of a digital mental health program designed for adolescents aged 13-17 years on perceived stress and rumination., Methods: This is a randomized controlled trial in which adolescents between the ages of 13 and 17 years, with elevated levels of perceived stress and brooding, will be randomly assigned to complete 8 weeks of a digital mental health program (Happify for Teens) or to a corresponding wait-list control group. The study will take place over 3 months, including the 8-week intervention period and 1-month postintervention follow-up. The primary outcome, perceived stress, along with secondary and exploratory outcomes (ie, brooding, optimism, sleep disturbance, and loneliness) will be assessed via self-report at baseline, 4 weeks, 8 weeks, and 12 weeks to compare changes in these outcomes across conditions., Results: Recruitment is expected to begin in the second quarter of 2021, with a target sample size of 800 participants (400 per condition). Participants will begin the study as they are recruited and will finish in waves, with the first wave of data expected 8 weeks after recruitment begins and the final wave of data expected by the end of the third quarter of 2021., Conclusions: Although school-based stress management programs for adolescents are common, research suggests that they may be limited in their reach and more effective for school-based stress than other types of stress. This trial will be one of the first attempts to examine the potential benefits of a digital mental health program on adolescents to address stress along with negative cognitive processes such as rumination. If successful, this would help introduce a more scalable alternative to school-based programs that offers adolescents greater privacy while also providing insight into novel ways to target adolescent mental health more generally., Trial Registration: ClinicalTrials.gov NCT04567888; https://clinicaltrials.gov/ct2/show/NCT04567888., International Registered Report Identifier (irrid): PRR1-10.2196/25545., (©Eliane M Boucher, Haley E Ward, Julia L Stafford, Acacia C Parks. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 19.04.2021.)

Background: Loneliness is a growing area of concern, attracting attention as a public health concern due to its association with a variety of psychological and physical health problems. However, interventions targeting loneliness are less common than interventions for other mental health problems, such as depression and anxiety, and existing interventions focus primarily on building social skills and increasing opportunities for social interaction despite research suggesting these techniques are not the most effective. Furthermore, although there is an increasing need for scalable and convenient interventions, digital interventions for loneliness are even less common., Objective: Using a qualitative approach, we explore how adults (18-64 years of age) who express wanting to be more connected to others experience loneliness and react to a digital mental health intervention targeting loneliness., Methods: A total of 11 participants were recruited from a pilot randomized controlled trial exploring the impact of a digital mental health intervention, Happify Health, on loneliness among adults aged 18-64 years who indicated wanting to feel more connected to others when signing up for the platform. Participants were invited to participate in a 3-day asynchronous focus group about their experiences with loneliness, with Happify Health, and with social distancing during the COVID-19 pandemic. All 11 participants completed the focus group in May 2020., Results: Participants' responses were coded using thematic analysis, which led to identifying five themes, each with separate subthemes, that could be applied across the 3-day focus group: loneliness, relationships, social distancing, skill acquisition, and coping. Overall, we observed variability across participants in terms of the source of their loneliness, their perceptions of their social connections, and their motivation to reduce feelings of loneliness; however, participants commonly referred to negative self-perceptions as a cause or consequence of loneliness. Participants also varied in the extent to which they felt social distancing increased or decreased feelings of loneliness. In regard to the intervention, participants showed evidence of adopting skills they used to address their loneliness, particularly mindfulness and gratitude, and then using these skills to shift toward more active coping strategies following the intervention, including during the COVID-19 pandemic., Conclusions: The heterogeneity in participants' experiences with loneliness described during this focus group emphasizes the subjective and complex nature of loneliness. This highlights the importance of developing loneliness interventions that use a variety of strategies, including both direct and indirect strategies for reducing loneliness. However, based on our data, a key component to loneliness interventions is incorporating strategies for addressing underlying negative self-perceptions that stem from, but also contribute to, loneliness. This data also provides preliminary evidence that digital platforms may be an effective tool for disseminating loneliness interventions while providing the added benefit of offering a productive distraction when feeling lonely., (©Eliane M Boucher, Emily C McNaughton, Nicole Harake, Julia L Stafford, Acacia C Parks. Originally published in JMIR Mental Health (http://mental.jmir.org), 08.02.2021.)

Leukocyte immune-type receptors (LITRs) are a large family of immunoregulatory receptor-types originally identified in the channel catfish (Ictalurus punctatus (Ip)LITRs). Phylogenetic analyses of LITRs show that they share distant evolutionary relationships with important mammalian immunoregulatory receptors belonging to the Fc receptors family and the leukocyte receptor complex (LRC), but their syntenic relationships with these immunoglobulin superfamily members have not been investigated. To further examine the possible evolutionary connections between teleost LITRs and various mammalian immunoregulatory receptor-types, we surveyed the genomic databases of representative vertebrate taxa and our results show that teleost LITRs generally exist in large genomic clusters, which are linked to vangl2, arhgef11, and slam family genes, features that are also shared by amphibian and mammalian Fc receptor-like molecules (FCRLs). Moreover, detailed phylogenetic comparisons between the individual Ig-like domains of LITRs and mammalian FCRLs shows that these receptors share related Ig-like domains indicative of their common ancestry. However, contrary to our previous reports, no supportive evidence for phylogenetic relationships between the Ig-like domains of LITRs with the Ig-like domains of LRC-encoded mammalian immunoregulatory receptors was found. We also identified an LRC-like region in the zebrafish genome, but no expanded litr-related genes were located in this region. Similarly, no lilr-related genes were found in spotted gar, a representative basal ray-finned fish. Finally, two distantly related fcrls and an LRC-like gene were identified in the elephant shark genome, suggesting that the loss of an immunoregulatory receptor-containing LRC region may be unique to ray-finned fish.

Nanoparticles (NPs) that are exposed to blood are coated with an assortment of proteins that establish their biological identity by forming the interface between the NP and the cells and tissues of the body. The biological relevance of this protein corona is often overlooked during toxicological assessments of NPs. However, accurate interpretation of biological outcomes following exposure to NPs, including activation of coagulation, opsonization of pathogens, and cellular phagocytosis, must take this adsorbed proteome into account. In this study, we examined protein coronas on the surface of five poly(acrylic acid) (PAA) metal-oxide NPs (TiO 2 , CeO 2 , Fe 2 O 3 , ZnO, and PAA-capsules) following exposure to human plasma for key markers of various host response pathways, including humoral immunity and coagulation. We also evaluated the impacts of pre-exposing serum proteins to PAA-NPs on the opsonization and phagocytosis of bacteria by two immune cell lines. Results demonstrated that each PAA-NP type adsorbed a unique profile of blood proteins and that protein-coated PAA-NPs significantly inhibited human plasma coagulation with PAA-zinc oxide NPs and their associated proteome fully abrogating clotting. Protein-coated PAA-NPs also resulted in a 50% increase in phagocytic activity of RBL-2H3 cells and a 12.5% increase in phagocytic activity in the RAW 264.7 cell line. We also identified numerous structural, coagulation, and immune-activating proteins in the adsorbed protein corona, which resulted in altered biological function. Overall, our findings demonstrate that the formation of protein coronas on the surface of NPs plays an important role in directing the biological outcomes of opsonization, cell phagocytosis, and blood coagulation.

Leukocyte immune-type receptors (LITRs) are a multigene family of teleost immunoregulatory proteins that share structural, phylogenetic, and likely functional relationships with several innate immune receptor proteins in other vertebrates, including mammals. Originally discovered in channel catfish (Ictalurus punctatus), representative IpLITR-types have been shown to regulate diverse innate immune cell effector responses including phagocytosis, degranulation, and cytokine secretion. To date, IpLITRs have been primarily characterized using mammalian cell line expression systems, therefore many unanswered questions remain regarding their actual regulatory roles in fish immunity. In the present study, we report on the preliminary molecular characterization of five goldfish (Carassius auratus) CaLITR-types and the identification of several putative splice variants of these receptors cloned from various goldfish tissues and primary myeloid cell cultures. In general, CaLITR mRNA transcripts were detected in all goldfish tissues tested, and also in primary kidney macrophage and neutrophil cultures. Specifically, CaLITR1 is a functionally ambiguous receptor with no charged amino acids in its transmembrane (TM) segment and is devoid of tyrosine-based signaling motifs in its short cytoplasmic tail (CYT) region. CaLITR2 is a putative activating receptor-type that contains immunotyrosine-based activation motifs (ITAMs) within its long CYT region, and CaLITR3 has a positively charged TM segment, suggesting that it may recruit intracellular stimulatory adaptor signaling molecules. CaLITR4 and CaLITR5 appear to have diverse signaling capabilities since they contain various immunoregulatory signaling motifs within their CYT regions including putative Nck and STAT recruitment motifs as well as ITAM-like and ITIM sequences. We also identified putative CaLITR splice variants with altered extracellular Ig-like domain compositions and variable CYT regions. Interestingly, this suggests that alternative splicing-mediated diversification of CaLITRs can generate receptor forms with possible variable binding and/or intracellular signaling abilities. Overall, these findings reveal new information about the teleost LITRs and sets the stage for exploring how alternative splicing leads to the functional diversification of this complex multigene immunoregulatory receptor family., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Diabetes is a leading cause of years of life lost and accounts for approximately one-fourth of health care dollars spent in the United States. Many of these costs are related to poor medication adherence and lack of self-care behaviors and are thus preventable. Depression, which is more prevalent among people with diabetes than in the general population, predicts poorer management of one's diabetes, whereas positive affect predicts engaging in more positive health behaviors. Consequently, interventions that improve depression and positive affect may also improve diabetes-related outcomes among people with diabetes. Although preliminary research on the impact of such interventions among people with diabetes is promising, these studies focused primarily on in-person interventions, have had small samples, and lack long-term follow-up., Objective: This study aims to examine the short- and long-term effects of a digital therapeutic platform focused on mental health among adults with poorly managed type 2 diabetes and elevated levels of depression., Methods: This is a randomized controlled trial in which adults with a type 2 diabetes diagnosis, elevated hemoglobin A 1c (HbA 1c ) levels (≧7), and moderate to severe depressive symptoms will be randomly assigned to a positive emotion regulation skills intervention group or a sham digital intervention with only psychoeducational content. The study will take place over 14 months, including the 8-week intervention (or control) delivered via a digital therapeutic platform (Happify Health) and follow-up assessments at 3, 6, and 12 months postintervention. Throughout the intervention and for 1 week at each postintervention follow-up, participants will complete daily assessments of diabetes-related distress, diabetes regimen adherence, and mood. Our primary outcome, HbA 1c , will be self-reported every 3 months throughout the study. Secondary and exploratory outcomes will be assessed at baseline; at 8 weeks; and at 3, 6, and 12 months postintervention., Results: Recruitment is expected to begin in June 2020. Participants will begin the study as they are recruited and will finish in waves. The final wave of data collection from the 8-week intervention is expected for winter 2020, with the completion of the 12-month follow-up in winter 2021., Conclusions: Although previous research suggests that in-person psychological interventions have promising effects on both psychological and physical outcomes among adults with diabetes, digital interventions can be advantageous because they are easily scalable and reduce many barriers that prevent people from seeking treatment. This trial will provide important information about the effects of a digital mental health intervention among adults with type 2 diabetes, assessing both short- and long-term effects of this intervention on HbA1c, depressive symptoms, and other diabetes-specific outcomes. If successful, this may introduce a scalable intervention that would help reduce some of the preventable costs associated with diabetes., Trial Registration: ClinicalTrials.gov NCT04068805; https://clinicaltrials.gov/ct2/show/NCT04068805., International Registered Report Identifier (irrid): PRR1-10.2196/18578., (©Eliane Boucher, Judith T Moskowitz, Gina M Kackloudis, Julia L Stafford, Ian Kwok, Acacia C Parks. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 04.08.2020.)

The focus of the present study was to examine the acute immunotoxic properties of oil sands process-affected waters (OSPW) using the RAW 264.7 macrophage cell line. Specifically, we used a quantitative PCR assay to monitor changes in the expression of stress, cytokine, and antimicrobial enzyme genes in activated macrophages following acute (i.e. < 24 h) exposure of the cells to whole OSPW and its fractions. Overall, our data shows that OSPW inorganic fraction (IF) significantly induces the expression of genes associated with oxidative stress and DNA damage and that the OSPW-IF also significantly augmented cytokine gene expression. These effects are similar to what was observed following whole OSPW exposures, which contrasts the minimal effects observed when cells were treated with equivalent doses of the OSPW organic fraction (OF). Surprisingly, OSPW-IF had reciprocal effects on gene and protein expression levels of two key macrophage enzymes (e.g. inducible nitric oxide (iNOS) synthase and arginase), which indicates that components within OSPW-IF have the unique ability to alter the overall functional states of macrophage by polarizing them towards an alternatively activated status; concomitant with the reciprocal depression of iNOS levels and enhanced expression and activity of arginase. Collectively, these findings show that at sub-lethal exposure doses, the inorganic constituents of OSPW have significant immunotoxicological properties that could potentially affect innate cellular defense responses of exposed animals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Channel catfish ( Ictalurus punctatus ) leukocyte immune-type receptors (IpLITRs) are a family of immunoregulatory proteins shown to regulate several innate immune cell effector responses, including phagocytosis. The precise mechanisms of IpLITR-mediated regulation of the phagocytic process are not entirely understood, but we have previously shown that different IpLITR-types use classical as well as novel pathways for controlling immune cell-mediated target engulfment. To date, all functional assessments of IpLITR-mediated regulatory actions have focused on the independent characterization of select IpLITR-types in transfected cells. As members of the immunoglobulin superfamily, many IpLITRs share similar extracellular Ig-like domains, thus it is possible that various IpLITR actions are influenced by cross-talk mechanisms between different IpLITR-types; analogous to the paired innate receptor paradigm in mammals. Here, we describe in detail the co-expression of different IpLITR-types in the human embryonic AD293 cell line and examination of their receptor cross-talk mechanisms during the regulation of the phagocytic response using imaging flow cytometry, confocal microscopy, and immunoprecipitation protocols. Overall, our data provides interesting new insights into the integrated control of phagocytosis via the antagonistic networking of independent IpLITR-types that requires the selective recruitment of inhibitory signaling molecules for the initiation and sustained cross-inhibition of phagocytosis.

Background: Cardiogenic shock (CS) is a state of critical end-organ hypoperfusion due to a primary cardiac disorder. For people with refractory CS despite maximal vasopressors, inotropic support and intra-aortic balloon pump, mortality approaches 100%. Mechanical assist devices provide mechanical circulatory support (MCS) which has the ability to maintain vital organ perfusion, to unload the failing ventricle thus reduce intracardiac filling pressures which reduces pulmonary congestion, myocardial wall stress and myocardial oxygen consumption. This has been hypothesised to allow time for myocardial recovery (bridge to recovery) or allow time to come to a decision as to whether the person is a candidate for a longer-term ventricular assist device (VAD) either as a bridge to heart transplantation or as a destination therapy with a long-term VAD., Objectives: To assess whether mechanical assist devices improve survival in people with acute cardiogenic shock., Search Methods: We searched CENTRAL, MEDLINE (Ovid), Embase (Ovid) and Web of Science Core Collection in November 2019. In addition, we searched three trials registers in August 2019. We scanned reference lists and contacted experts in the field to obtain further information. There were no language restrictions., Selection Criteria: Randomised controlled trials on people with acute CS comparing mechanical assist devices with best current intensive care management, including intra-aortic balloon pump and inotropic support., Data Collection and Analysis: We performed data collection and analysis according to the published protocol. Primary outcomes were survival to discharge, 30 days, 1 year and secondary outcomes included, quality of life, major adverse cardiovascular events (30 days/end of follow-up), dialysis-dependent (30 days/end of follow-up), length of hospital stay and length of intensive care unit stay and major adverse events. We used the five GRADE considerations (study limitations, consistency of effect, imprecision, indirectness, and publication bias) to assess the quality of a body of evidence as it relates to the studies which contribute data to the meta-analyses for the prespecified outcomes Summary statistics for the primary endpoints were risk ratios (RR), hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs)., Main Results: The search identified five studies from 4534 original citations reviewed. Two studies included acute CS of all causes randomised to treatment using TandemHeart percutaneous VAD and three studies included people with CS secondary to acute myocardial infarction who were randomised to Impella CP or best medical management. Meta-analysis was performed only to assess the 30-day survival as there were insufficient data to perform any further meta-analyses. The results from the five studies with 162 participants showed mechanical assist devices may have little or no effect on 30-day survival (RR of 1.01 95% CI 0.76 to 1.35) but the evidence is very uncertain. Complications such as sepsis, thromboembolic phenomena, bleeding and major adverse cardiovascular events were not infrequent in both the MAD and control group across the studies, but these could not be pooled due to inconsistencies in adverse event definitions and reporting. We identified four randomised control trials assessing mechanical assist devices in acute CS that are currently ongoing., Authors' Conclusions: There is no evidence from this review of a benefit from MCS in improving survival for people with acute CS. Further use of the technology, risk stratification and optimising the use protocols have been highlighted as potential reasons for lack of benefit and are being addressed in the current ongoing clinical trials., (Copyright © 2020 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd.)

Repeatable patient positioning is key to minimising the burden on planning radiotherapy treatment. There are very few materials commercially available which are suitable for use in all common imaging and treatment modalities such as magnetic resonance imaging (MRI), X-Ray computed tomography (CT) and radiotherapy. In this article, we present several such materials based on woven natural fibres embedded in a range of different resin materials which are suitable for such applications. By investigating a range of resins and natural fibre materials in combination and evaluating their performance in terms of MRI and X-Ray imaging, we show that a woven cotton material impregnated with a two-part epoxy resin provides a 15% improvement in passage of X-Rays and has no impact on the MRI signal (unlike the 40% MRI signal attenuation from carbon fibre), whilst also retaining a flexural modulus up to 71% of that of carbon fibre. These results demonstrate that natural fibre composites produced using such materials provide desirable properties for use in patient support and positioning devices for multi-modal imaging, without the need to significantly compromise on the strength of the material.

The ability of phagocytes to recognize, immobilize, and engulf extracellular targets are fundamental immune cell processes that allow for the destruction of a variety of microbial intruders. The phagocytic process depends onsignalling events that initiate dynamic changes in the plasma membrane architecture that are required to accommodate the internalization of large particulate targets. To better understand fundamental molecular mechanisms responsible for facilitating phagocytic receptor-mediated regulation of cytoskeletal networks, our research has focused on investigating representative immunoregulatory proteins from the channel catfish (Ictalurus punctatus) leukocyte immune-type receptor family (IpLITRs). Specifically, we have shown that a specific IpLITR-type can regulate the constitutive deployment of filopodial-like structures to actively capture and secure targets to the phagocyte surface, which is followed by F-actin mediated membrane dynamics that are associated with the formation of phagocytic cup-like structures that precede target engulfment. In the present study, we use confocal imaging to examine the recruitment of mediators of the F-actin cytoskeleton during IpLITR-mediated regulation of membrane dynamics. Our results provide novel details regarding the dynamic recruitment of the signaling effectors Nck and Syk during classical as well as atypical IpLITR-induced phagocytic processes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Chronic conditions account for 75% of health care costs, and the impact of chronic illness is expected to grow over time. Although subjective well-being predicts better health outcomes, people with chronic conditions tend to report lower well-being. Improving well-being might mitigate costs associated with chronic illness; however, existing interventions can be difficult to access and draw from a single theoretical approach. Happify, a digital well-being intervention program drawing from multiple theoretical traditions to target well-being, has already been established as an efficacious means of improving well-being in both distressed and nondistressed users., Objective: This study aimed to compare change in well-being over time after using Happify for users with and without a chronic condition., Methods: Data were obtained from Happify users, a publicly available digital well-being program accessible via website or mobile phone app. Users work on tracks addressing a specific issue (eg, conquering negative thoughts) composed of games and activities based on positive psychology, cognitive behavioral therapy, and mindfulness principles. The sample included 821 users receiving at least 6 weeks' exposure to Happify (ranging from 42 to 179 days) who met other inclusion criteria. As part of a baseline questionnaire, respondents reported demographic information (age and gender) and whether they had any of the prespecified chronic conditions: arthritis, diabetes, insomnia, multiple sclerosis, chronic pain, psoriasis, eczema, or some other condition (450 reported a chronic condition, whereas 371 did not). Subjective well-being was assessed with the Happify Scale, a 9-item measure of positive emotionality and life satisfaction. To evaluate changes in well-being over time, a mixed effects linear regression model was fit for subjective well-being, controlling for demographics and platform usage., Results: At baseline, users with a chronic condition had significantly lower subjective well-being (mean 38.34, SD 17.40) than users without a chronic condition (mean 43.65, SD 19.13). However, change trajectories for users with or without a chronic condition were not significantly different; both groups experienced equivalent improvements in well-being. We also found an effect for time from baseline (b=0.071; SE=0.010; P<.01) and number of activities completed (b=0.03; SE=0.009; P<.01), and a 2-way interaction between number of activities completed and time from baseline (b=0.0002; SE=0.00006; P<.01), such that completing more activities and doing so over increasingly longer periods produced improved well-being scores., Conclusions: Data from this study support the conclusion that users with a chronic condition experienced significant improvement over time. Despite reporting lower subjective well-being on the whole, their change trajectory while using Happify was equivalent to those without a chronic condition. Consistent with past research, users who completed more activities over a longer period showed the most improvement. In short, the presence of a chronic condition did not prevent users from showing improved well-being when using Happify., (©Acacia C Parks, Allison L Williams, Gina M Kackloudis, Julia L Stafford, Eliane M Boucher, Ryan D Honomichl. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 10.01.2020.)

Dissolved organic compounds are major contaminants in oil sands process-affected water (OSPW), of which naphthenic acids (NAs) are one of the main persistent toxicants. In the present study, we explore the toxic effects of the organic fraction extracted from OSPW (OSPW-OF) in mice during pregnancy and lactation. Here, we report that acute oral exposure of female Balb/c mice during gestation, and subchronic exposure throughout gestation and lactation to OSPW-OF (containing naturally occurring levels of NAs found in tailings ponds), had negligible effects on their reproductive performance. Specifically, mating behavior, pregnancy success, embryonic implantation, gestation period, litter size, and offspring viability were not affected by OSPW-OF containing up to 55 mg/L NAs. OSPW-OF exposure also did not affect plasma concentrations of pregnancy-associated hormones or pro- and anti-inflammatory cytokines, and it had minimal effects on liver stress gene expression. This study presents the first comprehensive in vivo analysis of mammalian toxicity associated with OSPW-OF exposure. Overall, our results suggest that the risk of acute and subchronic toxicity to mice exposed to OSPW-OF at environmentally relevant concentrations of NAs in contaminated drinking water is likely negligible.

Cells of the innate immune system rapidly detect and eliminate invading microbes using surface-expressed immunoregulatory receptors that translate extracellular binding events into potent effector responses. Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are a family of immunoregulatory proteins that have been shown to regulate several innate immune cell effector responses including the phagocytic process. The mechanisms by which these receptors regulate phagocytosis are not entirely understood but we have previously shown that different IpLITR-types use ITAM-dependent as well as ITAM-independent pathways for controlling target engulfment. The main objective of this study was to develop and use imaging flow cytometry and confocal microscopy-based assays to further examine both F-actin and phosphoinositide dynamics that occur during the different IpLITR-mediated phagocytic pathways. Results show that the ITAM-dependent IpLITR-induced phagocytic response promotes canonical changes in F-actin polymerization and PI(4,5)P 2 redistributions. However, the ITAM-independent IpLITR phagocytic response induced unique patterns of F-actin and PI(4,5)P 2 redistributions, which are likely due to its ability to regulate alternative signaling pathways. Additionally, both IpLITR-induced phagocytic pathways induced target internalization into PI(3)P-enriched phagosomes indicative of a maturing phagosome compartment. Overall, this imaging-based platform can be further applied to monitor the recruitment and distribution of signaling molecules during IpLITR-mediated phagocytic processes and may serve as a useful strategy for functional examinations of other immunoregulatory receptor-types in fish., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Phagocytosis evolved from a fundamental nutrient acquisition mechanism in primitive unicellular amoeboids, into a dynamic and complex component of innate immunity in multicellular organisms. To better understand the cellular mechanisms contributing to phagocytic processes across vertebrates, our research has focused on characterizing the involvement of innate immune proteins originally identified in channel catfish ( Ictalurus punctatus ) called leukocyte immune-type receptors (IpLITRs). These unique teleost proteins share basic structural as well as distant phylogenetic relationships with several immunoregulatory proteins within the mammalian immunoglobulin superfamily. In the present study, we use a combination of live-cell confocal imaging and high-resolution scanning electron microscopy to further examine the classical immunoreceptor tyrosine-based activation motif (ITAM)-dependent phagocytic pathway mediated by the chimeric construct IpLITR 2.6b/IpFcRγ-L and the functionally diverse immunoreceptor tyrosine-based inhibitory motif-containing receptor IpLITR 1.1b. Results demonstrate that IpLITR 1.1b-expressing cells can uniquely generate actin-dense filopodia-like protrusions during the early stages of extracellular target interactions. In addition, we observed that these structures retract after contacting extracellular targets to secure captured microspheres on the cell surface. This activity was often followed by the generation of robust secondary waves of actin polymerization leading to the formation of stabilized phagocytic cups. At depressed temperatures of 27°C, IpLITR 2.6b/IpFcRγ-L-mediated phagocytosis was completely blocked, whereas IpLITR 1.1b-expressing cells continued to generate dynamic actin-dense filopodia at this lower temperature. Overall, these results provide new support for the hypothesis that IpLITR 1.1b, but not IpLITR 2.6b/IpFcRγ-L, directly triggers filopodia formation when expressed in representative myeloid cells. This also offers new information regarding the directed ability of immunoregulatory receptor-types to initiate dynamic membrane structures and provides insights into an alternative ITAM-independent target capture pathway that is functionally distinct from the classical phagocytic pathways.

OSPW is a complex mixture of inorganic and organic substances and its principal toxic components have yet to be fully characterized. Previously, we showed in vitro that the oil sands process-affected water (OSPW) organic fraction (OF) caused a concentration-dependent immunotoxicity in mammals. In the present study we further explore the immunotoxicological properties of OSPW in mammals using a series of in vitro bioassays. Specifically, using the RAW 264.7 mouse macrophage cell line we show that whole OSPW containing naphthenic acid (NA) concentrations ranging from 12 to 18 mg/L, significantly inhibited cell proliferation, reduced cell viability, and was directly cytotoxic, whereas the exposure of cells to equivalent doses of the OSPW-OF had no measurable effects. Whole OSPW exposures also caused morphological changes in RAW 264.7 cells, and at sublethal doses (i.e., 10 mg/L) it induced the early expression of the stress genes hmox1 and gadd45. In addition, at NA concentrations of 10 mg/L, whole OSPW but not the OSPW-OF had significant effects on pro-inflammatory cytokine mRNA levels and cytokine protein secretion activities. Finally, whole OSPW also impaired the ability of RAW 264.7 cells to perform phagocytosis. Overall, we demonstrate that exposure to whole OSPW (at NA doses ranging from 10 to 20 mg/L), but not the OSPW-OF caused both cytotoxic and immunomodulatory changes in mouse macrophages. This suggests that the complex mixture of inorganic and organic components found in whole OSPW are acutely toxic at much lower doses than we previously reported for the OSPW-OF (i.e., 50 mg/L) due to unknown additive and/or synergistic interactions that likely occur between the various components present in whole OSPW.

While up to 25% of ovarian cancer (OVCA) cases are thought to be due to inherited factors, the majority of genetic risk remains unexplained. To address this gap, we sought to identify previously undescribed OVCA risk variants through the whole exome sequencing (WES) and candidate gene analysis of 48 women with ovarian cancer and selected for high risk of genetic inheritance, yet negative for any known pathogenic variants in either BRCA1 or BRCA2. In silico SNP analysis was employed to identify suspect variants followed by validation using Sanger DNA sequencing. We identified five pathogenic variants in our sample, four of which are in two genes featured on current multi-gene panels; (RAD51D, ATM). In addition, we found a pathogenic FANCM variant (R1931*) which has been recently implicated in familial breast cancer risk. Numerous rare and predicted to be damaging variants of unknown significance were detected in genes on current commercial testing panels, most prominently in ATM (n = 6) and PALB2 (n = 5). The BRCA2 variant p.K3326*, resulting in a 93 amino acid truncation, was overrepresented in our sample (odds ratio = 4.95, p = 0.01) and coexisted in the germline of these women with other deleterious variants, suggesting a possible role as a modifier of genetic penetrance. Furthermore, we detected loss of function variants in non-panel genes involved in OVCA relevant pathways; DNA repair and cell cycle control, including CHEK1, TP53I3, REC8, HMMR, RAD52, RAD1, POLK, POLQ, and MCM4. In summary, our study implicates novel risk loci as well as highlights the clinical utility for retesting BRCA1/2 negative OVCA patients by genomic sequencing and analysis of genes in relevant pathways.

Innate immune cell-mediated recognition, capture, and engulfment of large particulate targets such as bacteria is known as phagocytosis. This highly dynamic cellular process involves a series of steps including receptor-mediated target binding, phagocytic cup formation, pseudopod extension, and phagosome closure, which depend on distinct actin polymerization events. Using flow cytometry, precise determination of target locations relative to cell membranes (i.e., surface-bound vs. fully engulfed/internalized) during the phagocytic process is difficult to quantify. Here, we describe the application of new analysis features within the IDEAS® software to distinguish internalized and surface-bound particles on individual cells with a high degree of accuracy and reproducibility. Through the use of connected component masks, the accurate discrimination of surface-bound beads versus those internalized is clearly demonstrated. In addition, we were able to further analyze the ratio of beads that had been surface-bound or internalized within individual cells. This novel method of analyzing the phagocytic process provides more accurate determination of target-cell interactions that will assist in examination of the signalling events that occur during the various stages of phagocytosis. © 2017 International Society for Advancement of Cytometry., (© 2017 International Society for Advancement of Cytometry.)

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) control various innate immune cell effector responses including the phagocytic process. This large immunoregulatory receptor family also consists of multiple receptor-types with variable signaling abilities that is dependent on their inherent or acquired tyrosine-containing cytoplasmic tail (CYT) regions. For example, IpLITR 2.6b associates with the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor molecule IpFcRγ-L, and when expressed in mammalian cells it activates phagocytosis using a similar profile of intracellular signaling mediators that also regulate the prototypical mammalian Fc receptor (FcR) phagocytic pathway. Alternatively, IpLITR 1.1b contains a long tyrosine-containing CYT with multifunctional capabilities including both inhibitory and stimulatory actions. Recently, we demonstrated that IpLITR 1.1b activates a unique phagocytic pathway involving the generation of multiple plasma membrane extensions that rapidly capture extracellular targets and secure them on the cell surface in phagocytic cup-like structures. Occasionally, these captured targets are completely engulfed albeit at a significantly lower rate than what was observed for IpLITR 2.6b. While this novel IpLITR 1.1b phagocytic activity is insensitive to classical blockers of phagocytosis, its distinct target capture and engulfment actions depend on the engagement of the actin polymerization machinery. However, it is not known how this protein translates target recognition into intracellular signaling events during this atypical mode of phagocytosis. Using imaging flow cytometry and GST pulldown assays, the aims of this study were to specifically examine what regions of the IpLITR 1.1b CYT trigger phagocytosis and to establish what profile of intracellular signaling molecules likely participate in its actions. Our results show that in stably transfected AD293 cells, the membrane proximal and distal CYT segments of IpLITR 1.1b independently regulate its phagocytic activities. These CYT regions were also shown to differentially recruit various SH2 domain-containing intracellular mediators, which provides new information about the dynamic immunoregulatory abilities of IpLITR 1.1b. Overall, this work further advances our understanding of how certain immunoregulatory receptor-types link extracellular target binding events to the actin polymerization machinery during a non-classical mode of phagocytosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cocaine- and amphetamine-regulated transcript peptides (CARTp) are neuropeptides that act as neurotransmitters in the brain of vertebrates. The expression of CARTp has been characterized in teleosts, amphibians, and several mammalian species, but comparative data in reptiles and birds are nonexistent. In this study, we show the distribution of immunoreactivity against CART peptides (CARTp-ir) in the brains of two bird species: the pigeon (Columba livia) and zebra finch (Taeniopygia guttata). We found CARTp-ir cells and terminals in the brains of both, but no major differences between the two species. As in mammals, teleost fish, and amphibians, CARTp-ir terminals and cells were abundant in subpallial regions, particularly the striatum and nucleus accumbens. We also found CARTp-ir cells and terminals in the hypothalamus, and a large number of CARTp-ir terminals in the substantia nigra, ventral tegmental area, periaqueductal gray, parabrachial nucleus, and dorsal vagal complex. However, in contrast to other vertebrates, CARTp-ir was not found in the olfactory bulb. In addition there was almost no CARTp-ir in the pallium or the hippocampal formation, and little CARTp-ir in the cerebellum. The conserved expression of CARTp in the subpallium, hypothalamus, and dorsal vagal complex of birds suggests that some of the functions of CARTp, such as regulation of food intake and interactions with the social control network and mesolimbic reward system, are conserved among vertebrates. J. Comp. Neurol. 524:3747-3773, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are immunoregulatory proteins that control innate immune cellular responses. Previously, we demonstrated that two representative IpLITR forms, IpLITR 2.6b and IpLITR 1.1b, engage distinct components of the phagocytic machinery resulting in unique target capture and engulfment phenotypes. IpLITR-induced phagocytic mechanisms were also differentially susceptible to temperature and pharmacological inhibitors of canonical signaling mediators. In the present study, we examined the sensitivity of IpLITR-mediated phagocytosis to the endogenous serine-protease trypsin, a well-known mediator of immunoregulatory receptor functions. Trypsin selectively reduced IpLITR 1.1b cell surface expression and phagocytic activity in a dose-dependent manner. We also observed a significant alteration of the IpLITR 1.1b phagocytic phenotype post-trypsin exposure; whereas, the IpLITR 2.6b-mediated target engulfment phenotype was unchanged. Recovery experiments suggested that trypsin-induced inhibition of IpLITR 1.1b-dependent phagocytosis was reversible and that the re-establishment of phagocytic function was associated with a recovery of receptor surface expression. Cell-surface biotinylation and immunoprecipitation studies demonstrated that IpLITR 1.1b normally exists as a mature (∼70 kDa) protein on the cell surface. However, trypsin treatment reduced expression of the mature receptor and processed IpLITR 1.1b into an ∼60 kDa form. The trypsin-generated and putative immature IpLITR 1.1b form was not present on the cell surface; suggesting that the cleaved receptor may have been internalized, post-processing, by regulated endocytosis. Taken together, these results reveal a unique role for trypsin as a selective modulator of IpLITR-mediated phagocytosis and highlight a conserved role for serine proteases as potent immunomodulatory factors., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Across vertebrates, innate immunity consists of a complex assortment of highly specialized cells capable of unleashing potent effector responses designed to destroy or mitigate foreign pathogens. The execution of various innate cellular behaviors such as phagocytosis, degranulation, or cell-mediated cytotoxicity are functionally indistinguishable when being performed by immune cells isolated from humans or teleost fishes; vertebrates that diverged from one another more than 450 million years ago. This suggests that vital components of the vertebrate innate defense machinery are conserved and investigating such processes in a range of model systems provides an important opportunity to identify fundamental features of vertebrate immunity. One characteristic that is highly conserved across vertebrate systems is that cellular immune responses are dependent on specialized immunoregulatory receptors that sense environmental stimuli and initiate intracellular cascades that can elicit appropriate effector responses. A wide variety of immunoregulatory receptor families have been extensively studied in mammals, and many have been identified as cell- and function-specific regulators of a range of innate responses. Although much less is known in fish, the growing database of genomic information has recently allowed for the identification of several immunoregulatory receptor gene families in teleosts. Many of these putative immunoregulatory receptors have yet to be assigned any specific role(s), and much of what is known has been based solely on structural and/or phylogenetic relationships with mammalian receptor families. As an attempt to address some of these shortcomings, this review will focus on our growing understanding of the functional roles played by specific members of the channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs), which appear to be important regulators of several innate cellular responses via classical as well as unique biochemical signaling networks.

The lack of a reliable mammalian neutrophil in vitro culture system has restricted our ability to examine their precise roles in mycobacterial infections. Previously, we developed the procedures for the isolation and culture of primary kidney-derived neutrophil-like cells from goldfish that are functionally and morphologically similar to mammalian neutrophils. The cultured primary goldfish neutrophils exhibited prolonged viability and functional effector responses. In this study, we demonstrate that when exposed to live or heat-killed Mycobacterium marinum, goldfish neutrophils increased their mRNA levels for several pro-inflammatory cytokines (il-1β1, il-1β2, tnfα-1, tnfα-2) and the cytokine receptors (ifngr1-1, tnfr1, tnfr2). These neutrophils also exhibited chemotaxis toward live mycobacteria, internalized the bacilli, and produced reactive oxygen intermediates (ROI) in response to pathogen exposure. The survival of intracellular mycobacteria was significantly reduced in activated neutrophils, indicating a robust killing response by these teleost granulocytes. We suggest that this goldfish primary neutrophil in vitro model system will provide important information regarding neutrophil-mediated host defense mechanisms against mycobacteria in teleosts as well as in higher vertebrates., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In this study, the effects of rosette nanotube (RNT) exposure on immune cell viability and function were investigated in vitro using the rat basophilic leukemia (RBL)-2H3 cell line. RBL-2H3 viability was decreased in a dose- and time-dependent manner after lysine-functionalized RNT (K-RNT) exposure. In addition, K-RNTs had a significant effect on RBL-2H3 degranulation. When K-RNT exposure was concurrent with IgE sensitization, 50 and 100 mg l(-1) K-RNTs elicited a heightened degranulatory response compared with IgE alone. Exposure to 50 and 100 mg l(-1) K-RNTs also caused degranulation in RBL-2H3 cells not sensitized with IgE (0 ng ml(-1) IgE). Furthermore, in cells preexposed to K-RNTs for 2 h and subsequently washed, sensitized, and stimulated with IgE, a potentiated degranulatory response was observed. Using confocal laser scanning microscopy and a fluorescein isothiocyanate (FITC)-functionalized RNT construct (termed FITC(1)/TBL(19)-RNT), we demonstrated a strong and direct affiliation between RNTs and RBL-2H3 cell membranes. We also demonstrated cellular internalization of RNTs after 2 h of exposure. Together, these data demonstrate that RNTs may affiliate with the cellular membrane of RBL-2H3 cells and can be internalized. These interactions can affect viability and alter the ability of these cells to elicit IgE-FcεR mediated degranulation., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Channel catfish (Ictalurus punctatus) IpLITRs belong to the Ig superfamily and regulate innate immune cell effector responses. This study tested the hypothesis that ITAM-dependent and ITAM-independent phagocytic pathways are engaged by different subtypes of the IpLITR family. When stably expressed in RBL-2H3 cells, the ITAM-containing fusion-construct IpLITR 2.6b/IpFcRγ-L stimulated phagocytic responses that were abrogated at suboptimal incubation temperatures and by pharmacological inhibitors of the classic signaling components of the mammalian FcR-dependent phagocytic pathway. Interestingly, the ITIM-containing receptor IpLITR 1.1b also induced phagocytosis through an actin-dependent mechanism, but this process was insensitive to the pharmacological inhibitors tested and remained functional at temperatures as low as 22°C. The IpLITR 1.1b also displayed a unique target-acquisition phenotype that consisted of complex, membranous protrusions, which captured targets in phagocytic cup-like structures but often failed to completely engulf targets. Taken together, these findings suggest that teleost immunoregulatory receptors that associate with ITAM-containing adaptors can engage conserved components of the phagocytic machinery to engulf extracellular targets akin to the classic FcR-mediated response in mammals. Alternatively, IpLITR 1.1b displays a stalled phagocytic phenotype that is likely dependent on the selective recruitment of the minimal molecular machinery required for target capture but results in incomplete target engulfment. Overall, this study demonstrates that IpLITRs can selectively engage distinct components of the phagocytic process and provides important new information regarding the target acquisition as well as internalization mechanisms involved in controlling phagocytic responses across vertebrates., (© Society for Leukocyte Biology.)

Previous reports have shown that nanoparticles (NPs) can both enhance and suppress immune effector functions; however the mechanisms that dictate these responses are still unclear. Here, the effects of polyacrylic acid (PAA) functionalized metal-oxide NP are investigated on RBL-2H3 (representative mammalian granulocyte-like cell line) cell viability, cellular degranulation, immunoglobulin E (IgE) receptor binding, and cell signaling pathways related to immune function. The increasing development of PAA-NPs as pesticide dispersants and as drug carriers in therapeutics necessitates their investigation for safe production. Using two in vitro experimental approaches, this study demonstrates that pre-exposing RBL-2H3 cells, or IgE antibodies, to PAA-NPs (TiO 2 , CeO 2 , ZnO, Fe 2 O 3 , and PAA-Capsules (NP coating control) over 24 h, significantly decrease the binding capacity of IgE for Fcε receptors, inhibit the phosphorylation of intracellular signaling proteins (e.g., MAPK ERK) that mediate degranulation, and inhibited RBL-2H3 cell degranulation. In addition, and unlike the other NPs tested, PAA-TiO 2 significantly reduced RBL-2H3 viability, in a time (4-24 h) and dose-dependent manner (>50 μg mL -1 ). Together, these data demonstrate that PAA-NPs at sub-lethal doses can interact with cell surface structures, such as receptors, to suppress various stages of the RBL-2H3 degranulatory response to external stimuli, and modify immune cell functions that can impact host-immunity.

Exposure effects from polyacrylic acid (PAA) metal-oxide nanoparticles (TiO2, CeO2, Fe2O3, ZnO) on fish neutrophil viability and effector functions (degranulation, respiratory burst, inflammatory gene expression) were investigated using primary kidney goldfish (Carassius auratus L.) neutrophils as a model. Several studies have reported cytotoxic effects of NPs but there are limited reports on their potential to perturb the innate immune system of aquatic organisms. PAA-TiO2 significantly decreased neutrophil viability in a time and dose-dependent manner at all measured time points (0-48 h) and concentrations (0-200 µg/mL). Maximum viability decreased by (mean ± SEM): 67.1 ± 3.3%, 78.4 ± 4.2% and 74.9 ± 5.0% when exposed to 50, 100 and 200 µg/mL for 48 h, respectively. PAA-ZnO also significantly decreased neutrophil viability but only at 48 h exposures at higher concentrations. Neutrophil degranulation increased by approximately 3% after 30 min and by 8% after 4 h when exposed to sublethal doses (10 µg/mL) of PAA-CeO2 or PAA-Fe2O3. All PAA-NPs induced an increase in neutrophil respiratory burst when exposed to 10 µg/mL for 30 and 60 min, however, PAA-Fe2O3 was the only NP where the response was significant. Lastly, NPs altered the expression of a number of pro-inflammatory and immune genes, where PAA-TiO2 most significantly increased the mRNA levels of pro-inflammatory genes (il-1b, ifng) in neutrophils by 3 and 2.5 times, respectively. Together, these data demonstrate that goldfish neutrophils can be negatively affected from exposures to PAA-coated NPs and are functionally responsive to specific core-material properties at sublethal doses. These changes could perturb the innate response and affect the ability of fish to respond to pathogens.

In goldfish, 2 endogenous GnRH isoforms, GnRH2 and GnRH3, are released at the pituitary and directly stimulate LH and GH release using the same population of GnRH receptors (GnRHRs) but with GnRH-specific transduction mechanisms. Previously, we have shown that phosphoinositide 3-kinases (PI3Ks) mediate GnRH2- and GnRH3-stimulated LH and GH release. Among the 3 classes of PI3Ks, class I PI3Ks are the best characterized and consist of 4 110-kDa catalytic isoforms (p110α, p110β, p110γ, and p110δ). Importantly, p110β and p110γ, but not p110α or p110δ, can be directly activated by the Gβγ heterodimer of Gαβγ protein complexes. In the present study, we examined the expression of class I PI3K isoforms and the effects of selective inhibitors of p110α, p110β, p110γ, and p110δ catalytic activity on basal, as well as acute, GnRH2- and GnRH3-stimulated LH and GH release responses using primary cultures of dispersed goldfish pituitary cells in column perifusion. Results demonstrate that p110γ and p110δ are involved in the control of basal LH and GH release, whereas p110α and p110β only regulate basal LH secretion. However, p110β and p110γ both participated in GnRH3- and GnRH2-stimulated GH release, whereas p110β and p110γ mediated GnRH2- and GnRH3-induced LH release responses, respectively. GnRH2- and GnRH3-stimulated LH release, as well as GnRH3-elicited GH release, also required p110δ. These results constitute the first evidence for the differential involvement of class I PI3K catalytic subunits in GnRH actions, in general, and suggest that GnRH2 and GnRH3 binding to GnRHRs can bias the activation of class I PI3K signaling to mediate hormone release responses in 2 distinct pituitary cell types. The involvement of both class IA and IB PI3Ks implicates Gβγ subunits, as well as other known regulators of class I PI3Ks, as important components of GnRHR-mediated responses that could influence GnRH-selective signaling in other cell types.

Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines., (Published by Elsevier Ltd.)

Goldfish pituitary cells are exposed to two GnRHs, salmon (s)GnRH and chicken (c)GnRH-II. Phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) both participate in acute sGnRH- and cGnRH-II-stimulated LH and GH release. Using goldfish pituitary cells, we examined the relationship between PI3K and PKC in acute LH and GH secretion, and PI3K involvement in chronic hormone release and total LH and GH availability. The PI3K inhibitor LY294002 did not affect PKC agonists-induced LH or GH release, and PKC agonists did not alter PI3K p85 phosphorylation, suggesting PKC activation is not upstream of PI3K in acute hormone release. In 2, 6, 12 and 24h treatments, LY294002 did not affect LH release but stimulated total LH availability at 6h. sGnRH stimulatory actions on LH release and total availability at 12 and 24h, and cGnRH-II effects on these parameters at 6h were inhibited by LY294002. LY294002 enhanced basal GH release at 2 and 6h, but reduced total GH at 12 and 24h. Increased GH release was seen following 6, 12 and 24h of sGnRH, and 2, 6 and 24h of cGnRH-II treatment but total GH availability was only elevated by 24h cGnRH-II treatment. Whereas LY294002 inhibited GH release responses to sGnRH at 12h and cGnRH-II at 6h, it attenuated cGnRH-II-elicited, but not sGnRH-induced, effects on total GH. These results indicate that PI3K differentially modulates long-term basal and GnRH-stimulated hormone release, and total hormone availability, in a time-, cell-type-, and GnRH isoform-selective manner., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Context: Fetal growth involves highly complex molecular pathways. IGF2 is a key paternally expressed growth hormone that is critical for in utero growth in mice. Its role in human fetal growth has remained ambiguous, as it has only been studied in term tissues. Conversely the maternally expressed growth suppressor, PHLDA2, has a significant negative correlation between its term placental expression and birth weight., Objective: The aim of this study is to address the role in early gestation of expression of IGF1, IGF2, their receptors IGF1R and IGF2R, and PHLDA2 on term birth weight., Design: Real-time quantitative PCR was used to investigate mRNA expression of IGF1, IGF2, IGF1R, IGF2R and PHLDA2 in chorionic villus samples (CVS) (n = 260) collected at 11-13 weeks' gestation. Expression was correlated with term birth weight using statistical package R including correction for several confounding factors., Results: Transcript levels of IGF2 and IGF2R revealed a significant positive correlation with birth weight (0.009 and 0.04, respectively). No effect was observed for IGF1, IGF1R or PHLDA2 and birth weight. Critically, small for gestational age (SGA) neonates had significantly lower IGF2 levels than appropriate for gestational age neonates (p = 3.6 × 10(-7))., Interpretation: Our findings show that IGF2 mRNA levels at 12 weeks gestation could provide a useful predictor of future fetal growth to term, potentially predicting SGA babies. SGA babies are known to be at a higher risk for type 2 diabetes. This research reveals an imprinted, parentally driven rheostat for in utero growth.

Immunoregulatory receptors are categorized as stimulatory or inhibitory based on their engagement of unique intracellular signaling networks. These proteins also display functional plasticity, which adds versatility to the control of innate immunity. Here we demonstrate that an inhibitory catfish leukocyte immune-type receptor (IpLITR) also displays stimulatory capabilities in a representative myeloid cell model. Previously, the receptor IpLITR 1.1b was shown to inhibit natural killer cell-mediated cytotoxicity. Here we expressed IpLITR 1.1b in rat basophilic leukemia-2H3 cells and monitored intracellular signaling and functional responses. Although IpLITR 1.1b did not stimulate cytokine secretion, activation of this receptor unexpectedly induced phagocytosis as well as extracellular signal-related kinase 1/2- and protein kinase B (Akt)-dependent signal transduction. This novel IpLITR 1.1b-mediated response was independent of an association with the FcRγ chain and was likely due to phosphotyrosine-dependent adaptors associating with prototypical signaling motifs within the distal region of its cytoplasmic tail. Furthermore, compared to a stimulatory IpLITR, IpLITR 1.1b displayed temporal differences in the induction of intracellular signaling, and IpLITR 1.1b-mediated phagocytosis had reduced sensitivity to EDTA and cytochalasin D. Overall, this is the first demonstration of functional plasticity for teleost LITRs, a process likely important for the fine-tuning of conserved innate defenses., (© 2014 S. Karger AG, Basel.)

The toxicity of needle-(nHA-ND) and rod-shaped (nHA-RD) hydroxyapatite (HA) nanoparticles is evaluated in vitro on catfish B-cells (3B11) and catfish T-cells (28s.3) and in vivo on zebrafish embryos to determine if biological effects are similar to the effects seen in mammalian in vitro systems. Neither nHA-ND nor nHA-RD affect cell viability at concentrations of 10 to 300 μg mL(-1) . However, 30 μg mL(-1) needle-shaped nHA lower metabolic activity of the cells. Axial deformations are seen in zebrafish exposed to 300 μg mL(-1) needle shaped nHA after 120 h. For the first time, nHA is reported to cause zebrafish hatching delay. The lowest concentration (3 μg mL(-1) ) of both types of nHA cause the highest hatching inhibition and needle-shaped nHA exposed zebrafish exhibit the lowest hatch at 72 h post fertilization., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The role of lipid rafts in non-mammalian leukocytes has been scarcely investigated. We performed biochemical and functional analysis of lipid rafts in fish leukocytes. Fish Flotillin-1 and a fish GM1-like molecule (fGM1-L) were found in low density detergent-resistant membranes (LD-DRM) in goldfish macrophages and catfish B lymphocytes, similarly to mammals. The presence of flotillin-1 and fGM1-L in LD-DRM was sensitive to increased detergent concentrations, and cholesterol extraction. Confocal microscopy analysis of flotillin-1 and fGM1-L in fish leukocytes showed a distinctive punctuated staining pattern, suggestive of pre-existing rafts. Confocal microscopy analysis of macrophages showed that the membrane of phagosomes containing serum-opsonized zymosan was enriched in fGM1-L, and zymosan phagocytosis was reduced after cholesterol extraction. The presence of flotillin-1 and fGM1-L in LD-DRM, the microscopic evidence of flotillin-1 and fGM1-L on fish macrophages and B-cells, and the sensitivity of phagocytosis to cholesterol extraction, indicate that lipid rafts are biochemically and functionally similar in leukocytes from fish and mammals., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are immunoregulatory proteins belonging to the immunoglobulin superfamily that likely play an important role in the regulation of teleost immune cell effector responses. IpLITRs are expressed by myeloid and lymphoid subsets and based on their structural features can be classified as either putative stimulatory or inhibitory forms. We have recently demonstrated at the biochemical and functional levels that stimulatory IpLITR-types induced intracellular signaling cascades resulting in immune cell activation. Alternatively, we have shown that putative inhibitory IpLITRs may abrogate immune cell responses by recruiting teleost Src homology 2 (SH2) domain-containing cytoplasmic phosphatases (SHP) to their tyrosine-containing cytoplasmic tails. In the present study, we used vaccinia virus to express recombinant chimeric proteins encoding the extracellular and transmembrane regions of human KIR2DL3 fused with the cytoplasmic tails of two putative inhibitory IpLITRs (i.e. IpLITR1.2a and IpLITR1.1b) in mouse spleen-derived cytotoxic lymphocytes. This approach allowed us to study the specific effects of IpLITR-induced signaling on lymphocyte killing of B cell targets (e.g. 721.221 cells) using a standard chromium release assay. Our results suggest that both IpLITR1.2a and IpLITR1.1b are potent inhibitors of lymphocyte-mediated cellular cytotoxicity. Furthermore, using a catalytically inactive SHP-1 mutant in combination with site-directed mutagenesis and co-immunoprecipitations, we also demonstrate that the IpLITR1.2a-mediated functional inhibitory response is SHP-1-dependent. Alternatively, IpLITR1.1b-mediated inhibition of cellular cytotoxicity is facilitated by both SHP-1-dependent and independent mechanisms, possibly involving the C-terminal Src kinase (Csk). The involvement of this inhibitory kinase requires binding to a tyrosine residue encoded in the unique membrane proximal cytoplasmic tail region of IpLITR1.1b. Overall, this represents the first functional information for inhibitory IpLITR-types and reveals that catfish LITRs engage SHP-dependent and -independent inhibitory signaling pathways to abrogate lymphocyte-mediated killing., (Copyright © 2011 Elsevier Ltd. All rights reserved.)