Publications by authors named "Helen Dooley"

34 Publications

Plasma Proteome Responses in Salmonid Fish Following Immunization.

Front Immunol 2020 8;11:581070. Epub 2020 Oct 8.

Department of Microbiology and Immunology, Institute of Marine and Environmental Technology (IMET), University of Maryland School of Medicine, Baltimore, MD, United States.

Vaccination plays a critical role in the protection of humans and other animals from infectious diseases. However, the same vaccine often confers different protection levels among individuals due to variation in genetics and/or immunological histories. While this represents a well-recognized issue in humans, it has received little attention in fish. Here we address this knowledge gap in a proteomic study of rainbow trout (, Walbaum), using non-lethal repeated blood sampling to establish the plasma protein response of individual fish following immunization. Six trout were immunized with adjuvanted hen egg-white lysozyme (HEL) and peripheral blood sampled at ten time points from day 0 to day 84 post-injection. We confirm that an antigen-specific antibody response to HEL was raised, showing differences in timing and magnitude among individuals. Using label-free liquid chromatography-mass spectrometry, we quantified the abundance of 278 plasma proteins across the timecourse. As part of the analysis, we show that this approach can distinguish many (but not all) duplicated plasma proteins encoded by paralogous genes retained from the salmonid-specific whole genome duplication event. Global variation in the plasma proteome was predominantly explained by individual differences among fish. However, sampling day explained a major component of variation in abundance for a statistically defined subset of 41 proteins, representing 15% of those detected. These proteins clustered into five groups showing distinct temporal responses to HEL immunization at the population level, and include classical immune (e.g. complement system members) and acute phase molecules (e.g. apolipoproteins, haptoglobins), several enzymes and other proteins supporting the immune response, in addition to evolutionarily conserved molecules that are as yet uncharacterized. Overall, this study improves our understanding of the fish plasma proteome, provides valuable marker proteins for different phases of the immune response, and has implications for vaccine development and the design of immune challenge experiments.
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http://dx.doi.org/10.3389/fimmu.2020.581070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7579410PMC
October 2020

The immunoglobulins of cartilaginous fishes.

Dev Comp Immunol 2021 Feb 23;115:103873. Epub 2020 Sep 23.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA; Institute of Marine and Environmental Technology, Baltimore, MD, USA. Electronic address:

Cartilaginous fishes, comprising the chimeras, sharks, skates, and rays, split from the common ancestor with other jawed vertebrates approx. 450 million years ago. Being the oldest extant taxonomic group to possess an immunoglobulin (Ig)-based adaptive immune system, examination of this group has taught us much about the evolution of adaptive immunity, as well as the conserved and taxon-specific characteristics of Igs. Significant progress has been made analyzing sequences from numerous genomic and transcriptomic data sets. These findings have been supported by additional functional studies characterizing the Igs and humoral response of sharks and their relatives. This review will summarize what we have learned about the genomic organization, protein structure, and in vivo function of these Ig isotypes in cartilaginous fishes and highlight the areas where our knowledge is still lacking.
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http://dx.doi.org/10.1016/j.dci.2020.103873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708420PMC
February 2021

Atlantic salmon kidney (ASK) cells are an effective model to characterise interferon (IFN) and IFN-induced gene expression following salmonid alphavirus infection.

Fish Shellfish Immunol 2020 Nov 29;106:792-795. Epub 2020 Aug 29.

Dept Microbiology & Immunology, University of Maryland School of Medicine, Institute of Marine & Environmental Technology, Baltimore, USA. Electronic address:

Salmonid alphavirus (SAV), the causative agent of pancreas disease, is a serious pathogen of farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Given the economic impact of SAV outbreaks, much effort is focussed upon understanding the fish immune response following infection and the exploitation of this knowledge to reduce disease impact. Herein we examine the utility of the long-term Atlantic salmon kidney (ASK) cell line as a tool to study antiviral responses upon infection with SAV. Following infection with SAV subtype 1 (isolate V4640) we examined the kinetics and magnitude of induction of IFNa, IFN-regulatory factor (IRF) genes IRF1, IRF3, and IRF7b, as well as the antiviral effector Mx by RT-qPCR. SAV-1 non-structural protein (nsp1) transcript levels increased continuously over the experimental period, indicating viral replication, but cytopathic effect (CPE) was not observed. All the immune genes studied showed an increase in transcript levels over the 96-h study period following SAV infection, with strongest induction of Mx. Our data confirm that ASK cells are a suitable model to study the virus-associated immune responses of salmonids and may be a useful tool when assaying the effectiveness of potential prophylactic or antiviral treatments.
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http://dx.doi.org/10.1016/j.fsi.2020.08.043DOI Listing
November 2020

Proof of long-term immunological memory in cartilaginous fishes.

Dev Comp Immunol 2020 07 9;108:103674. Epub 2020 Mar 9.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA; Institute of Marine and Environmental Technology, Baltimore, MD, USA. Electronic address:

Immunological memory provides long-term protection against pathogen re-infection and is the foundation for successful vaccination. We have previously shown an antigen-specific recall response in nurse sharks almost one year after primary exposure. Herein, we extend the time between prime and successful recall to >8 years, the longest period for which immunological memory has been shown in any non-mammalian vertebrate. We confirm that antigen binding is mediated by monomeric IgM and IgNAR, but not pentameric IgM, in both the primary and recall phases. Our inability to find target-binding clones in recombinant VNAR expression libraries suggests that, at least in this instance, antigen-specific memory cells comprise a small fraction of the IgNAR-positive B cells in epigonal and spleen. Further, that the few memory cells present can generate a robust antigen-specific IgNAR titer following re-stimulation. Our results continue to challenge the long-held, but erroneous, belief that the shark adaptive immune system is 'primitive' when compared to that of mammals.
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http://dx.doi.org/10.1016/j.dci.2020.103674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7164379PMC
July 2020

Ocular torticollis: A tilt in perspective.

Aust J Gen Pract 2019 08;48(8):520-522

BBioMed, medical student, University of Melbourne, Vic.

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http://dx.doi.org/10.31128/AJGP-08-18-4671DOI Listing
August 2019

Discovery of All Three Types in Cartilaginous Fishes Enables Phylogenetic Resolution of the Origins and Evolution of Interferons.

Front Immunol 2019 12;10:1558. Epub 2019 Jul 12.

School of Biological Sciences, University of Aberdeen, Aberdeen, United Kingdom.

Interferons orchestrate host antiviral responses in jawed vertebrates. They are categorized into three classes; IFN1 and IFN3 are the primary antiviral cytokine lineages, while IFN2 responds to a broader variety of pathogens. The evolutionary relationships within and between these three classes have proven difficult to resolve. Here, we reassess interferon evolution, considering key phylogenetic pitfalls including taxon sampling, alignment quality, model adequacy, and outgroup choice. We reveal that cartilaginous fishes, and hence the jawed vertebrate ancestor, possess(ed) orthologs of all three interferon classes. We show that IFN3 groups sister to IFN1, resolve the origins of the human IFN3 lineages, and find that intronless IFN3s emerged at least three times. IFN2 genes are highly conserved, except for IFN-γ-rel, which we confirm resulted from a teleost-specific duplication. Our analyses show that IFN1 phylogeny is highly sensitive to phylogenetic error. By accounting for this, we describe a new backbone IFN1 phylogeny that implies several IFN1 genes existed in the jawed vertebrate ancestor. One of these is represented by the intronless IFN1s of tetrapods, including mammalian-like repertoires of reptile IFN1s and a subset of amphibian IFN1s, in addition to newly-identified intron-containing shark IFN1 genes. IFN-f, previously only found in teleosts, likely represents another ancestral jawed vertebrate IFN1 family member, suggesting the current classification of fish IFN1s into two groups based on the number of cysteines may need revision. The providence of the remaining fish IFN1s and the coelacanth IFN1s proved difficult to resolve, but they may also be ancestral jawed vertebrate IFN1 lineages. Finally, a large group of amphibian-specific IFN1s falls sister to all other IFN1s and was likely also present in the jawed vertebrate ancestor. Our results verify that intronless IFN1s have evolved multiple times in amphibians and indicate that no one-to-one orthology exists between mammal and reptile IFN1s. Our data also imply that diversification of the multiple IFN1s present in the jawed vertebrate ancestor has occurred through a rapid birth-death process, consistent with functional maintenance over a 450-million-year host-pathogen arms race. In summary, this study reveals a new model of interferon evolution important to our understanding of jawed vertebrate antiviral immunity.
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http://dx.doi.org/10.3389/fimmu.2019.01558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640115PMC
October 2020

Phylotranscriptomics suggests the jawed vertebrate ancestor could generate diverse helper and regulatory T cell subsets.

BMC Evol Biol 2018 11 15;18(1):169. Epub 2018 Nov 15.

School of Biological Sciences, University of Aberdeen, Aberdeen, AB24 2TZ, UK.

Background: The cartilaginous fishes diverged from other jawed vertebrates ~ 450 million years ago (mya). Despite this key evolutionary position, the only high-quality cartilaginous fish genome available is for the elephant shark (Callorhinchus milii), a chimaera whose ancestors split from the elasmobranch lineage ~ 420 mya. Initial analysis of this resource led to proposals that key components of the cartilaginous fish adaptive immune system, most notably their array of T cell subsets, was primitive compared to mammals. This proposal is at odds with the robust, antigen-specific antibody responses reported in elasmobranchs following immunization. To explore this discrepancy, we generated a multi-tissue transcriptome for small-spotted catshark (Scyliorhinus canicula), a tractable elasmobranch model for functional studies. We searched this, and other newly available sequence datasets, for CD4+ T cell subset-defining genes, aiming to confirm the presence or absence of each subset in cartilaginous fishes.

Results: We generated a new transcriptome based on a normalised, multi-tissue RNA pool, aiming to maximise representation of tissue-specific and lowly expressed genes. We utilized multiple transcriptomic datasets and assembly variants in phylogenetic reconstructions to unambiguously identify several T cell subset-specific molecules in cartilaginous fishes for the first time, including interleukins, interleukin receptors, and key transcription factors. Our results reveal the inability of standard phylogenetic reconstruction approaches to capture the site-specific evolutionary processes of fast-evolving immune genes but show that site-heterogeneous mixture models can adequately do so.

Conclusions: Our analyses reveal that cartilaginous fishes are capable of producing a range of CD4+ T cell subsets comparable to that of mammals. Further, that the key molecules required for the differentiation and functioning of these subsets existed in the jawed vertebrate ancestor. Additionally, we highlight the importance of considering phylogenetic diversity and, where possible, utilizing multiple datasets for individual species, to accurately infer gene presence or absence at higher taxonomic levels.
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http://dx.doi.org/10.1186/s12862-018-1290-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238376PMC
November 2018

Shark IgNAR-derived binding domains as potential diagnostic and therapeutic agents.

Dev Comp Immunol 2019 01 17;90:100-107. Epub 2018 Sep 17.

Dept. Microbiology & Immunology, University of Maryland School of Medicine, Institute of Marine & Environmental Technology (IMET), Baltimore, MD, 21202, USA. Electronic address:

Many of the most successful drugs generated in recent years are based upon monoclonal antibodies (mAbs). However, for some therapeutic and diagnostic applications mAbs are far from ideal; for example, while their relatively large size and inherent receptor binding aids their longevity in vivo it can also limit their tissue penetration. Further, their structural complexity makes them expensive to produce and prone to denaturation in non-physiological environments. Thus, researchers have been searching for alternative antigen-binding molecules that can be utilized in situations where mAbs are suboptimal tools. One potential source currently being explored are the shark-derived binding domains known as VNARs. Despite their small size VNARs can bind antigens with high specificity and high affinity. Combined with their propensity to bind epitopes that are inaccessible to conventional mAbs, and their ability to resist denaturation, VNARs are an emerging prospect for use in therapeutic, diagnostic, and biotechnological applications.
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http://dx.doi.org/10.1016/j.dci.2018.09.007DOI Listing
January 2019

Haptoglobin Is a Divergent MASP Family Member That Neofunctionalized To Recycle Hemoglobin via CD163 in Mammals.

J Immunol 2018 10 7;201(8):2483-2491. Epub 2018 Sep 7.

School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, United Kingdom;

In mammals, haptoglobin (Hp) is an acute-phase plasma protein that binds with high affinity to hemoglobin (Hb) released by intravascular hemolysis. The resultant Hp-Hb complexes are bound and cleared by the scavenger receptor CD163, limiting Hb-induced oxidative damage. In this study, we show that Hp is a divergent member of the complement-initiating MASP family of proteins, which emerged in the ancestor of jawed vertebrates. We demonstrate that Hp has been independently lost from multiple vertebrate lineages, that characterized Hb-interacting residues of mammals are poorly conserved in nonmammalian species maintaining Hp, and that the extended loop 3 region of Hp, which mediates CD163 binding, is present only in mammals. We show that the Hb-binding ability of cartilaginous fish (nurse shark, ; small-spotted catshark, ; and thornback ray, ) and teleost fish (rainbow trout, ) Hp is species specific, and where binding does occur it is likely mediated through a different structural mechanism to mammalian Hp. The continued, high-level expression of Hp in cartilaginous fishes in which Hb binding is not evident signals that Hp has (an)other, yet unstudied, role(s) in these species. Previous work indicates that mammalian Hp also has secondary, immunomodulatory functions that are independent of Hb binding; our work suggests these may be remnants of evolutionary more ancient functions, retained after Hb removal became the primary role of Hp in mammals.
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http://dx.doi.org/10.4049/jimmunol.1800508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6179929PMC
October 2018

Evolutionary history of the T cell receptor complex as revealed by small-spotted catshark (Scyliorhinus canicula).

Dev Comp Immunol 2017 09 19;74:125-135. Epub 2017 Apr 19.

School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, United Kingdom; Dept. Microbiology & Immunology, University of Maryland School of Medicine, Institute of Marine & Environmental Technology, Baltimore MD21202, USA.

In every jawed vertebrate species studied so far, the T cell receptor (TCR) complex is composed of two different TCR chains (α/β or γ/δ) and a number of CD3 subunits responsible for transmitting signals into the T cell. In this study, we characterised all of the TCR and CD3 genes of small-spotted catshark (Scyliorhinus canicula) and analysed their expression in a broad range of tissues. While the TCR complex is highly conserved across jawed vertebrates, we identified a number of differences in catshark, most notably the presence of two copies of both TCRβ and CD3γδ, and the absence of a functionally-important proline rich region from CD3ε. We also demonstrate that TCRβ has duplicated independently multiple times in jawed vertebrate evolution, bringing additional diversity to the TCR complex. This study reveals new insights about the evolutionary history of the TCR complex and raises new avenues for future exploration.
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http://dx.doi.org/10.1016/j.dci.2017.04.015DOI Listing
September 2017

Outgroup, alignment and modelling improvements indicate that two TNFSF13-like genes existed in the vertebrate ancestor.

Immunogenetics 2017 03 9;69(3):187-192. Epub 2017 Jan 9.

School of Biological Sciences, University of Aberdeen, Aberdeen, AB24 2TZ, UK.

The molecular machinery required for lymphocyte development and differentiation appears to have emerged concomitantly with distinct B- and T-like lymphocyte subsets in the ancestor of all vertebrates. The TNFSF superfamily (TNFSF) members BAFF (TNFSF13/Blys) and APRIL (TNFSF13) are key regulators of B cell development survival, and activation in mammals, but the temporal emergence of these molecules, and their precise relationship to the newly identified TNFSF gene BALM (BAFF and APRIL-like molecule), have not yet been elucidated. Here, to resolve the early evolutionary history of this family, we improved outgroup sampling and alignment quality, and applied better fitting substitution models compared to past studies. Our analyses reveal that BALM is a definitive TNFSF13 family member, which split from BAFF in the gnathostome (jawed vertebrate) ancestor. Most importantly, however, we show that both the APRIL and BAFF lineages existed in the ancestors of all extant vertebrates. This implies that APRIL has been lost, or is yet to be found, in cyclostomes (jawless vertebrates). Our results suggest that lineage-specific gene duplication and loss events have caused lymphocyte regulation, despite shared origins, to become secondarily distinct between gnathostomes and cyclostomes. Finally, the structure of lamprey BAFF-like, and its phylogenetic placement as sister to BAFF and BALM, but not the more slowly evolving APRIL, indicates that the primordial lymphocyte regulator was more APRIL-like than BAFF-like.
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http://dx.doi.org/10.1007/s00251-016-0967-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316386PMC
March 2017

Characterisation of the TNF superfamily members CD40L and BAFF in the small-spotted catshark (Scyliorhinus canicula).

Fish Shellfish Immunol 2015 Nov 16;47(1):381-9. Epub 2015 Sep 16.

Scottish Fish Immunology Research Centre (SFIRC), School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, United Kingdom.

The tumour necrosis factor superfamily (TNFSF) members CD40L and BAFF play critical roles in mammalian B cell survival, proliferation and maturation, however little is known about these key cytokines in the oldest jawed vertebrates, the cartilaginous fishes. Here we report the cloning of CD40L and BAFF orthologues (designated ScCD40L and ScBAFF) in the small-spotted catshark (Scyliorhinus canicula). As predicted both proteins are type II membrane-bound proteins with a TNF homology domain in their extracellular region and both are highly expressed in shark immune tissues. ScCD40L transcript levels correlate with those of TCRα and transcription of both genes is modulated in peripheral blood leukocytes following in vitro stimulation. Although a putative CD40L orthologue was identified in the elephant shark genome the work herein is the first molecular characterisation and transcriptional analysis of CD40L in a cartilaginous fish. ScBAFF was also cloned and its transcription characterised in an attempt to resolve the discrepancies observed between spiny dogfish BAFF and bamboo shark BAFF in previously published studies.
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http://dx.doi.org/10.1016/j.fsi.2015.09.033DOI Listing
November 2015

The CXC chemokine receptors of fish: Insights into CXCR evolution in the vertebrates.

Gen Comp Endocrinol 2015 May 23;215:117-31. Epub 2015 Jan 23.

Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen AB24 2TZ, UK; School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UK.

This article will review current knowledge on CXCR in fish, that represent three distinct vertebrate groups: Agnatha (jawless fishes), Chondrichthyes (cartilaginous fishes) and Osteichthyes (bony fishes). With the sequencing of many fish genomes, information on CXCR in these species in particular has expanded considerably. In mammals, 6 CXCRs have been described, and their homologues will be initially reviewed before considering a number of atypical CXCRs and a discussion of CXCR evolution.
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http://dx.doi.org/10.1016/j.ygcen.2015.01.004DOI Listing
May 2015

The immunoglobulins of cold-blooded vertebrates.

Biomolecules 2014 Nov 24;4(4):1045-69. Epub 2014 Nov 24.

School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UK.

Although lymphocyte-like cells secreting somatically-recombining receptors have been identified in the jawless fishes (hagfish and lamprey), the cartilaginous fishes (sharks, skates, rays and chimaera) are the most phylogenetically distant group relative to mammals in which bona fide immunoglobulins (Igs) have been found. Studies of the antibodies and humoral immune responses of cartilaginous fishes and other cold-blooded vertebrates (bony fishes, amphibians and reptiles) are not only revealing information about the emergence and roles of the different Ig heavy and light chain isotypes, but also the evolution of specialised adaptive features such as isotype switching, somatic hypermutation and affinity maturation. It is becoming increasingly apparent that while the adaptive immune response in these vertebrate lineages arose a long time ago, it is most definitely not primitive and has evolved to become complex and sophisticated. This review will summarise what is currently known about the immunoglobulins of cold-blooded vertebrates and highlight the differences, and commonalities, between these and more "conventional" mammalian species.
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http://dx.doi.org/10.3390/biom4041045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4279169PMC
November 2014

Kloss gibbon (Hylobates klossii) behavior facilitates the avoidance of human predation in the Peleonan forest, Siberut Island, Indonesia.

Am J Primatol 2015 Mar 8;77(3):296-308. Epub 2014 Oct 8.

School of Anatomy, Physiology and Human Biology M309, The University of Western Australia, Crawley, Australia.

Kloss gibbons (Hylobates klossii) are endemic to the Mentawai Islands in Indonesia and have been subject to human predation for more than 2000 years in the absence of any other significant predators. We investigate the behavior of Kloss gibbons that may be attributed to avoiding human predation. We observed Kloss gibbons in the Peleonan forest in the north of Siberut Island, the northernmost of the Mentawai island chain, over 18 months in 2007 and 2008, and collected data on their singing behavior, the number of individuals present during different conditions and their responses to humans. We examine behaviors that may reduce the risk of predation by humans during singing (the most conspicuous gibbon behavior), daily non-singing activities and encounters with humans. The individual risk of being stalked by hunters is reduced by singing in same-sex choruses and the risk of successful capture by hunters during singing is reduced by singing less often during daylight hours and by leaving the location of male pre-dawn singing before full light (reducing the visual signal to hunters). Groups in the Peleonan also fission during non-singing daily activity and rarely engage in play or grooming, enhancing the crypticity of their monochromatic black pelage in the canopy. We also observed a coordinated response to the presence of humans, wherein one adult individual acted as a "decoy" by approaching and distracting human observers, while other group members fled silently in multiple directions. "Decoy" behavior occurred on 31% of 96 encounters with unhabituated Kloss gibbons that detected our presence. "Decoy" individuals may put themselves at risk to increase the survival of related immatures (and adult females with infants) who have a greater risk of predation. We argue that, in combination, these behaviors are an evolved response to a long history of predation by humans.
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http://dx.doi.org/10.1002/ajp.22345DOI Listing
March 2015

A skin quandary in Fiji.

Aust Fam Physician 2014 May;43(5):297-8

MBBS, BaSci (Physiotherapy), Clinical Research Fellow, Royal Melbourne Hospital, VIC.

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May 2014

Noncoordinate expression of J-chain and Blimp-1 define nurse shark plasma cell populations during ontogeny.

Eur J Immunol 2013 Nov 27;43(11):3061-75. Epub 2013 Aug 27.

Department of Microbiology and Immunology, University of Maryland, Baltimore, MD, USA.

B-lymphocyte-induced maturation protein 1 (Blimp-1) is the master regulator of plasma cell development, controlling genes such as those encoding J-chain and secretory Ig heavy chain. However, some mammalian plasma cells do not express J-chain, and mammalian B1 cells secrete "natural" IgM antibodies without upregulating Blimp-1. While these results have been controversial in mammalian systems, here we describe subsets of normally occurring Blimp-1(-) antibody-secreting cells in nurse sharks, found in lymphoid tissues at all ontogenic stages. Sharks naturally produce large amounts of both pentameric (classically "19S") and monomeric (classically "7S") IgM, the latter an indicator of adaptive immunity. Consistent with the mammalian paradigm, shark Blimp-1 is expressed in splenic 7S IgM-secreting cells, though rarely detected in the J-chain(+) cells producing 19S IgM. Although IgM transcript levels are lower in J-chain(+) cells, these cells nevertheless secrete 19S IgM in the absence of Blimp-1, as demonstrated by ELISPOT and metabolic labeling. Additionally, cells in the shark BM equivalent (epigonal) are Blimp-1(-). Our data suggest that, in sharks, 19S-secreting cells and other secreting memory B cells in the epigonal are maintained for long periods without Blimp-1, but like in mammals, Blimp-1 is required for terminating the B-cell program following an adaptive immune response in the spleen.
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http://dx.doi.org/10.1002/eji.201343416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046329PMC
November 2013

B cell receptor accessory molecule CD79α: characterisation and expression analysis in a cartilaginous fish, the spiny dogfish (Squalus acanthias).

Fish Shellfish Immunol 2013 Jun 28;34(6):1404-15. Epub 2013 Feb 28.

Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen, UK.

CD79α (also known as Igα) is a component of the B cell antigen receptor complex and plays an important role in B cell signalling. The CD79α protein is present on the surface of B cells throughout their life cycle, and is absent on all other healthy cells, making it a highly reliable marker for B cells in mammals. In this study the spiny dogfish (Squalus acanthias) CD79α (SaCD79α) is described and its expression studied under constitutive and stimulated conditions. The spiny dogfish CD79α cDNA contains an open reading frame of 618 bp, encoding a protein of 205 amino acids. Comparison of the SaCD79α gene with that of other species shows that the gross structure (number of exons, exon/intron boundaries, etc.) is highly conserved across phylogeny. Additionally, analysis of the 5' flanking region shows SaCD79α lacks a TATA box and possesses binding sites for multiple transcription factors implicated in its B cell-specific gene transcription in other species. Spiny dogfish CD79α is most highly expressed in immune tissues, such as spleen, epigonal and Leydig organ, and its transcript level significantly correlates with those of spiny dogfish immunoglobulin heavy chains. Additionally, CD79α transcription is up-regulated, to a small but significant degree, in peripheral blood cells following stimulation with pokeweed mitogen. These results strongly indicate that, as in mammals, spiny dogfish CD79α is expressed by shark B cells where it associates with surface-bound immunoglobulin to form a fully functional BCR, and thus may serve as a pan-B cell marker in future shark immunological studies.
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http://dx.doi.org/10.1016/j.fsi.2013.02.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4034164PMC
June 2013

Humoral immune response of the small-spotted catshark, Scyliorhinus canicula.

Fish Shellfish Immunol 2013 May 21;34(5):1158-69. Epub 2013 Feb 21.

Global Biotherapeutics Technologies, Pfizer Inc., Foresterhill, Aberdeen AB25 2ZS, United Kingdom.

Cartilaginous fishes are the oldest group in which an adaptive immune system based on immunoglobulin-superfamily members is found. This manuscript compares humoral immune function in small-spotted catshark (Scyliorhinus canicula) with that described for spiny dogfish (Squalus acanthias), another member of the Squalomorphi superorder, and nurse shark, the model for humoral immunity in elasmobranchs and a member of the Galeomorphi superorder. Although small-spotted catshark and nurse shark are separated by over 200 million years we found that immunoglobulin isoforms are well conserved between the two species. However, the plasma protein profile of small-spotted catshark was most similar to that of spiny dogfish, with low levels of pentameric IgM, and IgNAR present as a multimer in plasma rather than a monomer. We show that an antigen-specific monomeric IgM response, with a profile similar to that described previously for nurse sharks, can be raised in small-spotted catshark. Lacking polyclonal or monoclonal antibody reagents for detecting catshark IgNAR we investigated phage-display and recombinant Fc-fusion protein expression as alternative methods to look for an antigen-specific response for this isotype. However, we could find no evidence of an antigen-specific IgNAR in the animals tested using either of these techniques. Thus, unlike nurse sharks where antigen-specific monomeric IgM and IgNAR appear together, it seems there may be a temporal or complete 'uncoupling' of these isotypes during a humoral response in the small-spotted catshark.
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http://dx.doi.org/10.1016/j.fsi.2013.01.025DOI Listing
May 2013

Singing by male and female Kloss gibbons (Hylobates klossii) in the Peleonan Forest, Siberut Island, Indonesia.

Primates 2013 Jan 2;54(1):39-48. Epub 2012 Sep 2.

School of Anatomy, Physiology and Human Biology M309, The University of Western Australia, Crawley, Perth, WA, Australia.

Kloss gibbons (Hylobates klossii) are endemic to the Mentawai Islands in Indonesia and are one of only two gibbon species in which mated pairs do not sing duets. This is the first long-term study of the factors influencing the singing activity of Kloss gibbons within a northern Siberut Island population and follows two previous studies in central Siberut nearly 30 years ago. We collected data on the presence/absence of male and female singing within the study area on 198 days and within a focal group on 47 days. Rainfall during the time period in which they normally sing inhibits singing in both males and females. Our study supports the hypothesis that male and female songs function in intrasexual resource defence, as singing is associated with singing by same-sex neighbours, and same-sex choruses are more likely to occur after one or more days of silence (from that sex), suggesting there is pressure for individuals to communicate with same-sex neighbours regularly. Singing was not coordinated within a mated pair, suggesting that vocal coordination of the pair has been lost with the loss of the duet and that Kloss gibbon songs do not convey information to neighbours about the strength of the pair bond. On days when males sang predawn, females were more likely to sing after dawn and earlier in the morning. Additionally, the number of groups singing in female choruses was positively associated with the number of males that had sung in the predawn male chorus. We suggest that female songs have an intersexual territory defence as well as an intrasexual function.
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http://dx.doi.org/10.1007/s10329-012-0326-2DOI Listing
January 2013

Generation and isolation of target-specific single-domain antibodies from shark immune repertoires.

Methods Mol Biol 2012 ;907:177-94

Molecular Partners AG, Zürich-Schlieren, Switzerland.

The drive to exploit novel targets and biological pathways has lead to the expansion of classical antibody research into innovative fragment adaptations and novel scaffolds. The hope being that alternative or cryptic epitopes may be targeted, tissue inaccessibility may be overcome, and easier engineering options will facilitate multivalent, multi-targeting approaches. To this end, we have been isolating shark single domains to gain a greater understanding of their potential as therapeutic agents. Their unique shape, small size, inherent stability, and simple molecular architecture make them attractive candidates from a drug discovery perspective. Here we describe protocols to capture the immune repertoire of an immunized shark species and to build and select via phage-display target-specific IgNAR variable domains (VNARs).
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http://dx.doi.org/10.1007/978-1-61779-974-7_9DOI Listing
December 2012

Characterisation and expression analysis of B-cell activating factor (BAFF) in spiny dogfish (Squalus acanthias): cartilaginous fish BAFF has a unique extra exon that may impact receptor binding.

Dev Comp Immunol 2012 Apr 3;36(4):707-17. Epub 2011 Dec 3.

Scottish Fish Immunology Research Centre, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen AB24 2TZ, Scotland, UK.

B-cell activating factor (BAFF), also known as tumour necrosis factor (TNF) ligand superfamily member 13B, is an important immune regulator with critical roles in B-cell survival, proliferation, differentiation and immunoglobulin secretion. A BAFF gene has been cloned from spiny dogfish (Squalus acanthias) and its expression studied. The dogfish BAFF encodes for an anchored type-II transmembrane protein of 288 aa with a putative furin protease cleavage site and TNF family signature as seen in BAFFs from other species. The identity of dogfish BAFF has also been confirmed by conserved cysteine residues, and phylogenetic tree analysis. The dogfish BAFF gene has an extra exon not seen in teleost fish, birds and mammals that encodes for 29 aa and may impact on receptor binding. The dogfish BAFF is highly expressed in immune tissues, such as spleen, and is up-regulated by PWM in peripheral blood leucocytes, suggesting a potentially important role in the immune system.
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http://dx.doi.org/10.1016/j.dci.2011.11.010DOI Listing
April 2012

Characterization of the immunoglobulin repertoire of the spiny dogfish (Squalus acanthias).

Dev Comp Immunol 2012 Apr 20;36(4):665-79. Epub 2011 Oct 20.

Global Biotherapeutics Technologies, Pfizer Inc., Foresterhill, Aberdeen AB25 2ZS, United Kingdom.

The cartilaginous fish (chimeras, sharks, skates and rays) are the oldest group relative to mammals in which an adaptive immune system founded upon immunoglobulins has been found. In this manuscript we characterize the immunoglobulins of the spiny dogfish (Squalus acanthias) at both the molecular and expressed protein levels. Despite the presence of hundreds of IgM clusters in this species the serum levels of this isotype are comparatively low. However, analysis of cDNA sequences and serum protein suggests microheterogeneity in the IgM heavy chains and supports the proposal that different clusters are preferentially used in the two forms (monomer or pentamer) of this isotype. We also found that the IgNAR isotype in this species exists in a previously unknown multimeric format in serum. Finally, we identified a new form of the IgW isotype (the shark IgD orthologue), in which the leader is spliced directly to the first constant domain, resulting in a molecule lacking an antigen-binding domain.
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http://dx.doi.org/10.1016/j.dci.2011.10.007DOI Listing
April 2012

Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

Mol Immunol 2011 Sep 12;48(15-16):2027-37. Epub 2011 Jul 12.

Defence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, UK.

Members of the genus Ebolavirus cause fulminating outbreaks of disease in human and non-human primate populations with a mortality rate up to 90%. To facilitate rapid detection of these pathogens in clinical and environmental samples, robust reagents capable of providing sensitive and specific detection are required. In this work recombinant antibody libraries were generated from murine (single chain variable domain fragment; scFv) and nurse shark, Ginglymostoma cirratum (IgNAR V) hosts immunised with Zaire ebolavirus. This provides the first recorded IgNAR V response against a particulate antigen in the nurse shark. Both murine scFv and shark IgNAR V libraries were panned by phage display technology to identify useful antibodies for the generation of immunological detection reagents. Two murine scFv were shown to have specificity to the Zaire ebolavirus viral matrix protein VP40. Two isolated IgNAR V were shown to bind to the viral nucleoprotein (NP) and to capture viable Zaire ebolavirus with a high degree of sensitivity. Assays developed with IgNAR V cross-reacted to Reston ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus. Despite this broad reactivity, neither of IgNAR V showed reactivity to Côte d'Ivoire ebolavirus. IgNAR V was substantially more resistant to irreversible thermal denaturation than murine scFv and monoclonal IgG in a comparative test. The demonstrable robustness of the IgNAR V domains may offer enhanced utility as immunological detection reagents in fieldable biosensor applications for use in tropical or subtropical countries where outbreaks of Ebolavirus haemorrhagic fever occur.
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http://dx.doi.org/10.1016/j.molimm.2011.06.437DOI Listing
September 2011

Social dynamics modify behavioural development in captive white-cheeked (Nomascus leucogenys) and silvery (Hylobates moloch) gibbons.

Primates 2011 Jul 18;52(3):271-7. Epub 2011 Mar 18.

School of Anatomy and Human Biology M309, The University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009, Australia.

Behavioural development was quantified in one family group of silvery gibbons (Hylobates moloch) and one of white-cheeked gibbons (Nomascus leucogenys) over 11 months during 2005 and 2008 at the Perth Zoo. Levels of locomotion, solo play and play solicitation peaked by 5 years of age but continued solo and social play in older immatures suggested that social development continued until at least 7 years of age. Mature offspring responded to play solicitations from younger siblings. The transition to sub-adulthood was marked by the presence of spatial peripheralisation from the parents, and coincided with aggression from the father to a sub-adult male. After the birth of a new infant, the male sub-adult stayed closer to his mother (and the infant) but not to his father; his juvenile brother was closer to both parents. Within-family observations of behaviour that is difficult to observe in the wild but can be observed in captivity contributes to our understanding of family dynamics in gibbons. Observations of these captive groups suggest that sub-adult peripheralisation may be influenced by family social dynamics as well as by local ecology, and that older offspring are responsive to the development of younger siblings.
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http://dx.doi.org/10.1007/s10329-011-0247-5DOI Listing
July 2011

Emergence of the acute-phase protein hemopexin in jawed vertebrates.

Mol Immunol 2010 Nov-Dec;48(1-3):147-52. Epub 2010 Sep 29.

Department of Microbiology & Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201, USA.

When released from damaged erythrocytes free heme not only provides a source of iron for invading bacteria but also highly toxic due to its ability to catalyze free radical formation. Hemopexin (Hx) binds free heme with very high-affinity and thus protects against heme toxicity, sequesters heme from pathogens, and helps conserve valuable iron. Hx is also an acute-phase serum protein (APP), whose expression is induced by inflammation. To date Hx has been identified as far back in phylogeny as bony fish where it is called warm-temperature acclimation-related 65 kDa protein (WAP65), as serum protein levels are increased at elevated environmental temperatures as well as by infection. During analysis of nurse shark (Ginglymostoma cirratum) plasma we isolated a Ni(2+)-binding serum glycoprotein and characterized it as the APP Hx. We subsequently cloned Hx from nurse shark and another cartilaginous fish species, the little skate Leucoraja erinacea. Functional analysis showed shark Hx, like that of mammals, binds heme but is found at unusually high levels in normal shark serum. As an Hx orthologue could not be found in the genomes of jawless vertebrates or lower deuterostomes it appears to have arisen just prior to the emergence of jawed vertebrates, coincident with the second round of genome-wide duplication and the appearance of tetrameric hemoglobin (Hb).
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http://dx.doi.org/10.1016/j.molimm.2010.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2993785PMC
January 2011

The generation and selection of single-domain, v region libraries from nurse sharks.

Methods Mol Biol 2009 ;562:71-82

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

The cartilaginous fish (sharks, skates, and rays) are the oldest phylogenetic group in which a human-type adaptive immune system and immunoglobulins (Igs) have been found. In addition to their conventional (heavy-light chain heterodimeric) isotypes, IgM and IgW, sharks produce the novel isotype, IgNAR, a heavy chain homodimer that does not associate with light chains. Instead, its variable (V) regions act as independent, soluble units in order to bind antigen. In this chapter, we detail our immunization protocol in order to raise a humoral IgNAR response in the nurse shark (Ginglymostoma cirratum) and the subsequent cloning of the single-domain V regions from this isotype in order to select antigen-specific binders by phage display.
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http://dx.doi.org/10.1007/978-1-60327-302-2_6DOI Listing
October 2009

Vocal responses of captive gibbon groups to a mate change in a pair of white-cheeked gibbons (Nomascus leucogenys).

Folia Primatol (Basel) 2007 11;78(4):228-39. Epub 2007 May 11.

School of Anatomy and Human Biology M309, University of Western Australia, Crawley, Australia.

The singing behaviour of 3 pairs of white-cheeked gibbons (Nomascus leucogenys) held at the Perth Zoo was observed for 6 months in 2005. These groups included a family (mated pair and 2 immature offspring) and a pair without offspring. During the study, the female without offspring was exchanged for an unpaired female from New Zealand. After the new pair had been released onto the island enclosure and began to duet, the duetting rate of the white-cheeked gibbon family increased. The increased singing began after the new female had started to sing solo female great calls. These observations support the hypothesis that duets have an intergroup communication function in white-cheeked gibbons. The pair that duetted most frequently also copulated most frequently but allogroomed the least. We suggest that duetting may be more important to intergroup relations than to pair bond maintenance in this species.
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http://dx.doi.org/10.1159/000102318DOI Listing
September 2007

Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding.

J Mol Biol 2007 Mar 22;367(2):358-72. Epub 2006 Dec 22.

Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.
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http://dx.doi.org/10.1016/j.jmb.2006.12.045DOI Listing
March 2007

First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate.

Proc Natl Acad Sci U S A 2006 Feb 30;103(6):1846-51. Epub 2006 Jan 30.

Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201, USA.

The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (Ginglymostoma cirratum) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the in vivo maturation mechanisms, in general, and for single-domain antibody fragments.
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http://dx.doi.org/10.1073/pnas.0508341103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1413636PMC
February 2006