Catherine J. Walsh

Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57–66 and 82–89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure (Cell 130: 1071–1082) of the ligand-bound TLR1/2 ectodomain heterodimer.

Evolutionary Origins of Lymphocytes: Ensembles of T Cell and B Cell Transcriptional Regulators in a Cartilaginous Fish

The evolutionary origins of lymphocytes can be traced by phylogenetic comparisons of key features. Homologs of rearranging TCR and Ig (B cell receptor) genes are present in jawed vertebrates, but have not been identified in other animal groups. In contrast, most of the transcription factors that are essential for the development of mammalian T and B lymphocytes belong to multigene families that are represented by members in the majority of the metazoans, providing a potential bridge to prevertebrate ancestral roles. This work investigates the structure and regulation of homologs of specific transcription factors known to regulate mammalian T and B cell development in a representative of the earliest diverging jawed vertebrates, the clearnose skate (Raja eglanteria). Skate orthologs of mammalian GATA-3, GATA-1, EBF-1, Pax-5, Pax-6, Runx2, and Runx3 have been characterized. GATA-3, Pax-5, Runx3, EBF-1, Spi-C, and most members of the Ikaros family are shown throughout ontogeny to be 1) coregulated with TCR or Ig expression, and 2) coexpressed with each other in combinations that for the most part correspond to known mouse T and B cell patterns, supporting conservation of function. These results indicate that multiple components of the gene regulatory networks that operate in mammalian T cell and B cell development were present in the common ancestor of the mammals and the cartilaginous fish. However, certain factors relevant to the B lineage differ in their tissue-specific expression patterns from their mouse counterparts, suggesting expanded or divergent B lineage characteristics or tissue specificity in these animals.

Effects of in vitro Brevetoxin Exposure on Apoptosis and Cellular Metabolism in a Leukemic T Cell Line (Jurkat)

Harmful algal blooms (HABs) of the toxic dinoflagellate, Karenia brevis, produce red tide toxins, or brevetoxins. Significant health effects associated with red tide toxin exposure have been reported in sea life and in humans, with brevetoxins documented within immune cells from many species. The objective of this research was to investigate potential immunotoxic effects of brevetoxins using a leukemic T cell line (Jurkat) as an in vitro model system. Viability, cell proliferation, and apoptosis assays were conducted using brevetoxin congeners PbTx-2, PbTx-3, and PbTx-6. The effects of in vitro brevetoxin exposure on cell viability and cellular metabolism or proliferation were determined using trypan blue and MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), respectively. Using MTT, cellular metabolic activity was decreased in Jurkat cells exposed to 5 – 10 μg/ml PbTx-2 or PbTx-6. After 3 h, no significant effects on cell viability were observed with any toxin congener in concentrations up to 10 μg/ml. Viability decreased dramatically after 24 h in cells treated with PbTx-2 or -6. Apoptosis, as measured by caspase-3 activity, was significantly increased in cells exposed to PbTx-2 or PbTx-6. In summary, brevetoxin congeners varied in effects on Jurkat cells, with PbTx-2 and PbTx-6 eliciting greater cellular effects compared to PbTx-3.

Sublethal red tide toxin exposure in free-ranging manatees (Trichechus manatus) affects the immune system through reduced lymphocyte proliferation responses, inflammation, and oxidative stress ☆

The health of many Florida manatees (Trichechus manatus latirostris) is adversely affected by exposure to blooms of the toxic dinoflagellate, Karenia brevis. K. brevis blooms are common in manatee habitats of Floridas southwestern coast and produce a group of cyclic polyether toxins collectively referred to as red tide toxins, or brevetoxins. Although a large number of manatees exposed to significant levels of red tide toxins die, several manatees are rescued from sublethal exposure and are successfully treated and returned to the wild. Sublethal brevetoxin exposure may potentially impact the manatee immune system. Lymphocyte proliferative responses and a suite of immune function parameters in the plasma were used to evaluate effects of brevetoxin exposure on health of manatees rescued from natural exposure to red tide toxins in their habitat. Blood samples were collected from rescued manatees at Lowry Park Zoo in Tampa, FL and from healthy, unexposed manatees in Crystal River, FL. Peripheral blood leukocytes (PBL) isolated from whole blood were stimulated with T-cell mitogens, ConA and PHA. A suite of plasma parameters, including plasma protein electrophoresis profiles, lysozyme activity, superoxide dismutase (SOD) activity, and reactive oxygen/nitrogen (ROS/RNS) species, was also used to assess manatee health. Significant decreases (p<0.05) in lymphocyte proliferation were observed in ConA and PHA stimulated lymphocytes from rescued animals compared to non-exposed animals. Significant correlations were observed between oxidative stress markers (SOD, ROS/RNS) and plasma brevetoxin concentrations. Sublethal exposure to brevetoxins in the wild impacts some immune function components, and thus, overall health, in the Florida manatee.

Cellular metabolism of brevetoxin (PbTx-2) by a monocyte cell line (U-937).

Blooms of Karenia brevis produce brevetoxins which cause neurotoxic shellfish poisoning and respiratory symptoms in humans as well as harmful effects on sea life. To investigate potential effects of brevetoxins on immune system components, a monocyte cell line (U-937) was exposed in vitro to PbTx-2. U-937 cells metabolized PbTx-2 through cellular detoxification mechanisms, as evidenced by depletion of intracellular glutathione and formation of glutathione and cysteine conjugates. Total intracellular glutathione was significantly decreased in toxin-treated cells compared to control cells, as measured using an enzymatic recycling method. LC/MS was used to detect the following brevetoxin metabolites: a cysteine-PbTx-2 conjugate (m/z 1018) and two putative glutathione-PbTx-2 conjugates (m/z 1204 and 1222). During 3h incubation, glutathione conjugates were detectable as early as 1h and increased in concentration after 2 and 3h. A cysteine-PbTx-2 conjugate appeared after 2h and increased in concentration after 3h. Detectable levels of brevetoxin conjugates were present in response to toxin concentrations of 1muM. Depletion of intracellular glutathione and formation of brevetoxin metabolites, with changes in concentrations over time, suggest immune cells (U-937) have important cellular detoxification pathways for PbTx-2.

Potential effects of brevetoxins and toxic elements on various health variables in Kemp's ridley (Lepidochelys kempii) and green (Chelonia mydas) sea turtles after a red tide bloom event

Natural biotoxins and anthropogenic toxicants pose a significant risk to sea turtle health. Documented effects of contaminants include potential disease progression and adverse impacts on development, immune function, and survival in these imperiled species. The shallow seagrass habitats of Floridas northwest coast (Big Bend) serve as an important developmental habitat for Kemps ridley (Lepidochelys kempii) and green (Chelonia mydas) sea turtles; however, few studies have been conducted in this area. Our objectives were (1) to evaluate plasma analytes (mass, minimum straight carapace length, body condition index [BCI], fibropapilloma tumor score, lysozyme, superoxide dismutase, reactive oxygen/nitrogen species, plasma protein electrophoresis, cholesterol, and total solids) in Kemps ridleys and green turtles and their correlation to brevetoxins that were released from a red tide bloom event from July-October 2014 in the Gulf of Mexico near Floridas Big Bend, and (2) to analyze red blood cells in Kemps ridleys and green turtles for toxic elements (arsenic, cadmium, lead, mercury, selenium, thallium) with correlation to the measured plasma analytes. Positive correlations were observed between brevetoxins and α2-globulins in Kemps ridleys and α2- and γ-globulins in green turtles, indicating potential immunostimulation. Arsenic, cadmium, and lead positively correlated with superoxide dismutase in Kemps ridleys, suggesting oxidative stress. Lead and mercury in green turtles negatively correlated with BCI, while mercury positively correlated with total tumor score of green turtles afflicted with fibropapillomatosis, suggesting a possible association with mercury and increased tumor growth. The total tumor score of green turtles positively correlated with total protein, total globulins, α2-globulins, and γ-globulins, further suggesting inflammation and immunomodulation as a result of fibropapillomatosis. Lastly, brevetoxin concentrations were positively related to tumor score, indicating potential tumor promotion by brevetoxin. These results signify that brevetoxins and toxic elements elicit various negative effects on sea turtle health, including immune function, oxidative stress, and possibly disease progression.

Epigonal Conditioned Media from Bonnethead Shark, Sphyrna tiburo, Induces Apoptosis in a T-Cell Leukemia Cell Line, Jurkat E6-1

Representatives of Subclass Elasmobranchii are cartilaginous fish whose members include sharks, skates, and rays. Because of their unique phylogenetic position of being the most primitive group of vertebrates to possess all the components necessary for an adaptive immune system, the immune regulatory compounds they possess may represent the earliest evolutionary forms of novel compounds with the potential for innovative therapeutic applications. Conditioned medium, generated from short term culture of cells from the epigonal organ of bonnethead sharks (Sphyrna tiburo), has been shown to have potent reproducible cytotoxic activity against a variety of human tumor cell lines in vitro. Existing data suggest that epigonal conditioned medium (ECM) exerts this cytotoxic activity through induction of apoptosis in target cells. This manuscript describes apoptosis induction in a representative tumor cell line, Jurkat E6-1, in response to treatment with ECM at concentrations of 1 and 2 mg/mL. Data indicate that ECM exposure initiates the mitochondrial pathway of apoptosis through activation of caspase enzymes. Future purification of ECM components may result in the isolation of an immune-regulatory compound with potential therapeutic benefit for treatment of human cancer.

Survey of Antibiotic-producing Bacteria Associated with the Epidermal Mucus Layers of Rays and Skates

Elasmobranchs represent a distinct group of cartilaginous fishes that harbor a remarkable ability to heal wounds rapidly and without infection. To date very little work has addressed this phenomenon although it is suggested that antibiotic capabilities associated with epidermal surfaces may be a factor. The study of benefits derived from mutualistic interactions between unicellular and multicellular organisms is a rapidly growing area of research. Here we survey and identify bacterial associates of three ray and one skate species in order to assess the potential for antibiotic production from elasmobranch associated bacteria as a novel source for new antibiotics.

Toxic elements and associations with hematology, plasma biochemistry, and protein electrophoresis in nesting loggerhead sea turtles (Caretta caretta) from Casey Key, Florida

Toxic elements (arsenic, cadmium, lead, mercury, selenium, thallium) are a group of contaminants that are known to elicit developmental, reproductive, general health, and immune system effects in reptiles, even at low concentrations. Reptiles, including marine turtles, are susceptible to accumulation of toxic elements due to their long life span, low metabolic rate, and highly efficient conversion of prey into biomass. The objectives of this study were to (1) document concentrations of arsenic, cadmium, lead, mercury, selenium, and thallium in whole blood and keratin from nesting loggerhead sea turtles (Caretta caretta) from Casey Key, Florida and document correlations thereof and (2) correlate whole blood toxic element concentrations to various hematological and plasma biochemistry analytes. Baselines for various hematological and plasma analytes and toxic elements in whole blood and keratin (i.e., scute) in nesting loggerheads are documented. Various correlations between the toxic elements and hematological and plasma biochemistry analytes were identified; however, the most intriguing were negative correlations between arsenic, cadmium, lead, and selenium with and α- and γ-globulins. Although various extrinsic and intrinsic variables such as dietary and feeding changes in nesting loggerheads need to be considered, this finding may suggest a link to altered humoral immunity. This study documents a suite of health variables of nesting loggerheads in correlation to contaminants and identifies the potential of toxic elements to impact the overall health of nesting turtles, thus presenting important implications for the conservation and management of this species.

Lack of cross-reactivity of human and porcine reagents to quantify manatee (Trichechus manatus) cytokines

Veterinary medical examinations, including both physical examination and diagnostic tests, are important to monitor the health of both managed-care and wild marine mammals. However, limited species-specific reagents and assays are available that may contribute to a broader medical examination. This project evaluated if commercially available human and porcine antibodies and reagents would cross-react with manatee (Trichechus manatus) cytokines as the first step to validate a new diagnostic tool for manatees. Overall, as a result of limited cross-reactivity, human and porcine commercial reagents did not allow for the quantification of manatee cytokines. At this point, caution must be exercised when using human or porcine immunoassay reagents to quantify manatee cytokines if the reagents have not been fully validated. Future efforts will continue to explore and test the cross-reactivity of reagents to measure manatee cytokines as new species-specific and commercial reagents become available.