An update on the development of a bottlenose dolphin, Tursiops truncatus, immune reagent toolkit.

There are extensive immunological reagents available for laboratory rodents and humans. However, for veterinary species there is a need for expansion of immunological toolkits, with this especially evident for marine mammals, such as cetaceans. In addition to their use in a research setting, immune assays could be employed to monitor the health status of cetaceans and serve as an adjunct to available diagnostic tests. Such development of specific and sensitive immune assays will enhance the proper care and stewardship of wild and managed cetacean populations. Our goal is to provide immune reagents and immune assays for the research community, clinicians, and others involved in care of bottlenose dolphins. This review will provide an update on our development of a bottlenose dolphin immunological toolkit. The future availability and continued development of these reagents is critical for improving wild and managed bottlenose dolphin population health through enhanced assessment of their responses to alterations in the marine environment, including pathogens, and improve our ability to monitor their status following vaccination.

Production and characterization of anti-porcine CXCL10 monoclonal antibodies.

Research on C-X-C motif chemokine ligand 10 (CXCL10) has been widely reported for humans and select animal species, yet immune reagents are limited for pig chemokines. Our goal is to provide veterinary immunologists and the biomedical community with new commercial immune reagents and standardized assays. Recombinant porcine CXCL10 (rPoCXCL10) protein was produced by yeast expression and used to generate a panel of α CXCL10 monoclonal antibodies (mAbs). All mAbs were assessed for cross-inhibition and reactivity to orthologous yeast expressed CXCL10 proteins. Characterization of a panel of nine α PoCXCL10 mAbs identified six distinct antigenic determinants. A sensitive quantitative sandwich ELISA was developed with anti-PoCXCL10-1.6 and -1.9 mAb; reactivity was verified with both rPoCXCL10 and native PoCXCL10, detected in supernatants of peripheral blood mononuclear cells stimulated with rPoIFNγ or PMA/Ionomycin. Immunostaining of in vitro rPoIFNγ stimulated pig spleen and blood cells verified CXCL10 + cells as CD3-CD4-CD172+, with occasional CD3-CD4 + CD172 + subsets. Comparison studies determined that α PoCXCL10-1.4 mAb was the ideal mAb clone for intracellular staining, whereas with α PoCXCL10-1.1 and -1.2 mAbs were best for immunohistochemistry analyses. These techniques and tools will be useful for evaluating swine immune development, responses to infectious diseases and vaccines, as well as for improving utility of pigs as an important biomedical model.

Development and Characterization of New Monoclonal Antibodies Against Porcine Interleukin-17A and Interferon-Gamma.

Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune reagents for pigs and pipeline them for marketing. Our efforts were aimed at the expression of soluble swine cytokines and the production of panels of monoclonal antibodies (mAbs) to these proteins. Swine interleukin-17A (IL-17A) and Interferon-gamma (IFNγ) recombinant proteins were produced using yeast expression and used for monoclonal antibody (mAb) production resulting in panels of mAbs. We screened each mAb for cross-species reactivity with orthologs of IL-17A or IFNγ and checked each mAb for inhibition by other related mAbs, to assign mAb antigenic determinants. For porcine IL-17A, the characterization of a panel of 10 mAbs identified eight different antigenic determinants; interestingly, most of the mAbs cross-reacted with the dolphin recombinant ortholog. Likewise, the characterization of a panel of nine anti-PoIFNγ mAbs identified four different determinants; most of the mAbs cross-reacted with dolphin, bovine, and caprine recombinant orthologs. There was a unique reaction of one anti-PoIFNγ mAb that cross-reacted with the zebrafish recombinant ortholog. The αIL-17A mAbs were used to develop a quantitative sandwich ELISA detecting the yeast expressed protein as well as native IL-17A in stimulated peripheral blood mononuclear cell (PBMC) supernatants. Our analyses showed that phorbol myristate acetate/ionomycin stimulation of PBMC induced significant expression of IL-17A by CD3+ T cells as detected by several of our mAbs. These new mAbs expand opportunities for immunology research in swine.

Development and testing of species-specific ELISA assays to measure IFN-γ and TNF-α in bottlenose dolphins (Tursiops truncatus).

Monitoring the immune status of cetaceans is important for a variety of health conditions. Assays to quantify cytokines, especially pro-inflammatory cytokines, could be employed, in addition to currently available diagnostic assays, to screen for alterations in the health status of an animal. Though a number of immunological assays are readily available for humans and mice, specific assays for many veterinary species, including cetaceans such as bottlenose dolphins (Tursiops truncatus), are more limited. Herein, we describe the development of IFN-gamma (IFN-γ) and TNF-alpha (TNF-α) enzyme-linked immunosorbent assays (ELISAs) specific to bottlenose dolphins. Utilizing these assays, we monitored the immune status of bottlenose dolphins from a managed population over a period of eleven months. The ELISA assays developed for bottlenose dolphins were used to measure IFN-γ and TNF-α in serum or in culture supernatants from peripheral blood mononuclear cells (PBMCs) stimulated with varying concentrations of mitogens concanavalin A (ConA) or phytohemagglutinin (PHA). Induction of TNF-α in PBMC cultures was consistently highest with 1 µg/mL ConA, while 1 µg/mL PHA induced the highest secretion of IFN-γ. Serum levels of TNF-α and IFN-γ remained relatively constant for each animal over the time period examined. CBC and plasma chemistry variables measured concurrently in the bottlenose dolphins were then examined as independent predictors of cytokine levels. We found these clinical variables were more likely to predict linear changes in serum IFN-γ and TNF-α levels compared to concentrations of these cytokines in mitogen-stimulated PBMC culture supernatants. Cytokine assays developed will be of substantial benefit in monitoring bottlenose dolphin health as an adjunct to currently available diagnostic tests.

Development and characterization of mouse monoclonal antibodies reactive with chicken CXCLi2.

Interleukin-8(IL-8)/CXCL8 is a CXC-family chemokine that attracts lymphocytes to sites of tissue damage and plays a role in the inflammatory response and wound healing. Chicken chemotactic and angiogenic factor was referred to as chCXCLi2 and has been studied as one of human CXCL8 homologue for more than 20 years. However, no monoclonal antibodies (mAbs) that specifically detect chCXCLi2 have been developed. Here, we developed and characterized mouse mAbs against chCXCLi2 to define its immunological properties. Two mouse mAbs against chCXCLi2 were generated and confirmed to display specific binding with not only recombinants, but endogenous chCXCLi2 by Western blot analysis, ELISA, and immunocytochemistry. Inhibition of chCXCLi2-induced chemotactic activity on peripheral blood lymphocytes, proliferation of chicken macrophage cells and expression of alpha smooth-muscle actin in chicken embryonic fibroblast cells by antibodies indicate that these antibodies are capable of blocking chCXCLi2 bioactivity. These chCXCLi2 mAbs will be useful reagents for future investigations of inflammation in poultry.

Development and characterization of mouse monoclonal antibodies reactive with chicken TL1A.

Tumor necrosis factor-like ligand 1A (TL1A) is a type II transmembrane protein predominantly expressed by endothelial cells that promotes the expansion of activated T cells and regulatory T cells, modulates inflammation, and regulates the production of a wide variety of T cell cytokines. However, there have not been any mAbs which specifically detect chTL1A and define its biochemical and immunological properties. So in this study, two mouse monoclonal antibodies (mAbs) which specifically detect chicken TL1A (chTL1A) were developed and characterized. Both mAbs identified a 32 kDa Escherichia coli-derived, poly-histidine-tagged fusion protein by Western blot analysis. The mAbs identified TL1A-secreting cells in the chicken thymus, cecal tonsil, and bursa of Fabricius by immunocytochemistry, and were used to measure serum TL1A levels in normal and necrotic enteritis (NE)-afflicted chickens by antigen capture ELISA. These mAbs inhibited chTL1A-induced spleen lymphocyte proliferation, nitric oxide production by chicken macrophage cells (HD11), and blocked the cytotoxic effect of chTL1A against lymphoblastoid chicken B tumor cells (LSCC-RP9). These new mAbs that detect chTL1A will be important immune reagents for basic and applied research in poultry immunology.

Is chest CT useful in newborn screened infants with cystic fibrosis at 1 year of age?

RATIONALE: Sensitive outcome measures applicable in different centres to quantify and track early pulmonary abnormalities in infants with cystic fibrosis (CF) are needed both for clinical care and interventional trials. Chest CT has been advocated as such a measure yet there is no validated scoring system in infants. OBJECTIVES: The objectives of this study were to standardise CT data collection across multiple sites; ascertain the incidence of bronchial dilatation and air trapping in newborn screened (NBS) infants with CF at 1 year; and assess the reproducibility of Brody-II, the most widely used scoring system in children with CF, during infancy. METHODS: A multicentre observational study of early pulmonary lung disease in NBS infants with CF at age 1 year using volume-controlled chest CT performed under general anaesthetic. MAIN RESULTS: 65 infants with NBS-diagnosed CF had chest CT in three centres. Small insignificant variations in lung recruitment manoeuvres but significant centre differences in radiation exposures were found. Despite experienced scorers and prior training, with the exception of air trapping, inter- and intraobserver agreement on Brody-II score was poor to fair (eg, interobserver total score mean (95% CI) κ coefficient: 0.34 (0.20 to 0.49)). Only 7 (11%) infants had a total CT score ≥ 12 (ie, ≥ 5% maximum possible) by either scorer. CONCLUSIONS: In NBS infants with CF, CT changes were very mild at 1 year, and assessment of air trapping was the only reproducible outcome. CT is thus of questionable value in infants of this age, unless an improved scoring system for use in mild CF disease can be developed.

Multislice spiral computed tomography for pediatric intracranial vascular pathophysiologies.

OBJECT: Spiral computed tomography (SCT) and, more recently, multislice SCT (MSCT) angiography have established roles in studying subarachnoid hemorrhage (SAH). Potential advantages in MSCT angiography include rapid acquisition, ready availability, ease of monitoring, high spatial resolution, some temporal resolution, and relative freedom from artifacts. The authors assert that these attributes make MSCT angiography the initial imaging method of choice in the assessment of not just SAH but all intracranial vascular pathophysiologies, particularly in children. METHODS: The installation of a MSCT unit sparked the authors' interest in using MSCT angiography and MSCT venography in cases in which they would have formerly performed magnetic resonance (MR) angiography, MR venography, or catheter angiography as an initial investigational method. They retrospectively evaluated seven cases in which they had used the former imaging techniques to study intracranial vascular pathophysiologies. All scans were obtained on a Siemens Sensation 16-slice scanner, and postprocessing was performed on a Leonardo Workstation. RESULTS: Multislice spiral CT consistently provided useful vascular imaging of a wide variety of intracranial vascular pathophysiologies and an alternative imaging modality in patients considered to be too unstable for more time-consuming investigations. CONCLUSIONS: Multislice spiral CT offers advantages over MR imaging in the assessment of intracranial vascular pathophysiologies and frequently allows complete avoidance or deferral of catheter angiography.

CD8+ T cells that express CD4 on their surface (CD4dimCD8bright T cells) recognize an antigen-specific target, are detected in vivo, and can be productively infected by T-tropic HIV.

CD4 can be up-regulated on CD8+ T cells generating a CD4dimCD8bright phenotype. We previously demonstrated that the CD4dimCD8bright phenotype constitutes an activated phenotype of CD8+ T cells. We demonstrate here that the activated CD4dimCD8bright T cells are not undergoing apoptosis and do not produce significant intracellular levels of interferon gamma (IFNgamma), interleukin 2 (IL-2), or IL-10 but express elevated levels of intracellular IL-4 in comparison to CD8+CD4- and CD4+ T cells. In response to cytomegalovirus (CMV) peptide (pp65) priming, CD4dimCD8bright cells recognized CMV pp65 tetramer approximately 19-fold higher than CD4-CD8+ T cells, indicating that these cells are capable of antigen-specific recognition to a far greater extent than CD4-CD8+ T cells. CD4dimCD8bright T cells also express both CXCR4 and CCR5 but are susceptible to T-tropic and not M-tropic HIV infection. A soluble factor believed to be beta-chemokine is responsible for the inhibition of M-tropic HIV infection in CD4dimCD8bright T cells. CD8+ T cells from HIV+ patients were capable of up-regulating CD4 on CD8+ T cells. We also provide evidence of the presence of peripheral blood CD4dimCD8bright T cells in HIV+ patients, albeit at low frequency. Collectively, these data suggest a role of CD4dimCD8bright T cells in both normal T-cell biology and HIV pathogenesis.