Developing a xenograft model of human vasculature in the mouse ear pinna.

Humanised xenograft models allow for the analysis of human tissue within a physiological environment in vivo. However, current models often rely on the angiogenesis and ingrowth of recipient vasculature to perfuse tissues, preventing analysis of biological processes and diseases involving human blood vessels. This limits the effectiveness of xenografts in replicating human physiology and may lead to issues with translating findings into human research. We have designed a xenograft model of human vasculature to address this issue. Human subcutaneous fat was cultured in vitro to promote blood vessel outgrowth prior to implantation into immunocompromised mice. We demonstrate that implants survived, retained human vasculature and anastomosed with the circulatory system of the recipient mouse. Significantly, by performing transplants into the ear pinna, this system enabled intravital observation of xenografts by multiphoton microscopy, allowing us to visualise the steps leading to vascular cytoadherence of erythrocytes infected with the human parasite Plasmodium falciparum. This model represents a useful tool for imaging the interactions that occur within human tissues in vivo and permits visualization of blood flow and cellular recruitment in a system which is amenable to intervention for various studies in basic biology together with drug evaluation and mechanism of action studies.

T Cell Response in the Lung Following Influenza Virus Infection.

Methods that enable the identification of virus-specific CD4 and CD8 T cells are key to our understanding of how the adaptive immune response controls viral infection. Here we describe two distinct methods to evaluate the T cell response to influenza A virus (IAV). The number and phenotype of T cells that respond to natural IAV epitopes can be assessed by flow cytometry using MHC class I and class II tetramers. Using this system, IAV-specific T cells can be tracked in various organs within the same animal, or, in different cohorts, the response can be evaluated at several time points following infection. While providing clear quantitative data, flow cytometry cannot provide any information about T cell location within the lung or interactions between responding T cells and other cell types. Here we also describe a method to examine activated CD4 T cells in the lungs of living animals using multiphoton intravital microscopy, thus providing real-time analysis of T cell behavior during an infection.

Using lymph node transplantation as an approach to image cellular interactions between the skin and draining lymph nodes during parasitic infections.

The growing use of protozoan parasites expressing fluorescent reporter genes, together with advances in microscopy, is enabling visualisation of their behaviour and functions within the host from the very earliest stages of infection with previously unparalleled spatiotemporal resolution. These developments have begun to provide novel insights, which are informing our understanding of where host immune responses may be initiated, which cells are involved and the types of response that are elicited. Here we will review some of these recent observations that highlight the importance of cellular communication between the site of infection and the draining lymph node (dLN) in establishing infection and immunity. We also highlight a number of remaining challenges and unknowns that arise through our inability to follow and fate map the journey of a single cell between spatially separated tissue sites. In response to these challenges, we review a recently described experimental strategy that extends the spatial and temporal limits of previous imaging approaches, most significantly allowing longitudinal analysis of cellular migration between the skin and draining lymph nodes in vivo, without the requirement for invasive surgery.

An evaluation of cause-effect relationships between polychlorinated biphenyl concentrations and sediment toxicity to benthic invertebrates.

Cause-effect sediment-quality benchmarks for the protection of benthic invertebrates are needed for polychlorinated biphenyls (PCBs) to support predictive risk assessments and retrospective evaluations of the causes of observed sediment toxicity. An in-depth evaluation of PCB aquatic toxicity and organic carbon partitioning was conducted to predict sediment effect concentrations using the equilibrium partitioning (EqP) approach. This evaluation was limited to invertebrate toxicity data, because PCBs may exert toxicity to invertebrates and fish via different toxicological mechanisms. As a result of differences in organic carbon partitioning among PCBs of differing levels of chlorination, the estimated EqP benchmarks increase with increasing degree of chlorination for various commercial and environmental PCB mixtures. Studies of spiked sediment toxicity using PCBs were reviewed, and their results generally were consistent with EqP predictions. Additionally, toxicity and benthic community data were reviewed for eight PCB-contaminated sites; these data also showed agreement with EqP predictions. None of these lines of evidence supports previously proposed, empirical sediment-quality guidelines for PCBs. Reasons for the lack of agreement between cause-effect and association-based benchmarks are discussed, and areas of future research to further refine EqP predictions for PCBs are identified.

Toxicity of atrazine to the juvenile hard clam, Mercenaria mercenaria.

The herbicide atrazine is one of the most heavily used pesticides in the United States. The effects of atrazine on the clam Mercenaria mercenaria were evaluated in aqueous and sediment laboratory assays. Juvenile clams of approximately 1mm in size were used for all experiments. An acute aqueous bio-assay was used to determine the 96-h LC(50) for the juvenile clams. A chronic aqueous bioassay was conducted at lower atrazine concentrations over a 10-day exposure period to examine both lethal and sublethal (dry mass, shell size, and condition index) endpoints. A chronic sediment bioassay examined mortality and sublethal endpoints in a 10-day exposure. The acute 96-h LC(50) was 5608 microg/L with 95% confidence intervals ranging from 5003 to 6287 microg/L. Results of the chronic aqueous assay indicated both lethal and sublethal (reduced shell size) effects at high atrazine concentrations. In the 10-day chronic aqueous assay, the no observable effect concentration was 500 microg/L, the lowest observable effect concentration was 1000 microg/L, and the maximum allowable toxicant concentration (MATC) was 707 microg/L. There were no significant effects of atrazine in the chronic sediment exposure. Safe concentrations for the aqueous experiments were estimated by applying an uncertainty factor of 10 to the calculated MATC values. While there were adverse effects of atrazine at high concentrations, these results suggest that atrazine is not directly toxic to M. mercenaria at environmentally relevant concentrations.