Extracellular adenosine has an important role in regulating the severity of inflammation during an immune response. Although there are four adenosine receptor (AR) subtypes, the A2AAR is both highly expressed on lymphocytes and known as a prime mediator of adenosine's anti-inflammatory effects. To define the importance of A2AAR signaling during neuroinflammatory disease progression, we used the experimental autoimmune encephalomyelitis (EAE) animal model for multiple sclerosis. In EAE induction experiments, A2AAR antagonist treatment protected mice from disease development and its associated CNS lymphocyte infiltration. However, A2AAR(-/-) mice developed a more severe acute EAE phenotype characterized by more proinflammatory lymphocytes and activated microglia/macrophages. Interestingly, very high levels of A2AAR were expressed on the choroid plexus, a well-established CNS lymphocyte entry point. To determine the contribution of A2AAR signaling in lymphocytes and the CNS during EAE, we used bone marrow chimeric mice. Remarkably, A2AAR(-/-) donor hematopoietic cells potentiated severe EAE, whereas lack of A2AAR expression on nonhematopoietic cells protected against disease development. Although no defect in the suppressive ability of A2AAR(-/-) regulatory T cells was observed, A2AAR(-/-) lymphocytes were shown to proliferate more and produced more IFN-γ following stimulation. Despite this more proinflammatory phenotype, A2AAR antagonist treatment still protected against EAE when A2AAR(-/-) lymphocytes were adoptively transferred to T cell-deficient A2AAR(+/+) mice. These results indicate that A2AAR expression on nonimmune cells (likely in the CNS) is required for efficient EAE development, while A2AAR lymphocyte expression is essential for limiting the severity of the inflammatory response.

The blood-brain barrier (BBB) is comprised of specialized endothelial cells that form the capillary microvasculature of the CNS and is essential for brain function. It also poses the greatest impediment in the treatment of many CNS diseases because it commonly blocks entry of therapeutic compounds. Here we report that adenosine receptor (AR) signaling modulates BBB permeability in vivo. A(1) and A(2A) AR activation facilitated the entry of intravenously administered macromolecules, including large dextrans and antibodies to β-amyloid, into murine brains. Additionally, treatment with an FDA-approved selective A(2A) agonist, Lexiscan, also increased BBB permeability in murine models. These changes in BBB permeability are dose-dependent and temporally discrete. Transgenic mice lacking A(1) or A(2A) ARs showed diminished dextran entry into the brain after AR agonism. Following treatment with a broad-spectrum AR agonist, intravenously administered anti-β-amyloid antibody was observed to enter the CNS and bind β-amyloid plaques in a transgenic mouse model of Alzheimer's disease (AD). Selective AR activation resulted in cellular changes in vitro including decreased transendothelial electrical resistance, increased actinomyosin stress fiber formation, and alterations in tight junction molecules. These results suggest that AR signaling can be used to modulate BBB permeability in vivo to facilitate the entry of potentially therapeutic compounds into the CNS. AR signaling at brain endothelial cells represents a novel endogenous mechanism of modulating BBB permeability. We anticipate these results will aid in drug design, drug delivery and treatment options for neurological diseases such as AD, Parkinson's disease, multiple sclerosis and cancers of the CNS.

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.

The blood-brain barrier (BBB) of the central nervous system (CNS) consists of a unique subset of endothelial cells that possess tight junctions which form a relatively impervious physical barrier to a large variety of blood components. Until recently, there have been no good in vitro models for studying the human BBB without the co-culture of feeder cells. The hCMEC/D3 cell line is the first stable, well-differentiated human brain endothelial cell line that grows independently in culture with characteristics that closely resemble those of resident human brain endothelial cells. As our previously published findings demonstrated the importance of adenosine receptor (AR) signaling for lymphocyte entry into the CNS, we wanted to determine if human brain endothelial cells possess the capacity to generate and respond to extracellular adenosine. Utilizing the hCMEC/D3 cell line, we determined that these cells express CD73, the cell surface enzyme that converts extracellular AMP to adenosine. When grown under normal conditions, these cells also express the A(1), A(2A), and A(2B) AR subtypes. Additionally, hCMEC/D3 cells are responsive to extracellular AR signaling, as cAMP levels increase following the addition of the broad spectrum AR agonist 5'-N-ethylcarboxamidoadenosine (NECA). Overall, these results indicate that human brain endothelial cells, and most likely the human BBB, have the capacity to synthesize and respond to extracellular adenosine.

The Aryl Hydrocarbon Receptor (AHR) is a basic helix-loop-helix transcription factor, implicated as an important modulator of the immune system and early thymocyte development. Previously we have shown that AHR activation by the environmental contaminant and potent AHR agonist, 2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) leads to a significant decline in the percentage of S phase cells in the CD3-CD4-CD8- triple negative stage 3 (TN3) and TN4 T-cell committed thymocytes 9-12 hours after exposure. In the more immature TN1 or TN2 stage cells no effect on cell cycle was observed. In order to identify early molecular targets, which could provide insight into how the AHR acts as a modulator of thymocyte development and cell cycle regulation, we performed gene profiling experiments using RNA isolated from four intrathymic progenitor populations where the AHR was activated for 6 or 12 hours. This microarray analysis of AHR activation identified 108 distinct gene probes that were significantly modulated in the TN1-4 thymocyte progenitor stages. While most of the genes identified have specific AHR recognition sequences, only seven genes were altered exclusively in the two T-cell committed stages of early thymocyte development (TN3 and TN4) in which the decline of S phase cells is seen. Moreover, all seven of these genes were reduced in expression and five of these seven are associated with cell cycle regulatory processes. These 7 genes are novel targets for modulation by the TCDD activated AHR and may be involved in the observed cell cycle arrest and suppression of early thymocyte development.

In 1937, Edward Mapother, Medical Superintendent of the Maudsley Hospital in London, took a trip around the mental hospitals of Britain's dominions in South Asia. The result was a series of documents that provide a snapshot of psychiatry in India and Ceylon in the twilight years of the British Empire. This chapter will consider Mapother's reports from a number of perspectives in order to assess the politics and the impact of an expert 'visitor' to a colonial medical system.

We examined the impact of co-expressing the inwardly rectifying potassium channel, Kir2.3, with the scaffolding protein, SAP97, and determined that co-expression of these proteins caused a ~2 fold increase in current density. A combination of techniques was used to determine if the SAP97-induced increase in Kir2.3 whole cell currents resulted from changes in the number of channels in the cell membrane, unitary channel conductance, or channel open probability. In the absence of SAP97, Kir2.3 was found predominantly in a cytoplasmic, vesicular compartment with relatively little Kir2.3 localized to the plasma membrane. The introduction of SAP97 caused a redistribution of Kir2.3 leading to prominent co-localization of Kir2.3 and SAP97 and a modest increase in cell surface Kir2.3. The median Kir2.3 single channel current in the absence of SAP97 was ~13 pS, while co-expression of SAP97 led to a wide distribution of channel events with three distinct peaks centered at 16 pS, 29 pS and 42 pS. These changes occurred without altering channel open probability, current rectification properties or pH sensitivity. Thus, association of Kir2.3 with SAP97 in HEK293 cells increased channel cell surface expression and unitary channel conductance. However, changes in single channel conductance play the major role in determining whole cell currents in this model system. We further suggest that the SAP97 effect results from SAP97 binding to the Kir2.3 C-terminal domain and altering channel conformation. Key words: Inward rectifier, PDZ domain, Channelosome, Membrane cytoskeleton.

We recently developed a phage-based system for the evolution of proteins in bacteria with expanded amino acid genetic codes. Here we demonstrate that the unnatural amino acid p-boronophenylalanine (BF) confers a selective advantage in the evolution of glycan-binding proteins. We show that an unbiased library of naive antibodies with NNK-randomized V(H) CDR3 loops converges upon mutants containing BF when placed under selection for binding to a model acyclic amino sugar. This work represents a first step in the evolution of carbohydrate-binding proteins that use a reactive unnatural amino acid "warhead" and demonstrates that a "synthetic" genetic code can confer a selective advantage by increasing the number of functional groups available to evolution.

Detection of amplitude modulation (AM) in 500 and 4000 Hz tonal carriers was measured as a function of modulation frequency from younger and older adults with normal hearing through 4000 Hz. The modulation frequency above which sensitivity to AM increased ("transition frequency") was similar for both groups. Temporal modulation transfer function shapes showed significant age-related differences. For younger subjects, AM detection thresholds were generally constant for low modulation frequencies. For a higher carrier frequency, AM detection thresholds then increased as modulation frequency further increased until the transition frequency. In contrast, AM detection for older subjects continuously increased with increasing modulation frequency, indicating an age-related decline in temporal resolution for faster envelope fluctuations. Significant age-related differences were observed whenever AM detection was dependent on temporal cues. For modulation frequencies above the transition frequency, age-related differences were larger for the lower frequency carrier (where both temporal and spectral cues were available) than for the higher frequency carrier (where AM detection was primarily dependent on spectral cues). These results are consistent with a general age-related decline in the synchronization of neural responses to both the carrier waveform and envelope fluctuation.