A Novel Rubric to Evaluate Wearable Cameras for Assessment of Interrater Reliability.

Research has reported on traditional methods of assessing interrater reliability but, currently, no such standard protocol exists for selection of alternative methods of assessing interrater reliability, such as wearable video cameras. The professions of occupational therapy and occupational science take a unique ecological perspective when evaluating individuals, which focuses on the naturalistic perspective of an individual, ideally resulting in optimal performance. Given current advancements in technology, wearable, low-cost, unobtrusive, first-person view digital video cameras are readily available for use in research. The researchers generated an original rubric for critiquing cameras, then trialed four cameras for use in a future interrater reliability study. This standardized protocol fills a gap in the field and can be used by future researchers searching for a standardized method of camera selection.

The GS Protein-coupled A2a Adenosine Receptor Controls T Cell Help in the Germinal Center.

T follicular helper (TFH) cells have been shown to be critically required for the germinal center (GC) reaction where B cells undergo class switch recombination and clonal selection to generate high affinity neutralizing antibodies. However, detailed knowledge of the physiological cues within the GC microenvironment that regulate T cell help is limited. The cAMP-elevating, Gs protein-coupled A2a adenosine receptor (A2aR) is an evolutionarily conserved receptor that limits and redirects cellular immunity. However, the role of A2aR in humoral immunity and B cell differentiation is unknown. We hypothesized that the hypoxic microenvironment within the GC facilitates an extracellular adenosine-rich milieu, which serves to limit TFH frequency and function, and also promotes immunosuppressive T follicular regulatory cells (TFR). In support of this hypothesis, we found that following immunization, mice lacking A2aR (A2aRKO) exhibited a significant expansion of T follicular cells, as well as increases in TFH to TFR ratio, GC T cell frequency, GC B cell frequency, and class switching of GC B cells to IgG1. Transfer of CD4 T cells from A2aRKO or wild type donors into T cell-deficient hosts revealed that these increases were largely T cell-intrinsic. Finally, injection of A2aR agonist, CGS21680, following immunization suppressed T follicular differentiation, GC B cell frequency, and class switching of GC B cells to IgG1. Taken together, these observations point to a previously unappreciated role of GS protein-coupled A2aR in regulating humoral immunity, which may be pharmacologically targeted during vaccination or pathological states in which GC-derived autoantibodies contribute to the pathology.

Germinal Center Hypoxia Potentiates Immunoglobulin Class Switch Recombination.

Germinal centers (GCs) are anatomic sites where B cells undergo secondary diversification to produce high-affinity, class-switched Abs. We hypothesized that proliferating B cells in GCs create a hypoxic microenvironment that governs their further differentiation. Using molecular markers, we found GCs to be predominantly hypoxic. Compared to normoxia (21% O2), hypoxic culture conditions (1% O2) in vitro accelerated class switching and plasma cell formation and enhanced expression of GL-7 on B and CD4+ T cells. Reversal of GC hypoxia in vivo by breathing 60% O2 during immunization resulted in reduced frequencies of GC B cells, T follicular helper cells, and plasmacytes, as well as lower expression of ICOS on T follicular helper cells. Importantly, this reversal of GC hypoxia decreased Ag-specific serum IgG1 and reduced the frequency of IgG1+ B cells within the Ag-specific GC. Taken together, these observations reveal a critical role for hypoxia in GC B cell differentiation.

Differential Tissue-Specific Function of Adora2b in Cardioprotection.

The adenosine A2b receptor (Adora2b) has been implicated in cardioprotection from myocardial ischemia. As such, Adora2b was found to be critical in ischemic preconditioning (IP) or ischemia/reperfusion (IR) injury of the heart. Whereas Adora2b is present on various cells types, the tissue-specific role of Adora2b in cardioprotection is still unknown. To study the tissue-specific role of Adora2b signaling on inflammatory cells, endothelia, or myocytes during myocardial ischemia in vivo, we intercrossed floxed Adora2b mice with Lyz2-Cre(+), VE-cadherin-Cre(+), or myosin-Cre(+) transgenic mice, respectively. Mice were exposed to 60 min of myocardial ischemia with or without IP (four times for 5 min) followed by 120 min of reperfusion. Cardioprotection by IP was abolished in Adora2b(f/f)-VE-cadherin-Cre(+) or Adora2b(f/f)-myosin-Cre(+), indicating that Adora2b signaling on endothelia or myocytes mediates IP. In contrast, primarily Adora2b signaling on inflammatory cells was necessary to provide cardioprotection in IR injury, indicated by significantly larger infarcts and higher troponin levels in Adora2b(f/f)-Lyz2-Cre(+) mice only. Cytokine profiling of IR injury in Adora2b(f/f)-Lyz2-Cre(+) mice pointed toward polymorphonuclear neutrophils (PMNs). Analysis of PMNs from Adora2b(f/f)-Lyz2-Cre(+) confirmed PMNs as one source of identified tissue cytokines. Finally, adoptive transfer of Adora2b(-/-) PMNs revealed a critical role of Adora2b on PMNs in cardioprotection from IR injury. Adora2b signaling mediates different types of cardioprotection in a tissue-specific manner. These findings have implications for the use of Adora2b agonists in the treatment or prevention of myocardial injury by ischemia.

Immunological mechanisms of the antitumor effects of supplemental oxygenation.

Antitumor T cells either avoid or are inhibited in hypoxic and extracellular adenosine-rich tumor microenvironments (TMEs) by A2A adenosine receptors. This may limit further advances in cancer immunotherapy. There is a need for readily available and safe treatments that weaken the hypoxia-A2-adenosinergic immunosuppression in the TME. Recently, we reported that respiratory hyperoxia decreases intratumoral hypoxia and concentrations of extracellular adenosine. We show that it also reverses the hypoxia-adenosinergic immunosuppression in the TME. This, in turn, stimulates (i) enhanced intratumoral infiltration and reduced inhibition of endogenously developed or adoptively transfered tumor-reactive CD8 T cells, (ii) increased proinflammatory cytokines and decreased immunosuppressive molecules, such as transforming growth factor-ß (TGF-ß), (iii) weakened immunosuppression by regulatory T cells, and (iv) improved lung tumor regression and long-term survival in mice. Respiratory hyperoxia also promoted the regression of spontaneous metastasis from orthotopically grown breast tumors. These effects are entirely T cell- and natural killer cell-dependent, thereby justifying the testing of supplemental oxygen as an immunological coadjuvant to combine with existing immunotherapies for cancer.

Systemic oxygenation weakens the hypoxia and hypoxia inducible factor 1α-dependent and extracellular adenosine-mediated tumor protection.

UNLABELLED: Intratumoral hypoxia and hypoxia inducible factor-1α (HIF-1-α)-dependent CD39/CD73 ectoenzymes may govern the accumulation of tumor-protecting extracellular adenosine and signaling through A2A adenosine receptors (A2AR) in tumor microenvironments (TME). Here, we explored the conceptually novel motivation to use supplemental oxygen as a treatment to inhibit the hypoxia/HIF-1α-CD39/CD73-driven accumulation of extracellular adenosine in the TME in order to weaken the tumor protection. We report that hyperoxic breathing (60 % O2) decreased the TME hypoxia, as well as levels of HIF-1α and downstream target proteins of HIF-1α in the TME according to proteomic studies in mice. Importantly, oxygenation also downregulated the expression of adenosine-generating ectoenzymes and significantly lowered levels of tumor-protecting extracellular adenosine in the TME. Using supplemental oxygen as a tool in studies of the TME, we also identified FHL-1 as a potentially useful marker for the conversion of hypoxic into normoxic TME. Hyperoxic breathing resulted in the upregulation of antigen-presenting MHC class I molecules on tumor cells and in the better recognition and increased susceptibility to killing by tumor-reactive cytotoxic T cells. Therapeutic breathing of 60 % oxygen resulted in the significant inhibition of growth of established B16.F10 melanoma tumors and prolonged survival of mice. Taken together, the data presented here provide proof-of principle for the therapeutic potential of systemic oxygenation to convert the hypoxic, adenosine-rich and tumor-protecting TME into a normoxic and extracellular adenosine-poor TME that, in turn, may facilitate tumor regression. We propose to explore the combination of supplemental oxygen with existing immunotherapies of cancer. KEY MESSAGES: Oxygenation decreases levels of tumor protecting hypoxia. Oxygenation decreases levels of tumor protecting extracellular adenosine. Oxygenation decreases expression of HIF-1alpha dependent tumor-protecting proteins. Oxygenation increases MHC class I expression and enables tumor regression.