Extracellular ATP and adenosine have immunoregulatory roles during inflammation. Elevated extracellular ATP is known to exacerbate GVHD, and the pharmacologic activation of the adenosine A 2A receptor is protective. However, the role of endogenous adenosine is unknown. We used gene-targeted mice and a pharmacologic inhibitor to test the role of adenosine generated by CD73/ecto-5-nucleotidase in GVHD. In allogeneic transplants, both donor and recipient CD73 were protective, with recipient CD73 playing the dominant role. CD73 deficiency led to enhanced T-cell expansion and IFN-␥ and IL-6 production, and the migratory capacity of Cd73 ؊/؊ T cells in vitro was increased. However, the number of regulatory T cells and expression of costimulatory molecules on antigen-presenting cells were unchanged. A 2A receptor deficiency led to increased numbers of allogeneic T cells, suggesting that signaling through the A 2A receptor via CD73-generated adenosine is a significant part of the mechanism by which CD73 limits the severity of GVHD. IntroductionPatients with hematologic malignancies that are refractory to conventional chemotherapy have a chance of cure by allogeneic hematopoietic stem cell transplantation (allo-HCT). 1 However, the success of this treatment is limited by GVHD. 2 We have recently shown that extracellular ATP, which is released from dying or stressed cells and serves as an endogenous danger signal to evoke systemic inflammatory responses, enhances GVHD by activation of the purinergic receptor P2X 7 R. 3,4 The abundance of extracellular ATP is regulated by ecto-nucleotidases, such as CD39, which dephosphorylates ATP to ADP and AMP. Extracellular AMP is dephosphorylated to adenosine via the action of CD73, a glycosyl phosphatidylinositol-anchored glycoprotein with ecto-5Ј-nucleotidase enzyme activity. 5-7 CD73 is expressed on many cell types, including subsets of T lymphocytes, endothelial cells, and epithelial cells. 7,8 It is also present as a secreted form lacking a glycosyl phosphatidylinositol anchor. 9 CD73-generated adenosine can activate any of 4 G-protein-coupled 7-transmembrane-spanning adenosine receptors (ARs; A 1 , A 2A , A 2B and A 3 ) and can act as either a pro-or anti-inflammatory mediator depending on the physiologic setting and the type of AR engaged. [10][11][12] In most circumstances, A 1 and A 3 receptor triggering is proinflammatory, whereas activation of A 2A and A 2B receptors is antiinflammatory or tolerogenic. 13,14 The importance of CD73 in producing adenosine for AR signaling has been revealed through studies with CD73-deficient mice. For example, CD73-generated adenosine reduces inflammation and fibrosis in lungs of bleomycin-treated mice 15 and is tolerogenic for cardiac and airway allografts. 16,17 CD73-dependent A 2B AR signaling protects mice during renal ischemia, 18 inhibits systemic vascular leakage during hypoxia, 19,20 and is also required for cardioprotection as a result of ischemic preconditioning. 21 Extracellular adenosine inhibits platelet activation and leukocyte...

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Objective To report a novel mutation in GHR and to characterize a novel mechanism of nonclassical growth hormone insensitivity. Context Laron syndrome (LS) is a well‐described disorder of growth hormone insensitivity due to mutations in the growth hormone receptor (GHR) that leads to short stature. Biochemically, LS patients classically have elevated levels of growth hormone (GH), but low levels of insulin‐like growth factor (IGF)‐1, IGF binding protein (IGFBP)‐3 and GH binding protein (GHBP). Design Case presentation with in vitro functional studies. Patients A young male Caucasian child with short stature was found to have growth hormone insensitivity manifested by elevated levels of GH and GHBP. Measurements Growth hormone stimulation tests revealed baseline GH level of 20.9 µg/L and maximum stimulated GH level of 52.7 µg/L and GHBP level of 4868 pmol/L. GHR gene sequencing revealed a novel heterozygous nonsense mutation (c.800G > A, p.Trp267*) in the transmembrane domain of the receptor. Immunoblot analysis of transfected GHR p.Trp267* in HEK293 revealed inhibition of GH‐induced STAT5 signalling that was overcome with increasing doses of recombinant human GH. Results Using an in vitro model, we show that elevated levels of GHBP inhibit the action of GH. Furthermore, our studies demonstrate that this inhibition by GHBP can be overcome by increasing doses of recombinant human GH. Conclusions To our knowledge, this is the first study to demonstrate in vitro that elevated levels of GHBP attenuate the effect of GH and inhibit GH‐induced signalling, thereby leading to short stature. Though this inhibition was overcome in vitro with supraphysiologic doses of GH, significantly above endogenously available GH, it remains to be seen whether such an effect can be replicated in vivo.

Pseudohypertriglyceridemia is an overestimation of serum triglyceride levels that may incorrectly lead to a diagnosis of hypertriglyceridemia. Glycerol kinase deficiency is a condition in which glycerol cannot be phosphorylated to glycerol-3-phosphate, resulting in elevated levels of serum glycerol. Laboratory assays that measure triglycerides indirectly may be affected by elevated glyerol levels and incorrectly report serum tryglyceride levels. We present a case of a novel missense mutation in the GK gene leading to isolated glycerol kinase deficiency and pseudohypertriglyceridemia in a male infant of a mother with gestational diabetes. This paper reviews glycerol kinase deficiency, describes the challenges in diagnosing pseudohypertriglyceridemia, and provides suggestions on improving diagnostic accuracy. Additionally, a potential maternal-fetal interaction between gestational diabetes and glycerol kinase deficiency is discussed.

312 Acute graft-versus-host disease (GvHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT). We have recently shown that extracellular adenosine triphosphate (ATP) enhances GvHD by activation of the purinergic receptor P2X7R. The abundance of extracellular ATP is regulated by ecto-nucleotidases such as CD39, which dephosphorylate ATP to ADP and AMP. Ecto-5'-nucleotidase (CD73) degrades AMP to adenosine, which can itself exert tolerogenic functions via activation of A2A and/or A2B adenosine receptors (AR). Extracellular ATP and adenosine have immunoregulatory roles under different inflammatory conditions. Deficiency of CD73, the enzyme that metabolizes AMP to adenosine, could therefore have pro- or antiinflammatory activity by reducing adenosine levels in the microenvironment. Currently, the roles of CD73 and adenosine in GvHD are unclear. By using cd73−/− donor or recipient mice and a specific CD73/ecto-5'-nucleotidase inhibitor we observed enhanced GvHD severity when CD73 was genetically deleted or pharmacologically blocked. CD73 was upregulated on CD4+ and CD11c+ cells after irradiation, suggesting an inducible rescue mechanism along with CD39 to counteract ATP accumulation resulting from tissue damage due to the preconditioning treatment. CD73 deficiency led to enhanced T cell expansion and IFN-γ and IL-6 production in GvHD mice. Migratory capacity of cd73−/− T cells was increased, compatible with the ability of adenosine to inhibit transendothelial migration. A2A receptor deficiency led to increased numbers of proliferating allogeneic T cells in allo-HCT recipient mice, suggesting that signaling through the A2A receptor via CD73-generated adenosine is a significant part of the mechanism by which CD73 limits the severity of GvHD. Furthermore, pharmacological blockade of CD73 improved graft-versus-tumor activity in an in vivo B cell leukemia model. In conclusion, we demonstrate that CD73 plays a protective role in GvHD with enhanced disease severity in its absence, while blocking CD73 led to increased graft-versus-tumor effect. These data have potential clinical implications as the alloimmune response could be enhanced by CD73 blocking in patients with residual tumor burden after transplantation. Conversely, immune modulation by addition of CD73 enzyme or an adenosine receptor agonist could improve the outcome of GvHD patients. Disclosures: No relevant conflicts of interest to declare.