Fracture of the vertebral endplates, but not equienergetic impact load, promotes disc degeneration in vitro

Vertebral endplate damage is associated with intervertebral disc (IVD) degeneration (DD) in vivo as confirmed by in‐vitro investigations. Our aims were to further characterize the process of DD using an in vitro full‐organ culture model and to elucidate whether significant endplate damage or impact loading alone is pivotal for the initiation of DD. Rabbit spinal segments (n = 80) were harvested, subjected to pure axial impact loading (n = 40) using a custom‐made device, and cultured for 28 days. The applied threshold energy (0.76 J) induced endplate fractures in 21 specimens (group A); 19 remained intact (group B). Markers for DD (cell viability, apoptosis, necrosis, matrix remodeling, and inflammation) were monitored for 28 days post‐trauma in the annulus fibrosus (AF) and nucleus pulposus and compared to non‐impacted control discs. Cell viability in both groups stayed at a control level. Group A compared to group B showed enhanced lactate dehydrogenase (LDH) and caspase‐3/7 activity, reduced glycosaminoglycan content, reduced aggrecan mRNA, but elevated mRNA for collagen‐2, catabolic enzymes (MMP‐1/‐3/‐13), and pro‐inflammatory (TNFα, IL‐6, IL‐8, MCP‐1) and pro‐apoptotic (fas ligand, caspase‐3) proteins. Group B compared to control only showed small changes in mRNA levels. Our findings demonstrate that burst endplates, but not equienergetic loading, promotes DD. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:809–816, 2012     

Acute oliguric renal failure in HELLP syndrome: case report and review of literature

We describe a 32-year-old female with past medical history of preeclampsia, who presented at 29th week of gestation of her second pregnancy with abdominal pain, emesis, and diarrhea. Initial evaluation revealed hypertension, placental abruption, and intrauterine fetal death. After spontaneous rupture of membranes, a stillborn fetus was delivered. The clinical course was complicated by seizures and acute kidney injury requiring hemodialysis. She also exhibited microangiopathic hemolytic anemia, thrombocytopenia, and elevated liver enzymes (consistent with HELLP syndrome). A biopsy showed acute renal cortical necrosis.     

Australian plantation inventory: ownership changes,availability and policy

The Australian National Plantation Inventory has collected and collated plantation information since 1993 and has periodically published forecasts of availability based on those data. This paper outlines the past methodology and summarises updates of the most recent forecasts. The failures of some managed investment scheme (MIS) forestry companies have drawn attention to the risks and difficulties involved in forecasting plantation wood yields for species and areas where few data are available. These issues have important implications for forecasts of availability but several unknowns still exist, especially in relation to future replanting. The accuracy of prospectus forecasts of yields from several MISs is examined. The methods used in national and selected regional forecasts are reviewed and some of the underlying policy issues for Australian plantations and forestry are critically examined.     

Dynamin 2–dependent endocytosis is required for sustained S1PR1 signaling

Sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) is critical for lymphocyte egress from lymphoid organs. Lymphocytes encounter low S1P concentrations near exit sites before transmigration, yet S1PR1 signaling is rapidly terminated after exposure to S1P. How lymphocytes maintain S1PR1 signaling in a low S1P environment near egress sites is unknown. Here we identify dynamin 2, an essential component of endocytosis, as a novel regulator of T cell egress. Mice with T cell–specific dynamin 2 deficiency had profound lymphopenia and impaired egress from lymphoid organs. Dynamin 2 deficiency caused impaired egress through regulation of S1PR1 signaling, and transgenic S1PR1 overexpression rescued egress in dynamin 2 knockout mice. In low S1P concentrations, dynamin 2 was essential for S1PR1 internalization, which enabled continuous S1PR1 signaling and promoted egress from both thymus and lymph nodes. In contrast, dynamin 2–deficient cells were only capable of a pulse of S1PR1 signaling, which was insufficient for egress. Our results suggest a possible mechanism by which T lymphocytes positioned at exit portals sense low S1P concentrations, promoting their egress into circulatory fluids.The egress of T lymphocytes from lymphoid organs is essential for adaptive immune responses. The exit of mature single-positive (SP) thymocytes from the thymus into blood establishes a pool of naive T cells with a diverse repertoire in peripheral organs. Egress from lymph nodes into lymph is required for the recirculation of T cells through secondary lymphoid organs and for immune surveillance. Egress from lymphoid organs is critically dependent on the binding of sphingosine-1-phosphate (S1P) to S1P receptor 1 (S1PR1) that is expressed on T cells (Matloubian et al., 2004; Pappu et al., 2007; Zachariah and Cyster, 2010; Cyster and Schwab, 2012). Sensing of S1P gradients that exist between lymphoid tissues (interstitial S1P concentration in low nanomolar range) and blood or lymph (plasma S1P concentration ∼100–1,000 nM) is required for egress (Schwab et al., 2005; Pappu et al., 2007; Cyster and Schwab, 2012). Beyond a requirement for S1PR1, the lymphocyte-intrinsic molecular mechanisms that regulate egress remain incompletely defined.S1PR1 is a G protein–coupled receptor (GPCR) with unique properties (Lee et al., 1996, 1998; Windh et al., 1999; Rivera et al., 2008; Rosen et al., 2009; Spiegel and Milstien, 2011; Cyster and Schwab, 2012). It is highly sensitive to desensitization and internalization in the continued presence of its ligand S1P (Liu et al., 1999; Schwab et al., 2005; Oo et al., 2007, 2011; Pappu et al., 2007; Arnon et al., 2011), particularly when compared with chemokine receptors and even when compared with members of the same receptor family, such as S1PR5 (Jenne et al., 2009). Receptor desensitization is mediated by GPCR kinase 2 (GRK2), which phosphorylates serine residues in the cytoplasmic tail of S1PR1 (Watterson et al., 2002; Arnon et al., 2011). Receptor phosphorylation recruits β-arrestins that sterically uncouple the receptor from heterotrimeric G proteins, thereby leading to the rapid loss of receptor responsiveness (“desensitization”). Arrestin binding also leads to GPCR internalization via clathrin-mediated endocytosis and either receptor degradation or recycling back to the cell surface (Ferguson, 2001; Pierce et al., 2002; Sorkin and von Zastrow, 2009). Receptor internalization can restore GPCR responsiveness (“resensitization”) as has been shown for the β₂-adrenergic receptor (Zhang et al., 1997).Although large S1P gradients exist between blood/lymph and lymphoid tissue, several data indicate that lymphocytes encounter small S1P gradients that likely instruct migration toward exit portals within lymphoid tissues. For example, thymocytes are attracted to egress sites at corticomedullary junctions in response to S1P produced locally by pericytes that ensheath thymic blood vessels (Zachariah and Cyster, 2010). Furthermore, S1PR1 signaling enforces internalization of the surface molecule CD69 (Shiow et al., 2006; Bankovich et al., 2010; Cyster and Schwab, 2012), a molecular timer which delays egress (Zachariah and Cyster, 2010). A prediction from these observations is the presence of an intrathymic gradient of low S1P concentration that guides thymocytes to exit sites, although technical limitations have not yet allowed direct visualization of S1P gradients within tissue (Cyster and Schwab, 2012). Given the rapid and sensitive down-regulation of S1PR1 signaling upon S1P engagement, this prediction also implies that S1PR1, after exposure to intrathymic S1P, maintains S1P responsiveness to promote thymocyte egress. However, the molecular requirements for, and the functional significance of, S1PR1 resensitization for T cell egress have not been defined.Intravital microscopy of S1PR1-deficient lymphocytes revealed that T cells approach lymph node egress sites (cortical lymphatic sinuses) efficiently, but S1PR1 is critical for the transendothelial migration step (Grigorova et al., 2009). The data on lymph node egress are consistent with a model in which a pulse of S1PR1 signaling, as opposed to sustained signaling, is sufficient for lymphocyte egress. Alternatively, egress requires sustained S1PR1 signaling, and therefore, lymphocytes need to maintain S1PR1 responsiveness even in the presence of low S1P concentrations. The latter mode of egress would predict that rapid S1PR1 resensitization mechanisms are essential for egress to occur.Binding of S1P to S1PR1 triggers both signaling through the receptor and its endocytosis (Liu et al., 1999; Oo et al., 2007, 2011), yet whether and how endocytosis regulates S1PR1 signaling are unclear. Given the critical role for dynamins in promoting the membrane scission step during endocytosis (Praefcke and McMahon, 2004; McMahon and Boucrot, 2011; Schmid and Frolov, 2011; Ferguson and De Camilli, 2012), we used mice genetically deficient in dynamin 2 (Dnm2; Ferguson et al., 2009), the dynamin isoform expressed in immune cells, to investigate the physiological function of endocytosis in T lymphocytes. We report here that dynamin 2 is critical for T cell egress from thymus and lymph nodes by directly regulating S1PR1 signaling. Surprisingly, dynamin 2–dependent endocytosis was not required for termination of S1PR1 signaling. Instead, dynamin 2 was required for S1PR1 resensitization in T lymphocytes, thereby promoting sustained S1PR1 signaling in vivo. We propose that dynamin 2–dependent endocytosis enables continuous S1PR1 signaling in T lymphocytes that are positioned near exit sites in a low S1P environment and promotes their egress into blood and lymph. Overall, our findings identify a previously unknown mechanistic link between endocytosis and T lymphocyte egress.     

Recurring Extracorporeal Circuit Clotting During Continuous Renal Replacement Therapy in Fungal Sepsis: Successful Treatment With Argatroban

The relative effectiveness of anticoagulation strategies during continuous renal replacement therapy (CRRT) may vary according to the clinical circumstances. In this study, the case of a 46-year-old man who developed fungal mediastinitis with the pathogen Scedosporium prolificans after coronary bypass surgery is reported. Numerous debridements and multiple antifungal agents were not effective in this patient. Miltefosine, a non-Food and Drug Administration-approved agent, was started after institutional review board request and approval. CRRT was initiated with regional citrate anticoagulation (RCA) for clinical sepsis with acute kidney injury. Subsequently, crescendo clotting of the extracorporeal circuit (ECC) occurred. Multiple interventions, including escalating RCA, adding increasing heparin to RCA and exchanging the dialysis catheter, were not effective. Argatroban anticoagulation was started without further ECC clotting, and the patient recovered from both acute kidney injury and septic shock, despite continued miltefosine administration. Sepsis may contribute to recurrent ECC clotting. Argatroban, a direct thrombin inhibitor, had a disproportionate effectiveness to maintain ECC patency in this patient.