Intravascular hemolytic diseases such as sickle cell anemia as well as diseases such as sepsis/DIC in which intravascular hemolysis occurs are frequently complicated by micro- and macrovascular thrombosis but mechanisms underlying this association are unclear. Plasma heme levels in sickle cell anemia are typically 5-30 µM. LPS is a prototypic and potent agonist for peripheral blood monocyte tissue factor expression and by this mechanism contributes to thrombosis in sepsis. Because heme, like LPS, signals by binding to and activating TLR4, we hypothesized that heme would also stimulate tissue factor expression in monocytes and so promote thrombosis in sickle cell anemia. We isolated human peripheral blood mononuclear cells (PBMC) and monocytes, incubated them 4 hours in the presence or absence of 10 µM hemin or 10 ng/ml LPS, made whole cell lysates by freeze-thawing and sonication and measured tissue factor activity using a one-stage clotting assay employing recalcified citrated human plasma. The assay was standardized with recombinant human tissue factor and the tissue factor dependence of the assay was confirmed using the neutralizing anti-human tissue factor antibody ATF. Hemin stimulated PBMC TF activity ca. 40-fold (range 6- to 296-fold) from 53 to 1885 pg/ml/million cells, an extent comparable to that of LPS (1939 pg/ml/million cells). In isolated monocytes heme increased TF activity ca. 70-fold (range 25-115-fold) to 9800 pg/ml/million cells, again comparable to LPS (8500 pg/ml/million cells.) Heme stimulation of monocyte TF expression did not reflect endotoxin contamination of our hemin preparation because it was 1) reproduced with a preparation of hemin made for infusion into patients with intermittent porphyria that had no detectable endotoxin; 2) unaffected by addition of 1 µg polymyxin B, which abrogated LPS stimulation; 3) blocked by 15 µM hemopexin, a high affinity heme-binding protein. qRT-PCR of TF expression in monocytes shows a 140-350-fold increase in TF mRNA levels over baseline between 2 and 4 hours after exposure to heme. Examining pathways for heme signaling, we demonstrate that heme stimulation of TF expression in monocytes is impaired > 90% by inhibitors of TLR4 (TAK242), NADPH oxidase (diphenylene iodonium), PKC (5 µM calphostin) and ERK ½ (10 µM U-0126), impaired 75% by the p50 NF-kB inhibitor 10 µg/ml andrographolide and unaffected by 100 nM wortmannin, an AKT/PI3K pathway inhibitor. We conclude that heme, at concentrations found in intravascular hemolytic diseases such as sickle cell anemia, is a highly potent stimulant of blood monocyte TF transcriptional expression dependent upon signaling through TLR4, PKC, NADPH oxidase, ERK ½ and NF-kB. By this direct mechanism heme may promote thrombosis and contribute to the pathogenesis of sickle cell anemia and other diseases characterized by intravascular hemolysis, including sepsis/DIC. Disclosures: No relevant conflicts of interest to declare.

PMN and eosinophils (EO) activated by physiologic agonist such as LPS, C5a, GM-CSF and IL-5 form extracellular traps (ET) comprised of extracellular DNA, histones, and active secondary granule constituents that retain microbicidal capacity and thus contribute to host defense but also participate in the pathogenesis of sepsis, microangiopathy, vasculitis, asthma and thrombosis. Previous studies have implicated superoxide, H2O2 and myeloperoxidase (MPO) in PMN ET formation, which reflects a novel death pathway dubbed “ETosis.” Thiocyanate (SCN-) is the principle physiologic substrate for eosinophil peroxidase (EPO) and a major substrate for MPO. Because previous studies of ET were all performed in the absence of SCN- and HOSCN, the product of SCN- peroxidation, is a weak, sulfhydryl-specific oxidant markedly less toxic than HOCl or HOBr, we hypothesized SCN- blocks PMN and EO ET formation. We also hypothesized that heme, elevated levels of which occur in intravascular hemolytic states such as sickle cell disease, induces PMN ETosis because both LPS and heme signal by engaging TLR4. We assessed ET formation by human PMN stimulated with either GM-CSF + C5a or 10 µM heme in RPMI one hour using glass slide-attached leukocytes with both cell impermeant (Sytox Orange) and permeant (Syto13) DNA stains and IF localization of secondary granule proteins using confocal microscopy. ET formation in response to to both stimuli was 5-20% of PMN in stimulated cells (vs. 0% in control). The NADPH oxidase inhibitor DPI, MPO inhibitor 4-ABAH and 1 mM SCN- all diminished ET formation by >80% in response to both stimuli. Heme-dependent ET formation was inhibited 80% by the TLR4 antagonist TAK242 (see figure below in which intracellular DNA stains green and extracellular and membrane-compromised PMN DNA stains orange). PMA-induced PMN ET formation requires singlet O2 resulting from the secondary reaction of HOCl with excess H2O2. In PMN stimulated with GM-CSF + C5a, the singlet O2 scavenger edaravone (10 µM) inhibited ETs by 80%. ETs also formed in 4-8% of EOs stimulated by IL-5 + C5a, vs. 0% in control EO and 22.5% in unstimulated EO supplemented with the alternative EPO substrate 1 mM Br-. The EPO inhibitor resorcinol decreased by >90% EO ET formation stimulated with by IL-5 + C5a or by adding Br-, as did addition of SCN- or edaravone. These studies show that both PMN and EO ETosis depend upon MPO- and EPO- generated oxidants, respectively, and the presence of an alternative physiologic substrate, SCN-, markedly antagonizes ETosis. We propose that SCN- inhibits ET formation by generating HOSCN that reacts with H2O2 to yield cyanate, not singlet O2. In addition, we also demonstrate that heme is a potent inducer of PMN ETosis through a mechanism dependent upon TLR4, NADPH oxidase and MPO, raising the possibility that this pathway may directly promote the inflammation and thrombosis that contribute of the pathogenesis of sickle cell disease. Based on these findings, we speculate that dietary augmentation of SCN- to normal or supranormal levels might have clinically beneficial therapeutic effects in a variety of inflammatory states, including sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

Objectives:Tibial tubercle osteotomy (TTO) is a common procedure that is frequently used in the treatment of recurrent patellar instability and/or patellar chondrosis. Medialization of the tubercle decreases the lateral quadriceps vector of the patella resulting in load shifting away from the lateral patella. Distalization of the tubercle decreases patella height and allows for earlier containment of the patella in the bony walls of the trochlear groove. Anteriorization has been shown to be an effective treatment to unload the inferior lateral patella when chondrosis of the patella is present in this region. Current estimates of this procedure’s complication rates range from 0% to 11%. The purpose of this study was to review the complication rate following TTO performed within an academic sports medicine practice. The hypothesis was that complication rate for TTO is greater than 10% and that the rate of complications with distalization exceeds that of medialization alone.Methods:All patients between May 2009 and May 2015 who underwent a TTO were retrospectively identified. Those with at least 6 months of follow up or a complication within the first 3 months were included for data analysis. Complications were identified and labeled as either major or minor. Major complications were defined as fracture of the tibia, deep infection requiring surgical debridement, nonunion requiring revision fixation, delayed union requiring bone graft, bone stimulation, or screw exchange, arthrofibrosis requiring manipulation under sedation and/or open lysis of adhesions, loss of fixation of the tubercle fragment, and deep vein thrombosis (DVT) whereas minor complications were defined as removal of symptomatic hardware, superficial wound infection, disturbance of cutaneous sensation, and delay in wound healing not requiring surgery.Results:During the study period, 126 TTO were performed. Representing the study cohort are 111 patients, who have at least 6 months of follow up or a complication within 3 months. The mean follow up was 23 months. There were 62 of 126 (49.2%) TTO performed for patellofemoral instability and 23 of 126 (18.2%) for patellofemoral chondral damage. Thirty-eight osteotomies were performed for both instability and cartilage damage (30.2%). Two osteotomies were performed solely for patella alta and one TTO was performed for unspecified reason (2.4%). Of the complications, 28 came following distalization of the tubercle and 4 of these complications represent subsequent tibia fracture. Overall, the complication rate was 28.7 percent; major (17.1%) and minor (11.6%) complication rates are shown in Table 1. Subgroup analysis shows a complication rate of 54% for tubercles that were distalized versus 46% for medialization alone.Conclusion:The rate of total complication for TTO was 28.7%, this is greater than the estimated rate of complication in the current literature. Further, the rate of complications when the tibial tubercle was distalized was greater than when medialized alone suggesting that special considerations...

Background There is uncertainty about the long-term risks of living kidney donation. Well-designed studies with controls well-matched on risk factors for kidney disease are needed to understand the attributable risks of kidney donation. Methods The goal of the Minnesota Attributable Risk of Kidney Donation (MARKD) study is to compare the long-term (> 50 years) outcomes of living donors (LDs) to contemporary and geographically similar controls that are well-matched on health status. University of Minnesota (n = 4022; 1st transplant: 1963) and Mayo Clinic LDs (n = 3035; 1st transplant: 1963) will be matched to Rochester Epidemiology Project (REP) controls (approximately 4 controls to 1 donor) on the basis of age, sex, and race/ethnicity. The REP controls are a well-defined population, with detailed medical record data linked between all providers in Olmsted and surrounding counties, that come from the same geographic region and era (early 1960s to present) as the donors. Controls will be carefully selected to have health status acceptable for donation on the index date (date their matched donor donated). Further refinement of the control group will include confirmed kidney health (e.g., normal serum creatinine and/or no proteinuria) and matching (on index date) of body mass index, smoking history, family history of chronic kidney disease, and blood pressure. Outcomes will be ascertained from national registries (National Death Index and United States Renal Data System) and a new survey administered to both donors and controls; the data will be supplemented by prior surveys and medical record review of donors and REP controls. The outcomes to be compared are all-cause mortality, end-stage kidney disease, cardiovascular disease and mortality, estimated glomerular filtration rate (eGFR) trajectory and chronic kidney disease, pregnancy risks, and development of diseases that frequently lead to chronic kidney disease (e.g. hypertension, diabetes, and obesity). We will additionally evaluate whether the risk of donation differs based on baseline characteristics. Discussion Our study will provide a comprehensive assessment of long-term living donor risk to inform candidate living donors, and to inform the follow-up and care of current living donors.