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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) may manifest as thrombosis, stroke, renal failure, myocardial infarction, and thrombocytopenia, reminiscent of other complement- mediated diseases. Multiple clinical and preclinical studies have implicated complement in the pathogenesis of COVID-19 illness. We previously found that the SARS-CoV-2 spike protein activates the alternative pathway of complement (APC) in vitro through interfering with the function of complement factor H, a key negative regulator of APC. Here, we demonstrated that serum from 58 COVID-19 patients (32 patients with minimal oxygen requirement, 7 on high flow oxygen, 17 requiring mechanical ventilation and 2 deaths) can induce complementmediated cell death in a functional assay (the modified Ham test) and increase membrane attack complex (C5b-9) deposition on the cell surface. A positive modified Ham assay (>20% cell-killing) was present in 41.2% COVID-19 patients requiring intubation (n=7/17) and only 6.3% in COVID-19 patients requiring minimal oxygen support (n=2/32). C5 and factor D inhibition effectively mitigated the complement amplification induced by COVID-19 patient serum. Increased serum factor Bb level was associated with disease severity in COVID-19 patients, suggesting that APC dysregulation plays an important role. Moreover, SARS-CoV-2 spike proteins directly block complement factor H from binding to heparin, which may lead to complement dysregulation on the cell surface. Taken together, our data suggest that complement dysregulation contributes to the pathogenesis of COVID-19 and may be a marker of disease severity.

Objective To investigate the incidence and potential risk factors for development of fenofibrate-associated nephrotoxicity in gout patients. Methods A total of 983 gout patients on fenofibrate treatment who visited the dedicated Gout Clinic at the Affiliated Hospital of Qingdao University between September 2016 and June 2020 were retrospectively enrolled from the electronic records system. Fenofibrate-associated nephrotoxicity was defined as an increase in serum creatinine (SCr) ≥0.3 mg/dl within 6 months of fenofibrate initiation. The change trend of SCr and uric acid levels during the treatment period were assessed by a generalised additive mixed model (GAMM). Multivariate analysis was performed for risk factors affecting elevated SCr. Results A total of 100 (10.2%) patients experienced an increase in SCr ≥0.3 mg/dl within 6 months after fenofibrate initiation. The median change of SCr in the whole cohort was 0.11 mg/dl [interquartile range (IQR) 0.03–0.20], whereas it was 0.36 (0.33–0.45) in the fenofibrate-associated nephrotoxicity group. In a multivariable regression model, chronic kidney disease (CKD) [odds ratio (OR) 2.39 (95% CI 1.48, 3.86)] and tophus [OR 2.29 (95% CI 1.39, 3.78)] were identified to be risk predictors, independent of measured covariates, of fenofibrate-associated nephrotoxicity. During the treatment period, although SCr temporarily increased, serum urate and triglyceride concentrations decreased using the interaction analysis of GAMM. Of those with fenofibrate withdrawal records, the SCr increase in 65% of patients was reversed after an average of 49 days off the drug. Conclusions This observational study implied that fenofibrate-associated nephrotoxicity occurs frequently in gout patients, especially in patients with tophi or CKD. The potential renal risks of fenofibrate usage in gout needs additional research.

Terminal complement inhibition is the standard of care for atypical hemolytic uremic syndrome (aHUS). The optimal duration of complement inhibition is unknown, although indefinite therapy is common. Here, we present the outcomes of a physician-directed eculizumab discontinuation and monitoring protocol in a prospective cohort of 31 patients that started eculizumab for acute aHUS (and without a history of renal transplant). Twenty-five (80.6%) discontinued eculizumab therapy after a median duration on therapy of 2.37 (interquartile range: 1.06, 9.70) months. Eighteen patients discontinued per protocol and 7 because of nonadherence. Of these, 5 (20%) relapsed; however, relapse rate was higher in the case of nonadherence (42.8%) vs clinician-directed discontinuation and monitoring (11.1%). Four of 5 patients who relapsed were successfully retreated without a decline in renal function. One patient died because of recurrent aHUS and hypertensive emergency in the setting of nonadherence. Nonadherence to therapy (odds ratio, 8.25; 95% confidence interval, 1.02-66.19; P = .047) was associated with relapse, whereas the presence of complement gene variants (odds ratio, 1.39; 95% confidence interval, 0.39-4.87; P = .598) was not significantly associated with relapse. Relapse occurred in 40% (2 of 5) with a CFH or MCP variant, 33.3% (2 of 6) with other complement variants, and 0% (0 of 6) with no variants (P = .217). There was no decline in mean glomerular filtration rate from the date of stopping eculizumab until end of follow-up. In summary, eculizumab discontinuation with close monitoring is safe in most patients, with low rates of aHUS relapse and effective salvage with eculizumab retreatment in the event of recurrence.

There is a Blood Commentary on this article in this issue., Key Points SARS-CoV-2 spike proteins bind heparan sulfate and activate the alternative complement pathway on cell surfaces. Factor D inhibitor (ACH145951) blocks the complement activation induced by SARS-CoV-2 spike proteins., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious respiratory virus that can lead to venous/arterial thrombosis, stroke, renal failure, myocardial infarction, thrombocytopenia, and other end-organ damage. Animal models demonstrating end-organ protection in C3-deficient mice and evidence of complement activation in humans have led to the hypothesis that SARS-CoV-2 triggers complement-mediated endothelial damage, but the mechanism is unclear. Here, we demonstrate that the SARS-CoV-2 spike protein (subunit 1 and 2), but not the N protein, directly activates the alternative pathway of complement (APC). Complement-dependent killing using the modified Ham test is blocked by either C5 or factor D inhibition. C3 fragments and C5b-9 are deposited on TF1PIGAnull target cells, and complement factor Bb is increased in the supernatant from spike protein–treated cells. C5 inhibition prevents the accumulation of C5b-9 on cells, but not C3c; however, factor D inhibition prevents both C3c and C5b-9 accumulation. Addition of factor H mitigates the complement attack. In conclusion, SARS-CoV-2 spike proteins convert nonactivator surfaces to activator surfaces by preventing the inactivation of the cell-surface APC convertase. APC activation may explain many of the clinical manifestations (microangiopathy, thrombocytopenia, renal injury, and thrombophilia) of COVID-19 that are also observed in other complement-driven diseases such as atypical hemolytic uremic syndrome and catastrophic antiphospholipid antibody syndrome. C5 inhibition prevents accumulation of C5b-9 in vitro but does not prevent upstream complement activation in response to SARS-CoV-2 spike proteins., Visual Abstract

The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies, including anti-β2-glycoprotein-I (anti-β2GPI), that are considered central to APS pathogenesis. Based on animal studies showing a role of complement in APS-related clinical events, we used the modified Ham (mHam) assay (complement-dependent cell killing) and cell-surface deposition of C5b-9 to test the hypothesis that complement activation is associated with thrombotic events in APS. A positive mHam (and corresponding C5b-9 deposition) were present in 85.7% of catastrophic APS (CAPS), 35.6% of APS (and 68.5% of samples collected within 1 year of thrombosis), and only 6.8% of systemic lupus erythematosus (SLE) sera. A positive mHam assay was associated with triple positivity (for lupus anticoagulant, anticardiolipin, and anti-β2GPI antibodies) and recurrent thrombosis. Patient-derived anti-β2GPI antibodies also induced C5b-9 deposition, which was blocked completely by an anti-C5 monoclonal antibody, but not by a factor D inhibitor, indicating that complement activation by anti-β2GPI antibodies occurs primarily through the classical complement pathway. Finally, patients with CAPS have high rates of rare germline variants in complement regulatory genes (60%), compared with patients with APS (21.8%) or SLE (28.6%) or normal controls (23.3%), and have mutations at a rate similar to that of patients with atypical hemolytic uremic syndrome (51.5%). Taken together, our data suggest that anti-β2GPI antibodies activate complement and contribute to thrombosis in APS, whereas patients with CAPS have underlying mutations in complement regulatory genes that serve as a “second hit,” leading to uncontrolled complement activation and a more severe thrombotic phenotype.

IntroductionAchieving a goal of serum urate levels in patients with gout is an important way to prevent gout and its complications while it remains difficult with a low targeting rate worldwidely. Currently, hyperuricemia classification has not been widely applied to the management of gout owing to insufficient clinical evidences. This study aimed to evaluate the effectiveness of achieving target urate based on hyperuricemia classification in Chinese patients with gout.MethodsIn this prospective study, patients with gout receiving urate lowering therapy with benzbromarone were assigned to two groups, a renal underexcretion and an unclassified type. The primary endpoint was the proportion of patients achieving the serum urate target (ResultsTarget serum urate level was achieved in 60.5% of underexcretors compared with 39.0% of patients of the unclassified type at week 12 (P = 0.002). Blood glucose and cholesterol levels were lower in the underexcretor group compared with the unclassified type group at the end of the trial, without significant different frequencies in gout flare during the study. In subgroup analysis, stratified by body mass index and estimated glomerular filtration rate, the proportion of patients with serum urate ConclusionsThe increased achievement of target serum urate in the underexcretion group supports the use of a clinical hyperuricemia typing treatment strategy for gout.

Background Low doses of febuxostat or benzbromarone are widely used in Asian countries, but lacking studies to compare the efficacy and safety of the two urate-lowering drugs. Methods To compare the efficacy and safety of low-dose febuxostat with low-dose benzbromarone in patients with primary gout, a randomized controlled, open-label trial was performed among male patients with primary gout for urate-lowering therapy (ULT) in a dedicated gout clinic in China. Randomization was carried out by a third-party institution according to random number table. Patients were randomly assigned 1:1 to febuxostat group (Feb group) (20 mg daily) or benzbromarone group (Ben group) (25 mg daily) and treated for 12 weeks. General information and biochemical data were collected at baseline and at every visit monthly. Clinical characteristics before and after the ULT were analyzed in the two groups by SPSS and EmpowerStats software. Results Two hundred forty patients were enrolled and randomized in the two groups, with 214 patients completing 12 weeks’ ULT (105 in the Feb group and 109 in the Ben group). After 12 weeks, substantial percentages of patients in both Feb and Ben group achieved the target serum uric acid (sUA) (

Hyperuricemia (HU) is a cause of gout. Clinical studies show a link between HU and cardiovascular disease. However, the role of soluble serum urate (SU) on atherosclerosis development remains elusive. We aimed to use a new HU mouse model [Uricase/Uox knockout (KO)] to further investigate the relationship between HU and atherosclerosis. A mouse model by perivascular collar placement of induced carotid atherosclerosis was established in male Uox‐ KO mice. The Uox‐ KO mice had elevated SU levels and enhanced levels of atherosclerosis inflammatory response proteins. In contrast, Uox‐ KO mice with carotid atherosclerosis showed severe neointimal changes in histology staining consistent with increases in intimal area and increases in proliferating cell nuclear antigen (PCNA)‐ and F4/80‐positive cells. Allopurinol reduced neointimal areas induced by the perivascular collar in hyperuricemic mice, accompanied by decreased expression of PCNA‐ and F4/80‐positive cells. Urate‐lowering treatment alleviated atherosclerosis inflammatory response factors and reactive oxygen species (ROS) intensities in both collar placement Uox‐ KO mice and urate‐stimulated human umbilical vein endothelial cells (HUVECs). In vitro results using HUVECs showed ROS was induced by urate and ROS induction was abrogated using antioxidants. These data demonstrate that urate per se does not trigger atherosclerosis intima lesions in male mice. Urate worsens carotid neointimal lesions induced by the perivascular collar and urate‐lowering therapy partially abrogates the effects. The current study warrants clinical studies on the possible benefits of urate‐lowering therapy in atherosclerosis patients with HU., Clinical studies show a link between hyperuricemia (HU) and cardiovascular disease. However, the role of soluble urate on atherosclerosis development remains unclear. A mouse model of induced carotid atherosclerosis was established in a spontaneous HU mouse with the uricase gene inactivated. This study demonstrates that urate worsens carotid neointimal lesions induced by the perivascular collar and that urate‐lowering therapy partially abrogates the effects.

The increasingly growing epidemics of multidrug-resistant bacteria are becoming severe public health threat. There is in an urgent need to develop new antibacterial agents with broad-spectrum antibacterial activity and high selectivity. Here, a series of N-terminal dipeptide mimetics with an aromatic amide moiety were synthesized from amino acids. The effects of amino acid type and aromatic moiety on the biological activities of the mimetics were evaluated. The dipeptide mimetics not only showed significant broad-spectrum antibacterial activity against Gram-negative (Escherichia coli and Klebsiella pneumoniae), Gram-positive (Staphylococcus aureus) and drug-resistant bacterium MRSA (methicillin-resistant S. aureus) but also demonstrated high selectivity for S. aureus versus mammalian erythrocytes. The coupling product of L-valine with p-alkynylaniline (dipeptide mimetic 7) exhibited the best antibacterial activities with minimum inhibitory concentration (MIC) ranging from 2.5 to 5 μg/mL. Moreover, the bactericidal kinetics and multi-passage resistance tests indicated that the mimetic 7 both rapidly killed bacteria and had a low probability of emergence of antimalarial resistance. Meanwhile, the mimetic 7 possessed the ability to both inhibit bacterial biofilm formation and eradicate mature biofilm. The depolarization and destruction of the bacterial cell membrane is the main sterilization mechanism, which hinders the propensity to develop bacterial resistance. Furthermore, the mimetic 7 also showed good antineoplastic activity against gastric cancer cell (SGC 7901, IC50 = 70.8 μg/mL), while it had very low toxicity to mammalian cell (L929). The mimetics bear considerable potential to be used as antibacterial and anticancer agents to combat antibiotic resistance.

Adult-onset Still's disease (AOSD) is a rare autoinflammatory condition with unclear aetiology and highly heterogeneous manifestations, such as spiking fever, skin rash and arthralgia (or arthritis).1 2 AOSD is usually the result of both genetic and environmental factors. However, only a few genetic loci have been associated with AOSD, and none of them have reached the threshold for genome-wide significance (GWS) of p

BACKGROUND Chronic rhinosinusitis with nasal polyps (CRSwNP) is a highly heterogeneous disease and can be categorized into eosinophilic CRSwNP (eCRSwNP) and non-eosinophilic CRSwNP (neCRSwNP). Exploring effective biomarkers to distinguish endotypes is important for personalized therapies. The present study aims to evaluate the predictive value of serum MIF in CRSwNP endotypes. METHODS One hundred and twenty CRSwNP patients, including 51 eCRSwNP and 69 neCRSwNP, 40 chronic rhinosinusitis without nasal polyps (CRSsNP) patients and 40 healthy controls (HC) were enrolled. Serum MIF levels were determined by enzyme-linked immunosorbent assay (ELISA). The patients' clinical variables were analyzed for correlations with serum MIF. Receiver operating characteristic (ROC) curve and multivariate analysis were utilized to assess the predictive value of serum MIF in CRSwNP endotypes. RESULTS The serum MIF levels were significantly higher in CRSwNP group than CRSsNP group and HC group (P

Cadmium (Cd) and lead (Pb) are toxic heavy metals with endocrine-disrupting properties. We investigated the associations of low-level environmental exposure to Cd/Pb and gout status (intercritical gout, gout flare and combined gout) in a cohort study. We measured by ICP-MS the levels of Cd and Pb in blood (Cd-B and Pb-B) and urine (Cd-U and Pb-U) from 408 participants with blood and 346 participants with urine samples recruited from a hospital gout clinic. The median levels of Cd-B and Pb-B (in μg/L) in the gout flare group were 0.87 (range 0.41-2.49) and 31.54 (25.38-41.46), respectively, and the median levels of Cd-U and Pb-U in the gout flare group were 1.05 (0.69-1.91) and 3.86 (3.49-4.44), respectively. These medians were significantly higher than those in the control or intercritical groups (P 0.05). For Cd-B in tertile 2 (T2) and Cd-U in tertile 3, Cd levels were significantly associated with gout flare status compared to the reference tertile 1 (OR = 4.3, P = 0.041 and OR = 25.1, P = 0.002, respectively) after adjustment under Model 3. For Pb-U, the risk of gout flare status was significantly higher in T2 (OR = 51.0, P = 0.002) compared to the T1 under Model 3. Our results show that median levels of Cd-B, Pb-B, Cd-U and Pb-U in the gout flare group were significantly higher than participants without gout or with gout but in the intercritical period. We provide evidence that the risk of gout flare status is associated with increased Cd levels, and that blood and urine levels of Cd are a risk factor for gout flare status.

Publisher's Note: There is an Inside Blood Commentary on this article in this issue.

Gout is a chronic disease resulting from elevated serum urate (SU). Previous genome-wide association studies (GWAS) have identified dozens of susceptibility loci for SU/gout, but few have been conducted for Chinese descent. Here, we try to extensively investigate whether these loci contribute to gout risk in Han Chinese. A total of 2255 variants in linkage disequilibrium (LD) with GWAS identified SU/gout associated variants were analyzed in a Han Chinese cohort of 1255 gout patients and 1848 controls. Cumulative genetic risk score analysis was performed to assess the cumulative effect of multiple “risk” variants on gout incidence. 23 variants (41%) of LD pruned variants set (n = 56) showed nominal association with gout in our sample (p ALDH16A1), including ABCG2, SLC2A9, GCKR, ALDH2 and CNIH2, were replicated. Cumulative genetic risk score analyses showed that the risk of gout increased for individuals with the growing number (≥8) of the risk alleles on gout associated loci. Most of the gout associated loci identified in previous GWAS were confirmed in an independent Chinese cohort, and the SU associated loci also confer susceptibility to gout. These findings provide important information of the genetic association of gout.

Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome are diseases of excess activation of the alternative pathway of complement that are treated with eculizumab, a humanized monoclonal antibody against the terminal complement component C5. Eculizumab must be administered intravenously, and moreover some patients with paroxysmal nocturnal hemoglobinuria on eculizumab have symptomatic extravascular hemolysis, indicating an unmet need for additional therapeutic approaches. We report the activity of two novel small-molecule inhibitors of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 μM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA-null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome. Finally, ACH-4471 blocked alternative pathway activity when administered orally to cynomolgus monkeys. In conclusion, the small-molecule Factor D inhibitors show potential as oral therapeutics for human diseases driven by the alternative pathway of complement, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

Thirteen cationic peptidomimetics derived from amino acids bearing an alkyl or ethynylphenyl moiety that mimic the structure of cationic antibacterial peptides were designed and synthesized using a simple coupling reaction of an amino acid with a substituted amine. Antibacterial activities of the resulting peptidomimetics against drug-sensitive bacteria, such as Gram-positive Staphylococcus aureus (S. aureus) and Bacillus subtilis, Gram-negative Escherichia coli (E. coli) and Salmonella enterica, and a drug-resistant bacterium, methicillin-resistant S. aureus (MRSA), were systematically evaluated. Most peptidomimetics show significant broad-spectrum antibacterial activity. A-L-Iso-C12 (isoleucine derivative bearing a dodecyl moiety) show MICs of 2.5 μg/mL against S. aureus and 4 μg/mL against MRSA and A-L-Val-C12 (valine derivative bearing a dodecyl moiety) show MICs of 1.67 μg/mL against E. coli and 8.3 μg/mL against MRSA. A-L-Val-C12 showed low cytotoxicity toward L929 cells in comparison with SGC 7901 cells, indicating tumor-directed killing by peptidomimetics while avoiding toxicity to normal cells. The influences of type of amino acid and substituent, length of substituent, and stereochemistry of amino acids on antibacterial activity and cytotoxicity of peptidomimetics were systematically investigated. The results indicate that this series of cationic peptidomimetics derived from amino acids display antitumor activity and may be useful for treatment of bacterial infections.

Background: As a special medicinal plant in Xinjiang province, Elaeagnus angustifolia L. plays an important role in windbreak and sand fixation. Its leaves are widely used in the field of traditional Chinese medicine treatment, mainly including diarrhea, dysentery, coronary heart disease and arrhythmia. Objective: In this study, our primary goal was to understand the mechanism of action of Elaeagnus angustifolia L. leaves (EALs) in protecting Rat Schwann cells (RSC-96) against H2O2-induced oxidative stress. Materials and Methods: The study material, EALs, was collected from Xinjiang Province in China. RSC-96 cells were stimulated by H2O2. Cell Counting Kit-8 assay was used in the detection of cell viability, and the cellular apoptosis and reactive oxygen species (ROS) levels were assessed by flow cytometry. The level of nerve growth factor (NGF) was assayed through enzyme-linked immunosorbent assay, and the expression of p-Akt BAX, PI3K, Bcl-2, and Akt was analyzed by Western blot. In addition, we detected the expression level of NGF. Results: According to our results, its levels were increased after stimulation with H2O2, but the levels of ROS and apoptosis were found to be reduced. Simultaneously, after induction with H2O2, the expression of Bcl-2, PI3K, p-Akt, and Akt was increased due to the presence of EALs extract, but the expression level of BAX was reduced. According to our results, EALs protects RSC-96 cells from hydrogen peroxide stress and its antiapoptotic and antioxidant effects are mainly mediated through PI3K/Akt signaling pathway. Conclusion: EALs extracts protected RSC-96 cells against H2O2-induced oxidative stress by exerting antioxidative and antiapoptotic effects through PI3K/Akt signaling pathways.

Novel antibacterial and drug-release dual-function membranes based on the cross-linked hyperbranched cationic polymers (c-HCPM) were synthesized by cross-linkage of hyperbranched poly(4-chloromethylstyrene) (PVBC) followed by quaternary amination or quaternary phosphonium salinization. The resulting c-HCPMs have a good hydrophilicity and swelling ability and show excellent broad-spectrum antibacterial and anti-bacterial adhesion properties against gram negative Escherichia coli and gram positive Staphylococcus aureus. Meanwhile, curcumin, a medicine that can promote wound healing, was successfully loaded in the c-HCPM membranes. The curcumin release performance of the c-HCPM membranes was investigated by an in vitro drug release experiment. The curcumin loaded c-HCPMs exhibited a significant performance of controlled curcumin release. These results suggest that the dual-function of antibacterial and controlled drug-release were successfully realized by the c-HCPMs. The hyperbranched structure of PVBC having abundant terminal cations and intramolecular cavities was postulated to afford c-HCPMs the excellent antibacterial and controlled drug release performances. In this study, the influences of the kind and substituent of the cations in the membrane on the antibacterial and drug release performances of c-HCPMs were systematically investigated. In addition, hemcompatibility and cytotoxicity studies were performed on c-HCPMs, the results indicate that c-HCPMs have good biocompatibility and can be used as a potential antibacterial dressing for wounds. The curcumin loaded c-HCPM can be a potential wound dressing material that has the dual function of anti-bacterial infection and promoting wound healing.

In complement-driven thrombotic microangiopathies, failure to regulate complement activation leads to end-organ damage. The modified Ham (mHam) test measures complement-mediated killing of a nucleated cell in vitro but lacks a confirmatory assay and reliable positive controls. We demonstrate that C5b-9 accumulation on the surface of TF1 PIGAnull cells correlates with cell killing in the mHam. We also show that Sialidase treatment of cells or addition of Shiga toxin 1 to human serum serve as a more reliable positive control for the mHam than cobra venom factor or lipopolysaccharide. Simultaneously performing the mHam and measuring C5b-9 accumulation either in GVB(++) or GVB(0) MgEGTA buffer with the addition of complement pathway specific inhibitors (anti-C5 antibody or a factor D inhibitor, ACH-145951) can be used to localize defects in complement regulation. As more targeted complement inhibitors become available, these assays may aid in the selection of personalized treatments for patients with complement-mediated diseases.

BACKGROUND. HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome is a severe variant of hypertensive disorders of pregnancy affecting approximately 1% of all pregnancies, and has significant maternal and fetal morbidity. Previously, we showed that upregulation of the alternative pathway of complement (APC) plays a role in HELLP syndrome. We hypothesize that HELLP syndrome follows a 2-hit disease model similar to atypical hemolytic uremic syndrome (aHUS), requiring both genetic susceptibility and an environmental risk factor. Our objective was to perform a comparative analysis of the frequency of APC activation and germline mutations in affected women and to create a predictive model for identifying HELLP syndrome. METHODS. Pregnant women with HELLP syndrome, and healthy controls after 23 weeks of gestation were recruited, along with aHUS and thrombotic thrombocytopenic purpura participants. We performed a functional assay, the mHam, and targeted genetic sequencing in all groups. RESULTS. Significantly more participants with rare germline mutations in APC genes were present in the HELLP cohort compared with controls (46% versus 8%, P = 0.01). In addition, significantly more HELLP participants were positive for the mHam when compared with controls (62% versus 16%, P = 0.009). Testing positive for both a germline mutation and the mHam was highly predictive for the diagnosis of HELLP syndrome. CONCLUSION. HELLP syndrome is characterized by both activation of the APC and frequent germline mutations in APC genes. Similar to aHUS, treatment via complement inhibition to mitigate maternal and fetal morbidity and mortality may be possible. FUNDING. National Heart Lung and Blood Institute grants T32HL007525 and R01HL133113.

The current study investigated the mechanisms by which licochalcone C induces apoptosis of T24 human malignant bladder cancer cells. Cell viability was evaluated using an MTT assay. Apoptosis was investigated using a morphological assay, flow cytometry and a caspase‑3 activity assay. Alterations in the gene expression levels of Bcl‑2 family members were measured by semi‑quantitative reverse transcription‑polymerase chain reaction assays. The protein levels of pro‑caspase‑3 and cleaved poly(ADP ribose) polymerase were measured using western blotting. The results indicated that licochalcone C induced T24 cell apoptosis in a concentration‑dependent manner. Licochalcone C treatment reduced the levels of the anti‑apoptotic mRNAs (Bcl‑2, Bcl‑w and Bcl‑XL) and increased expression of the pro‑apoptotic mRNAs (Bax and Bim). The Bcl‑2 family inhibitor (ABT‑737) reduced apoptosis induced by licochalcone C in T24 cells. The current study demonstrated that licochalcone C may be a potential adjuvant therapeutic agent for bladder cancer.

Aims The alteration of ROS level is frequently observed in the course of morphine addiction, and ROS is proverbially involved in this process. This study aims to explore the relationship among morphine addiction, reactive oxygen species (ROS) and expression of μ-opioid receptor (MOR) in differentiated SH-SY5Y cells. Main methods SH-SY5Y cells were induced to differentiation by treatment with retinoic acid (RA); the activity of lactate dehydrogenase (LDH) and the nitro blue tetrazolium (NBT) reduction were assessed by spectrophotometry. Intracellular reactive oxygen species (ROS) was measured with the 2,7-dichlorofluorescin diacetate (DCFH-DA) assay. Cellular cAMP was determined by using a competitive protein binding kit. The mRNA expression of μ-opioid receptor (MOR) was evaluated by qRT-PCR. Key findings Morphine-induced ROS are generated in a concentration- and time-dependent manner and inhibited by naloxone. Exogenous oxidants increase the level of ROS and aggravate morphine addiction, while the exogenous antioxidants efficiently reverse these effects. Morphine decreases the mRNA level of MOR in a concentration-dependent manner. And the mRNA level of MOR is remarkably reduced in the presence of exogenous oxidants and effectively promoted by antioxidants. Significance This study indicates that ROS can affect morphine addiction through involving MOR. Treatment with ROS scavenging can serve as a medical therapy for morphine addiction.

Air pollution data, air quality index (AQI) data and L-band sounding data of Wuhan City from January 1 to February 28, 2015, were used in this study. Since air quality is mainly determined by the condition of the atmospheric boundary layer structure (ABLS), a detailed analysis was carried out in order to determine the evolution of this layer and its relationship with air quality. During the investigation period, the highest value of AQI was 307 on January 26 and the lowest was 33 on February 28 indicating ‘severe pollution’ and ‘excellent’ air quality, respectively. The concentrations of PM2.5 during the days with the highest and lowest AQI were 142.61 and 9.78 μg/m3, respectively. The percentage of days in which the ratio of PM2.5 in PM10 was more than half was 83.05% which means that the greater portion of pollutants were composed of smaller particles. Moreover, four PM2.5 episodes (three or more consecutive days of PM2.5 ≥ 75 μg/m3) were identified and the average percentage of elementary carbon (EC) in PM2.5 during episode 1 (prior to the episode) was 6.274% (6.276%), episode 2 was 5.634% (7.174%), and episode 4 was 4.067% (7.785%). Higher concentrations of EC prior to episodes suggest biomass burning to be one of the reasons for episodes occurrence. Analysis of the ABLS during polluted days show that the boundary layer was dry and warm and had weak low-level wind and dominance of northerly winds. A different scenario is seen on clean days as the boundary layer is observed to be wet and cool, and there is dominance of strong winds. Back trajectory analysis results show that polluted days were dominated by air mass from north China while on clean days, the dominant air masses were from East China Sea, Mongolia, and west China.

Background Defects in the human glycosylphosphatidylinositol anchor biosynthetic pathway are associated with inherited glycosylphosphatidylinositol (GPI)-deficiencies characterized by a broad range of clinical phenotypes including multiple congenital anomalies, dysmorphic faces, developmental delay, hypotonia, and epilepsy. Biallelic variants in PIGN, encoding phosphatidylinositol-glycan biosynthesis class N have been recently associated with multiple congenital anomalies hypotonia seizure syndrome. Case presentation Our patient is a 2 year old male with hypotonia, global developmental delay, and focal epilepsy. Trio whole-exome sequencing revealed heterozygous variants in PIGN, c.181G > T (p.Glu61*) and c.284G > A (p.Arg95Gln). Analysis of FLAER and anti-CD59 by flow-cytometry demonstrated a shift in this patient’s granulocytes, confirming a glycosylphosphatidylinositol-biosynthesis defect, consistent with PIGN-related disease. Conclusions To date, a total of 18 patients have been reported, all but 2 of whom have congenital anomalies and/or obvious dysmorphic features. Our patient has no significant dysmorphic features or multiple congenital anomalies, which is consistent with recent reports linking non-truncating variants with a milder phenotype, highlighting the importance of functional studies in interpreting sequence variants. Electronic supplementary material The online version of this article (10.1186/s12881-017-0481-9) contains supplementary material, which is available to authorized users.

Mutations in genes involved in glycosylphosphatidylinositol (GPI) anchor biosynthesis underlie a group of congenital syndromes characterized by severe neurodevelopmental defects. GPI anchored proteins have diverse roles in cell adhesion, signaling, metabolism and complement regulation. Over 30 enzymes are required for GPI anchor biosynthesis and PIGA is involved in the first step of this process. A hypomorphic mutation in the X-linked PIGA gene (c.1234C>T) causes multiple congenital anomalies hypotonia seizure syndrome 2 (MCAHS2), indicating that even partial reduction of GPI anchored proteins dramatically impairs central nervous system development, but the mechanism is unclear. Here, we established a human induced pluripotent stem cell (hiPSC) model containing the PIGAc.1234C>T mutation to study the effects of a hypomorphic allele of PIGA on neuronal development. Neuronal differentiation from neural progenitor cells generated by EB formation in PIGAc.1234C>T is significantly impaired with decreased proliferation, aberrant synapse formation and abnormal membrane depolarization. The results provide direct evidence for a critical role of GPI anchor proteins in early neurodevelopment. Furthermore, neural progenitors derived from PIGAc.1234C>T hiPSCs demonstrate increased susceptibility to complement-mediated cytotoxicity, suggesting that defective complement regulation may contribute to neurodevelopmental disorders.

This study evaluates antioxidative and cardioprotective effects of total flavonoids extracted from Dracocephalum moldavica L. (DML). The total flavonoids showed remarkable scavenging effects against 1,1-diphenyl-2-picrylhydrazyl, hydroxyl and superoxide anion radicals in vitro. Compared with the ischemia/reperfusion (I/R) group as demonstrated by the use of improved Langendorff retrograde perfusion technology, the total flavonoids (5 μg/mL) pretreatment improved the heart rate and coronary flow, rised left ventricular developed pressure and decreased creatine kinase, lactate dehydrogenase levels in coronary flow. The infarct size/ischemic area at risk of DML-treated hearts was smaller than that of I/R group; the superoxide dismutase activity and glutathione/glutathione disulfide ratio increased and malondialdehyde content reduced obviously (P < 0.01) in total flavonoids treatment groups. In conclusion, the total flavonoids possess obvious protective effects on myocardial I/R injury, which may be related to the improvement of myocardial oxidative stress states.

The aim of this study was to determine the relationship between proliferation inhibition and the production of reactive oxygen species (ROS) induced by Licochalcone A (LCA). Cell viability was evaluated using sulforhodamine B (SRB) assay. Intracellular ROS level was assessed using the 2, 7-dichlorofluorescein diacetate (H2DCFDA) probe and dihydroethidium (DHE) probe assay. The results indicate that LCA inhibits human bladder cancer T24 proliferation in a concentration-dependent manner, with an IC50 value of approximately 55 μM. The LCA-induced ROS production is inhibited by the co-treatment of LCA and free radical scavenger N-acetyl-cysteine (NAC), on the contrary, the proliferation rate and ROS production increase when treated by the combination of LCA and L-buthionine-(S,R)-sulfoximine (BSO). The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) decreases in a concentration-dependent manner. The results suggest that LCA inhibits proliferation by increasing intracellular ROS levels resulted in an oxidative stress status in T24 cells.

Introduction: The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity along with persistent antiphospholipid antibodies (aPL). Despite adequate anticoagulation, 10-30% of patients have recurrent thrombosis. Catastrophic APS (CAPS) is associated with approximately 40% mortality despite treatment. The pathogenesis of APS complications is incompletely understood. Recent animal studies indicate that complement is required for aPL-associated thrombosis, and complement has emerged as an attractive therapeutic target for refractory thrombotic APS and CAPS. Methods: We first evaluated complement activation in sera of patients with thrombotic APS by ISTH criteria (N=53), catastrophic APS (CAPS; N=8, sera available for 6), and systemic lupus erythematosus (SLE; N=74) who presented to our institution from June 2015 to June 2019 (and four patients with CAPS from other institutions). We used the modified Ham (mHam) test, a functional assay for complement activation as described previously (Gavriilaki et al. Blood 2015). The mHam assay is based on the principle that a PNH cell line (PIGA-null TF-1 cells) lacking the cell surface complement regulators CD55 and CD59 undergoes lysis in serum containing activated complement. Cell death (measured by a cell viability assay) is a measure of complement activation. Cell surface deposition of complement products (C3c, C5b-9) is also detected by flow cytometry. We then evaluated whether adding purified patient-derived aPL (anti-β2 glycoprotein IgG) to normal serum induced complement activation. Finally, we performed targeted sequencing of 15 complement genes in the study subjects, as well as 22 patients with aHUS and 36 healthy individuals as positive and negative controls, respectively. Results: (A) Complement activation is associated with thrombotic APS. A positive mHam assay (>20% cell killing) was detected in 32.1% (17 of 53) patients with thrombotic APS and 100% (6 of 6 with available sera) of CAPS compared with 6.8% (5 of 74) with SLE, (P (B) aPL from patients activate complement in vitro. Patient-derived anti- β2 glycoprotein IgG from all four patients induced complement activation in the mHam assay (Fig 2A). Flow cytometry confirmed cell surface deposition of complement activation products (C4d, C5b-9), which was inhibited by adding anti-C5 monoclonal Ab or a factor D inhibitor (representative sample in fig. 1B). (C) Catastrophic APS is associated with complement mutations. Rare (minor allele frequency Conclusions: APS serum activates complement in vitro shown by a functional assay (mHam) and increased C5b-9 deposition on the cell surface. A positive mHam test strongly associates with both recent thrombosis and triple positive APS. Purified human anti-B2GPI antibody from APS patients activates complement when added to normal human serum, suggesting that complement activation plays a pathophysiologic role in APS associated thrombosis. Finally, CAPS patients have a high rate of mutations in complement genes, which likely serves as a 'second-hit' (in addition to aPL) leading to uncontrolled complement activation and a more severe phenotype (Figure 3). Taken together, our results provide a rationale for complement inhibition as a therapeutic strategy in patients with CAPS and refractory thrombotic APS. Disclosures Chaturvedi: Shire/Takeda: Research Funding; Sanofi: Consultancy; Alexion: Consultancy. Streiff:Pfizer: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Portola: Consultancy, Honoraria; Roche: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria. Petri:Astellas: Consultancy; Novartis: Consultancy; Exagen: Consultancy, Research Funding; Glenmark Pharmaceuticals: Consultancy; EMD Serono: Consultancy; Bristol-Myers Squibb: Consultancy; IQVIA: Consultancy; Janssen Pharmaceuticals: Consultancy; Aleon Pharmaceuticals: Consultancy; Momenta Pharmaceuticals: Consultancy; Blackrock Pharmaceuticals: Consultancy; Astrazeneca: Consultancy, Research Funding; UCB Pharmaceuticals: Consultancy; GSK: Consultancy; Qiagen: Consultancy; Abbive: Consultancy; Amgen: Consultancy; Decision Resources: Consultancy; Principia Biopharma: Consultancy; Eli Lilly: Consultancy; Kezaar Life Sciences: Consultancy. McCrae:Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Brodsky:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: Grant funding; Achillion: Research Funding.

Low non-freezing temperature is one of the major environmental factors affecting growth, development and geographical distribution of chilling-sensitive plants, Jatropha curcas is considered as a sustainable energy plants with great potential for biodiesel production. In this study, chilling shock at 5 °C followed by recovery at 26 °C for 4 h significantly improved survival percentage of J. curcas seedlings under chilling stress at 1 °C. In addition, chilling shock could obviously enhance the activities of antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR), and the levels of antioxidants ascorbic acid (AsA) and glutathione (GSH), as well as the contents of osmolytes proline and betaine in leaves of seedlings of J. curcas compared with the control without chilling shock. During the process of recovery, GR activity, AsA, GSH, proline and betaine contents sequentially increased, whereas SOD, APX and CAT activities gradually decreased, but they markedly maintained higher activities than those of control. Under chilling stress, activities of SOD, APX, CAT, GR and GPX, and contents of AsA, GSH, proline and betaine, as well as the ratio of the reduced antioxidants to total antioxidants [AsA/(AsA + DHA) and GSH/(GSH + GSSG)] in the shocked and non-shock seedlings all dropped, but shocked seedlings sustained significantly higher antioxidant enzyme activity, antioxidant and osmolyte contents, as well as ratio of reduced antioxidants to total antioxidants from beginning to end compared with control. These results indicated that the chilling shock followed by recovery could improve chilling tolerance of seedlings in J. curcas, and antioxidant enzymes and osmolytes play important role in the acquisition of chilling tolerance.

The urate oxidase ( Uox ) gene encodes uricase that in the rodent liver degrades uric acid into allantoin, forming an obstacle for establishing stable mouse models of hyperuricemia. The loss of uricase in humans during primate evolution causes their vulnerability to hyperuricemia. Thus, we generated a Uox- knockout mouse model on a pure C57BL/6J background using the transcription activator-like effector nuclease (TALEN) technique. These Uox -knockout mice spontaneously developed hyperuricemia (over 420 μmol/l) with about 40% survival up to 62 weeks. Renal dysfunction (elevated serum creatinine and blood urea nitrogen) and glomerular/tubular lesions were observed in these Uox -knockout mice. Male Uox -knockout mice developed glycol-metabolic disorders associated with compromised insulin secretion and elevated vulnerability to streptozotocin-induced diabetes, whereas female mice developed hypertension accompanied by aberrant lipo-metabolism. Urate-lowering drugs reduced serum uric acid and improved hyperuricemia-induced disorders. Thus, uricase knockout provides a suitable mouse model to investigate hyperuricemia and associated disorders mimicking the human condition, suggesting that hyperuricemia has a causal role in the development of metabolic disorders and hypertension.

Introduction: HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) is a severe variant of preeclampsia that leads to severe morbidity and mortality to both the mother and fetus. Delivery is the treatment of choice at or beyond 34 weeks, but there are no good therapies for pregnant women with HELLP before 34 weeks of pregnancy. Although there are no strict criteria for its diagnosis, the Tennessee and Mississippi classifications have been proposed using platelet count (600 IU/L), bilirubin (>1.2 mg/dL) and aspartate aminotransferase (AST) with or without alanine aminotransferase (ALT) levels (AST >40 or 70 IU/L, ALT>40 IU/L). However, LDH, AST and bilirubin are not specific for liver dysfunction and their elevation may be caused by intravascular hemolysis alone. Recent evidence and clinical similarities suggest a link to atypical hemolytic uremic syndrome (aHUS), a disease of excessive alternative complement pathway (APC) activation. To test this hypothesis we utilized a functional complement assay, the modified Ham test recently described for aHUS diagnosis. Method: Women with classic HELLP, atypical HELLP, preeclampsia with severe features, and women with normal pregnancies were recruited for the study from September 1, 2014 to May 31, 2015. Women with known sickle cell disease, systemic lupus erythematous, antiphospholipid antibody syndrome, or previous diagnosed microangiopathic and hemolytic diseases were excluded. All participants were greater than 23 weeks pregnant. Classic HELLP syndrome was defined as satisfying all Mississippi or Tennessee criteria for HELLP syndrome; while atypical HELLP as having at least one laboratory abnormality in the Mississippi or Tennessee criteria. Sera was collected and sent blinded to the laboratory. APC activation was detected in the modified Ham test and compared to previously described marker of complement activation, serum C5b-9 levels. Furthermore, we tested the in vitro ability of eculizumab to inhibit APC activation. Eculizumab containing serum was collected from a patient with paroxysmal nocturnal hemoglobinuria (PNH) within 60 minutes after the infusion. Results: Serum from 9 women with classic or atypical HELLP, 7 women with severe preeclampsia, and 11 controls (healthy pregnancy) were tested. We found no significant difference in serum C5b-9 levels among patients with HELLP, preeclampsia and controls (p=0.808). However, increased complement activation in the modified Ham test, represented as significantly higher percentage of non-viable cells/cell killing (25.7±19.8% versus 4.1±7.3%, p=0.005), was found in participants with classic or atypical HELLP compared to participants with healthy pregnancy (Figure 1A). Participants with classic HELLP demonstrated significantly more cell killing when compared to severe preeclampsia (38.7+9.8% versus 13.0+11.7, p=0.048). In the ROC (receiver operating curve) analysis, a percentage of killing higher than 20.5% was determined as a cut-off value for the diagnosis of HELLP with 66.7% sensitivity and 88.9% specificity. Importantly, mixing sera from 4 HELLP patients with eculizumab containing serum in different percentages resulted in a significant decrease of cell killing compared to HELLP serum alone (Figure 1B, p=0.007). Conclusions: We have shown that preeclampsia with severe features along with classic and atypical HELLP syndrome may be considered, at least in part, a disease of excessive complement activation. The modified Ham test is a serum-based assay that does not theoretically detect increased complement activation caused by mutations in cell membrane factors. Other pathophysiological mechanisms beyond complement activation may also account for the negative results in 3 HELLP participants. Importantly, the modified Ham test seems a promising tool to identify patients with increased complement activation who may benefit from complement inhibition. If confirmed in a larger cohort, this rapid, inexpensive and highly specific assay may be valuable to select patients for such a clinical trial. Disclosures Brodsky: Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Phosphatidylinositol glycan class A (PIGA) is involved in the first step of glycosylphosphatidylinositol (GPI) biosynthesis. Many proteins, including CD55 and CD59, are anchored to the cell by GPI. Loss of CD55 and CD59 on erythrocytes causes complement-mediated lysis in paroxysmal nocturnal hemoglobinuria (PNH), a disease that manifests after clonal expansion of hematopoietic cells with somatic PIGA mutations. Although somatic PIGA mutations have been identified in many PNH patients, it has been proposed that germline mutations are lethal. We report a family with an X-linked lethal disorder involving cleft palate, neonatal seizures, contractures, central nervous system (CNS) structural malformations, and other anomalies. An X chromosome exome next-generation sequencing screen identified a single nonsense PIGA mutation, c.1234CT, which predicts p.Arg412(∗). This variant segregated with disease and carrier status in the family, is similar to mutations known to cause PNH as a result of PIGA dysfunction, and was absent in 409 controls. PIGA-null mutations are thought to be embryonic lethal, suggesting that p.Arg412(∗) PIGA has residual function. Transfection of a mutant p.Arg412(∗) PIGA construct into PIGA-null cells showed partial restoration of GPI-anchored proteins. The genetic data show that the c.1234CT (p.Arg412(∗)) mutation is present in an affected child, is linked to the affected chromosome in this family, is rare in the population, and results in reduced, but not absent, biosynthesis of GPI anchors. We conclude that c.1234CT in PIGA results in the lethal X-linked phenotype recognized in the reported family.

INTRODUCTION: Atypical hemolytic uremic syndrome (aHUS) is a complement mediated thrombotic microangiopathy that predominantly affects the kidneys although extra-renal manifestations are common. In the pre-eculizumab era, 40-65% of patients either died or had end stage renal disease (ESRD) at 1 year. Long-term renal and cardiovascular outcomes are less well described in the eculizumab era. We conducted this cohort study to describe the renal and cardiovascular outcomes of adult survivors of aHUS, both on and off continued eculizumab therapy. METHODS: Patients with aHUS were identified from the prospective Complement Associated Disease Registry and through the Center for Clinical Data Analysis at Johns Hopkins University. Demographic and clinical data were abstracted, including details of aHUS diagnosis, laboratory studies, treatment, and outcomes including renal function, hypertension and echocardiographic studies. The prevalence of hypertension was compared between patients with and without chronic kidney disease (CKD) using the chi squared test. RESULTS: 45 individuals with aHUS were followed at Johns Hopkins Hospital with a median [interquartile range (IQR)] time since diagnosis of 37.4 [IQR 20.7, 62.6] months. Median age at diagnosis was 32.5 [IQR 23.2, 49.2] years and 71.1% were female. Acute kidney injury was present in 98% (44/45); however, neurologic (64.4%), gastrointestinal (68.8%), and cardiovascular (55.5%) involvement was also common (Table 1). Hypertensive urgency or emergency was present in 40% (18/45), while 13.3% (6/45) had an acute coronary syndrome during the acute episode (2 ST elevation myocardial infarctions and 4 non-ST elevation myocardial infarctions). Complement gene sequencing was completed for 34 patients, of which 8 had variants in CFH, one in CFH and CD46, 5 in other genes (MCP1, CFHR1 homozygous deletion, DGKE, THBD, and THBD with del(CFH-SCR20-CFHR1-int5)]and 20 patients had no pathogenic variants. Thirty-two (71.1%) patients were treated initially with plasma exchange (median 6 [3, 12] exchanges). Thirty-nine (86.7%) received eculizumab (5 started at the time of renal transplant after developing ESRD), and 20 of these (51%) have since discontinued therapy. Median duration of eculizumab therapy was 2.7 [0.9-11.3] months for those who stopped therapy and 29.5 [8.8-55] months for those who continued. One patient died due to a myocardial infarction during the aHUS episode. Of the 44 survivors, 15 (34.1%) had complete renal recovery, 9 (20.5%) had chronic kidney disease (CKD) stage 1-4, and 20 (45.5%) developed CKD stage 5 requiring dialysis at 3 months after the acute episode. Fifteen patients underwent subsequent renal transplantation. At the end of follow up, 23 (52.2%) had CKD [2.2% stage 2, 15.6% stage 3, 4.4% stage 4 and 28.9% stage 5) (Figure 1A). Although not statistically significant, there was a higher rate of CKD (63.1% versus 52.6%, P=0.511) among those not on eculizumab; however, this primarily reflects eculizumab being stopped after ESRD. Hypertension was present in 35 (79.5%) survivors (Figure 1B), of which 14 (40%) had incident hypertension. The prevalence of hypertension was not significantly different between patients with CKD and normal renal function (87% versus 71.4%, P=0.202). Thirty-one (70.4%) were on antihypertensive therapy, and 67% (21 of 31) of these were not controlled to CONCLUSION: Malignant hypertension and cardiac involvement are common during acute aHUS. aHUS survivors also have a high prevalence of hypertension, including a notable incidence of new onset as well as uncontrolled hypertension following aHUS diagnosis. CKD is present in the majority of survivors, and structural cardiopulmonary disease is common. Complement activation has been implicated in the pathogenesis of cardiovascular disease. Further investigation is needed to evaluate the epidemiology of cardiovascular sequelae in aHUS, their associations with specific complement mutations, and optimal management. Disclosures No relevant conflicts of interest to declare.

The aim of our study was to investigate the neuroprotective properties of shikonin, a naphthoquinone pigment isolated from the roots of the traditional Chinese herb Lithospermum erythrorhizon. In the present study, mice were divided randomly into sham, model, shikonin and edaravone-treated groups. Shikonin (50, 25, and 12.5 mg/kg, i.g.) or maize oil was administered three times before ischemia and once at 2 h after the onset of ischemia. Mice were anesthetized with chloral hydrate and subjected to middle cerebral artery 2 h of occlusion and then 22 h of reperfusion. Different antioxidant assays were employed in order to evaluate the antioxidant activities of shikonin in vitro. Neurological deficit, infarct size, histopathology changes and oxidative stress markers were evaluated after 22 h of reperfusion. In comparison with the model group, treatment with shikonin significantly decreased neurological deficit scores, infarct size, the levels of malondialdehyde(MDA), carbonyl and reactive oxygen species, and attenuated neuronal damage, up-regulated superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) activities and reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio. Taken together, these results suggested that the neuroprotective effects of shikonin against cerebral ischemia/reperfusion injury may be attributed to its antioxidant effects.

Recent characterization of hemangioblasts differentiated from human embryonic stem cells (hESC) has further confirmed evidence from murine, zebrafish and avian experimental systems that hematopoietic and endothelial lineages arise from a common progenitor. Such progenitors may provide a valuable resource for delineating the initial developmental steps of human hemato-endotheliogenesis, which is a process normally difficult to study due to the very limited accessibility of early human embryonic/fetal tissues. Moreover, efficient hemangioblast and hematopoietic stem cell (HSC) generation from patient-specific pluripotent stem cells has enormous potential for regenerative medicine, since it could lead to strategies for treating a multitude of hematologic and vascular disorders. However, significant scientific challenges remain in achieving these goals, and the generation of transplantable hemangioblasts and HSC derived from hESC currently remains elusive. Our previous work has suggested that the failure to derive engraftable HSC from hESC is due to the fact that current methodologies for differentiating hESC produce hematopoietic progenitors developmentally similar to those found in the human yolk sac, and are therefore too immature to provide adult-type hematopoietic reconstitution. Herein, we outline the nature of this challenge and propose targeted strategies for generating engraftable human pluripotent stem cell-derived HSC from primitive hemangioblasts using a developmental approach. We also focus on methods by which reprogrammed somatic cells could be used to derive autologous pluripotent stem cells, which in turn could provide unlimited sources of patient-specific hemangioblasts and HSC.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy (TMA) characterized by excessive activation of the alternative pathway of complement (APC). Atypical HUS is frequently a diagnosis of exclusion. Differentiating aHUS from other TMAs, especially thrombotic thrombocytopenic purpura (TTP), is difficult due to overlapping clinical manifestations. We sought to develop a novel assay to distinguish aHUS from other TMAs based on the hypothesis that paroxysmal nocturnal hemoglobinuria cells are more sensitive to APC-activated serum due to deficiency of glycosylphosphatidylinositol- anchored complement regulatory proteins (GPI-AP). Here, we demonstrate that phosphatidylinositol-specific phospholipase C-treated EA.hy926 cells and PIGA-mutant TF-1 cells are more susceptible to serum from aHUS patients than parental EA.hy926 and TF-1 cells. We next studied 31 samples from 25 patients with TMAs, including 9 with aHUS and 12 with TTP. Increased C5b-9 deposition was evident by confocal microscopy and flow cytometry on GPI-AP-deficient cells incubated with aHUS serum compared with heat-inactivated control, TTP, and normal serum. Differences in cell viability were observed in biochemically GPI-AP-deficient cells and were further increased in PIGA-deficient cells. Serum from patients with aHUS resulted in a significant increase of nonviable PIGA-deficient TF-1 cells compared with serum from healthy controls (P < .001) and other TMAs (P < .001). The cell viability assay showed high reproducibility, sensitivity, and specificity in detecting aHUS. In conclusion, we developed a simple, rapid, and serum-based assay that helps to differentiate aHUS from other TMAs.

Flavonoids are important components of ‘functional foods’, with beneficial effects on cardiovascular function. The present study was designed to investigate whether licochalcone D (LD) could be a cardioprotective agent in ischemia/reperfusion (I/R) injury and to shed light on its possible mechanism. Compared with the I/R group, LD treatment enhanced myocardial function (increased LVDP, dp/dt max, dp/dt min, HR and CR) and suppressed cardiac injury (decreased LDH, CK and myocardial infarct size). Moreover, LD treatment reversed the I/R-induced cleavage of caspase-3 and PARP, resulting in a significant decrease in proinflammatory factors and an increase in antioxidant capacity in I/R myocardial tissue. The mechanisms underlying the antiapoptosis, antiinflammation and antioxidant effects were related to the activation of the AKT pathway and to the blockage of the NF-κB/p65 and p38 MAPK pathways in the I/R-injured heart. Additionally, LD treatment markedly activated endothelial nitric oxide synthase (eNOS) and reduced nitric oxide (NO) production. The findings indicated that LD had real cardioprotective potential and provided support for the use of LD in myocardial I/R injury.

Neurodevelopmental disorders (NDDs) affect more than 3% of children and are attributable to single-gene mutations at more than 1000 loci. Traditional methods yield molecular diagnoses in less than one-half of children with NDD. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) can enable diagnosis of NDD, but their clinical and cost-effectiveness are unknown. One hundred families with 119 children affected by NDD received diagnostic WGS and/or WES of parent-child trios, wherein the sequencing approach was guided by acuity of illness. Forty-five percent received molecular diagnoses. An accelerated sequencing modality, rapid WGS, yielded diagnoses in 73% of families with acutely ill children (11 of 15). Forty percent of families with children with nonacute NDD, followed in ambulatory care clinics (34 of 85), received diagnoses: 33 by WES and 1 by staged WES then WGS. The cost of prior negative tests in the nonacute patients was $19,100 per family, suggesting sequencing to be cost-effective at up to $7640 per family. A change in clinical care or impression of the pathophysiology was reported in 49% of newly diagnosed families. If WES or WGS had been performed at symptom onset, genomic diagnoses may have been made 77 months earlier than occurred in this study. It is suggested that initial diagnostic evaluation of children with NDD should include trio WGS or WES, with extension of accelerated sequencing modalities to high-acuity patients.

This study investigated the mechanisms by which licochalcone B (LCB) inhibits the adhesion,invasion and metastasis of human malignant bladder cancer T24 cells. Cell viability was evaluated using a sulforhodamine B (SRB) assay. Cell migration and invasion ability were conducted using wound-healing assay and matrigel transwell invasion assay. The activities of matrix metalloproteinases (MMP)-2 and MMP-9 were measured by gelatin zymography protease assays. The expression in protein level of NF-κBP65 and AP-1 was determined using the ELISA method; the protein levels of MMP-9, NF-κBP65, IκBα and P-IκBα were detected by Western blot. The expression in mRNA level of MMP-9 was assessed using quantitative real-time polymerase chain reaction (PCR) and reverse transcription PCR. The results indicated that LCB attenuated T24 cell migration, adhesion and invasion in a concentration-dependent manner. LCB treatment down-regulated the mRNA expression, protein expression and activity of MMP-9 but had no effect on MMP-2. In addition, LCB treatment decreased the protein level of NF-кBP65 and nuclear translocation of NF-кB. These findings suggested that LCB attenuated migration of bladder cancer T24 cells and adhesion and invasion accompanied with down-regulated protein expression of MMP-9 and the nuclear translocation of NF-кB. Our results provide support that LCB may be a potent adjuvant therapeutic agent in the prevention and therapy of bladder cancer.

Introduction: Antiphospholipid antibody syndrome (APS) is characterized by recurrent thrombosis and pregnancy loss, in the presence of antibodies directed against phospholipid-binding proteins. Although persistence of antiphospholipid antibodies (aPL), including lupus anticoagulant (LA), anti-cardiolipin (aCL) or anti-β2-glycoprotein (aβ2-GPI) antibodies, is a key diagnostic feature of APS and its severe variant, catastrophic APS (CAPS), the mechanism by which aPL contribute to the clinical manifestations of these disorders remains unclear. There is evidence that excessive activation of complement plays a role in the pathophysiology of APS and CAPS, and limited case reports suggest a therapeutic role for complement inhibition; however, a clear mechanistic understanding of this process has not been elucidated. The modified Ham (mHam) is a functional assay for detection of systemic activation of the alternative pathway of complement (APC). Here, we show that the APC is active in serum from patients with APS and CAPs using the mHam assay, and provide evidence that inhibition of complement factor H (CFH), a homolog of β2-GPI, by aβ2-GPI antibodies is a plausible mechanistic link between APS/CAPS and excessive APC activation. Methods: The patient population studied included adults with APS who presented to our institution between January 2015 and January 2016. A diagnosis of APS or CAPS was based on diagnostic criteria from 2002 and 2006. Patients with a CAPS-like phenotype, who did not meet all criteria for definite or probable CAPS (Asherson et al. 2003), were also included. Patient serum was evaluated in the mHam using the PIGA-null TF1 cell line and percentage of cell killing was measured using a cell proliferation assay. Additional studies testing the addition of purified phospholipids or commercially available β2-GPI antibodies (and isotype controls) also utilized the mHam. Serum from 5 patients with atypical hemolytic uremic syndrome (aHUS) was used as a control in the phospholipid study. It was hypothesized that CFH, the principal negative regulator of the APC, may be the target of pathogenic aPL given its homology to β2-GPI. To test this hypothesis, competitive binding assays were performed with commercially available recombinant β2-GPI and purified CFH. Results: Serum from 18 patients with APS, 1 patient with definite CAPS, 3 patients with CAPS-like phenotype, 5 healthy volunteers and 14 patients with other hypercoagulable states (9 patients with lupus who did not meet criteria for APS, 4 patients with TTP and 1 patient with DIC) was analyzed in the mHam. APC-mediated cell killing in the mHam was higher in the serum from patients with APS and CAPS, compared to normal controls and patients with other hypercoagulable states (mean of 27.9% and 36.2% versus 5.2% and 12.2%, respectively; see Figure). Addition of purified phospholipids to serum from patients with APS reduced APC-mediated cell killing, but had no effect on serum from patients with aHUS, consistent with divergent mechanisms for APC activation in these disorders. Addition of a β2-GPI IgG antibody to normal human serum resulted in increased APC-mediated cell killing in the mHam, relative to addition of a β2-GPI IgM antibody or isotype controls, suggesting that antibodies directed against β2-GPI may directly activate the APC. Finally, the addition of CFH blocked binding of a commercially available β2-GPI IgG antibody to purified β2-GPI, supporting the hypothesis that aβ2-GPI antibodies may promote excessive activation of the APC in APS and CAPS by binding to and inhibiting CFH. Conclusions: Systemic activation of the APC can be demonstrated by the mHam test in serum from patients with APS and CAPS. This suggests that, in at least a subset of patients with APS and CAPS, excessive activation of the APC may be an important driver of the disease manifestations. Further study is required to determine whether there is a relationship between seropositivity for LA, aCL and aβ2-GPI and activation of the APC. Autoantibodies directed against β2-GPI may directly activate the APC by inhibiting CFH, providing further rationale for the potential use of therapies aimed at complement inhibition in these devastating disorders. Figure Figure. Disclosures Streiff: Janssen: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding; Portola: Research Funding; Roche: Research Funding. Brodsky:Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Achillion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees.

Introduction: The complement system can be activated via three pathways: classical pathway (CP), lectin pathway (LP) and alternative pathway (AP). While the CP and LP are triggered in the solid phase upon interaction of a pattern-recognition molecule with a target surface, the AP can be activated in the fluid phase. In fact, under normal physiological conditions, the AP is constitutively activated at a low level in the fluid phase via a mechanism of "C3 tickover" which leads to production of the C3b that can bind covalently to adjacent target cells or its binding activity is lost very rapidly. On the normal red cells, the bound C3b molecules are rapidly inactivated by an array of membrane-expressed or fluid phase-recruited complement regulators. However, due to the deficiency of two membrane-expressed negative regulators CD55 and CD59, the bound C3b molecules on the PNH red cells are not only amplified via the AP loop but also proceed to form the C3 convertase, the C5 convertase and ultimately the membrane attack complex, which causes hemolysis of PNH red cells. Hence, the blockade of both AP activation in the fluid phase and AP amplification on the surface of PNH red cells with a complement factor D (CFD) inhibitor is expected to be efficacious for PNH indication since hemolysis is due to the constitutive fluid phase AP activation. In this report, we evaluated whether hemolysis of PNH red cells occurs under the conditions in which the CP, LP and/or AP are activated by various pathogens in vitro. Our objective was to determine whether there is "bystander" hemolysis due to massive pathogen-driven complement activation and the potential for resulting C3b to bind PNH red cells and lead to complement-mediated hemolysis through AP amplification. Methods: Blood was obtained from PNH patients with written informed consent. Pathogen inoculums, such as E. coli and Neisseria meningitidis(NM), were prepared by standard methods. Hemolysis of PNH red cells was assessed with ~80% ABO blood type-matched pooled normal human serum (NHS) in GVB0-Mg-EGTA buffer (pH6.4) as well as in GVB++ buffer (pH7.3) in the presence and absence of a small molecule CFD inhibitor (ACH-4471). The extent of bystander hemolysis of PNH red cells was assessed with ~80% ABO blood type-matched pooled NHS in GVB++ buffer (pH7.3) for all pathogens for all pathogens except the NM isolates, where individual sera with bactericidal activity were used. Parallel evidence of complement activation by the pathogens was evaluated by 1) bactericidal activity of E. coli with pooled NHS-depleted of C1q, C2, CFD or C5 in GVB++ buffer (pH7.3), 2) bactericidal activity of NM isolates with the sera in GVB++ buffer (pH7.3) in the presence of ACH-4471, a CFD inhibitor; 3) measurement of the complement components and their activation products. Results: The extent of hemolysis of red cells harvested from PNH subjects was approximately equal to the Type II/III clone size when assessed with NHS in GVB0-Mg-EGTA buffer (pH6.4) ranging from ~30% to 90%; hemolysis was effectively blocked by ACH-4471, consistent with the data shown previously(Gavriilaki et. al. ASH 2015, Abstract No 275). No significant hemolysis of red cells harvested from PNH subjects was seen when assessed with NHS in GVB++ buffer (pH7.3) and furthermore hemolysis was not increased upon addition of bacteria (Fig. A). For E. coli, the bactericidal activity was unaffected by disruption of the CP (C1q depletion), the AP (CFD depletion), the CP and LP (C2 depletion) although, as expected, bactericidal activity was abrogated by disruption of the terminal pathway (C5 depletion) (Fig. B), confirming that complement activation was initiated via multiple pathways by E. coli. For NM isolates, the bactericidal activity was unaffected by disruption of the AP (Fig. B), confirming that the complement activation is initiated via CP or LP by NM isolates. Complement activation for other pathogens will be presented. Conclusion: We demonstrated that PNH red cells were not subjected to theoretical "bystander hemolysis" when incubated with the bacteria tested herein, suggesting no increased risk of pathogen-induced hemolytic breakthrough in PNH patients if treated with a complement alternative pathway inhibitor. Figure Figure. Disclosures Yang: Achillion: Employment, Equity Ownership. Thanassi:Achillion: Employment, Equity Ownership. Galvan:Achillion: Employment, Equity Ownership. Podos:Achillion: Employment, Equity Ownership. Huang:Achillion: Employment, Equity Ownership. Brodsky:Achillion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.