A state-mutating genetic algorithm to design ion-channel models

Realistic computational models of single neurons require component ion channels that reproduce experimental findings. Here, a topology-mutating genetic algorithm that searches for the best state diagram and transition-rate parameters to model macroscopic ion-channel behavior is described. Important features of the algorithm include a topology-altering strategy, automatic satisfaction of equilibrium constraints (microscopic reversibility), and multiple-protocol fitting using sequential goal programming rather than explicit weighting. Application of this genetic algorithm to design a sodium-channel model exhibiting both fast and prolonged inactivation yields a six-state model that produces realistic activity-dependent attenuation of action-potential backpropagation in current-clamp simulations of a CA1 pyramidal neuron.The importance of modeling ion channels has been understood since Hodgkin and Huxley''s seminal work with the squid giant axon (1). Subsequently, the development of the patch-clamp method (2) enabled the characterization of the properties of a wide range of channels, and intensive efforts followed to produce quantitative models that could predict and explain specific ion-channel behavior (3–7). Such efforts have led to two broad classes of models: those describing single-channel and gating currents, and those describing macroscopic currents.Models of single-channel and gating currents can be used to analyze properties such as open-channel probabilities, dwell times, and activation kinetics; they therefore facilitate an improved understanding of channel biophysics (3–5, 7–9). By contrast, models of macroscopic currents are usually intended as empirical tools as part of larger compartmental models of neurons. Such a macroscopic model may not necessarily describe the actual molecular state changes of the channel; the goal is rather for it to function as a “black-box” that reproduces the mean behavior of a population of channels. Hodgkin and Huxley''s (1) original formulation of sodium- and potassium-channel models is a prime example of this case, and its basic formalism continues to be widely used to generate empirical ion-channel models.An alternative to the Hodgkin–Huxley formalism is the state-dependent modeling approach (3). In reality, state-dependent models subsume Hodgkin–Huxley models because the latter can be recast as the former (3). State-dependent models are more general, however, because they can describe certain behaviors more easily than Hodgkin–Huxley type models, such as having widely different transition rates into and out of a given state (10). In general, the gold standard for the use of state-dependent models is single-channel recording (3, 5, 11), but the state-dependent formalism is also often employed in models of macroscopic currents (12–15) because of the generality and flexibility it affords.Methods to make empirical state-dependent models conform to data have been studied extensively and have involved a multitude of techniques such as hand fitting (12–14, 16), principal-axis fitting (17), maximum-likelihood estimation (5, 7, 17, 18), and genetic algorithms (15), among others. Here, we present a new fitting technique based on a topology-mutating genetic algorithm. Genetic algorithms have a number of useful characteristics: First, they have been shown to explore a large area of parameter space with relatively quick convergence, especially for problems with many parameters (19). Second, they are easily parallelizable. Third, they have been successfully applied to neuronal modeling, both for Hodgkin–Huxley-type ion-channel parameters and for compartmental neuronal models with voltage-activated conductances (15). Here we show that if the ion-channel model is formulated properly, genetic algorithms provide a natural way to incorporate changes in model topology as mutations.The algorithm presented here has several key features. Most notably, whereas other published optimization algorithms fix the model topology and optimize the rate constants, our algorithm searches over the space of model topologies and the space of rate parameters simultaneously. In order to design such an algorithm appropriate for state-dependent ion-channel models, we formulated an automated, computationally efficient method to satisfy the principle of microscopic reversibility, an equilibrium condition that imposes constraints on topologies with loops (20, 21). Finally, our algorithm uses a sequential approach, also known as goal programming (22), to optimize multiple protocols without the need to assign weights to each of the objective functions. The ability of this genetic algorithm to select and examine topologies not previously explored demonstrates its flexibility in developing working empirical models.We applied this genetic algorithm to devise a sodium-channel model that exhibits both fast and slow inactivation. Fast inactivation refers to a nonconducting channel state that follows quickly after depolarization and activation (within milliseconds) and from which channels recover quickly when the voltage is restored to resting levels (1). In response to either sustained depolarization (23, 24) or a train of depolarizing pulses (16, 25), however, the fraction of sodium channels available for activation also decreases rapidly, but in this case recovery occurs much more slowly, on the order of seconds rather than milliseconds. This form of inactivation has therefore been called “prolonged” or “slow” (16, 25). The presence of such widely disparate time scales makes the creation of state-dependent models of these channels a challenge. At the same time, the effect of prolonged inactivation on processes such as action-potential backpropagation (26, 27), transitions from bursting to spiking (28), and dendritic spiking (29, 30) makes the development of accurate models of prolonged inactivation important for computational simulations of neuronal function.     

The Origin of Multiple Molecular Forms in Urine of HNL/NGAL

Background and objectives: Several molecular forms of human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), a novel biomarker for acute kidney injury (AKI), have been found in urine. The origin of these different forms and the effect of antibody configuration on assay performances were investigated in this report.Design, setting, participants, & measurements: The molecular forms of HNL/NGAL from human neutrophils and present in urine obtained from cardiac surgery patients and patients with urinary tract infection (UTI), as well as secreted from HK-2 cells, were studied by Western blotting. The levels of HNL/NGAL in urine were measured by ELISAs. Kidney injury was simulated by incubation of HK-2 cells under stressful conditions.Results: The major molecular form of HNL/NGAL secreted by neutrophils is dimeric, whereas the major form secreted by HK-2 cells is monomeric. This was reflected by a predominance of the monomeric form in urine from patients with AKI and the dimeric form in patients with UTIs. The epitope specificities of the antibody used in the ELISAs had a profound effect on assay performance and paralleled differences of the antibodies to identify the different forms of urine HNL/NGAL.Conclusions: The monomeric form is the predominant form secreted by tubular epithelial cells, and the dimeric form is the predominant form secreted by neutrophils. The development of molecular form-specific assays for HNL/NGAL may be a means to identify the origin of HNL/NGAL in urine and construct more specific tools for the diagnosis of AKI.Human neutrophil lipocalin(HNL) (1), also named neutrophil gelatinase-associated lipocalin (NGAL) (2), is a ubiquitous glycoprotein originally isolated from human neutrophils and localized in their specific granules. HNL/NGAL exists as a 25-kD monomer, or as a 45-kD disulfide-linked homodimer, and it is covalently conjugated with gelatinase (matrix metalloproteinase 9) via an intermolecular disulfide bridge as a 135-kD heterodimeric form (2).Although HNL/NGAL was originally identified in and purified from human neutrophils, it is also expressed in kidney, liver, and epithelial cells under certain conditions (3,4). Pathologic or stressful conditions such as inflammation, infection, cancer, intoxication, ischemia, kidney injury, and cardiac surgery can induce the upregulation of the synthesis of HNL/NGAL (5–13). In addition, several studies have shown that upregulation of HNL/NGAL in human cell lines (A459 (14), MCF-7 (15), and HepG2 (11)) is induced by oxidative stress, cytokines, or other stimuli.HNL/NGAL has recently been highlighted as a novel and early biomarker of acute kidney injury (AKI) (12,13,16–19). Thus, the levels of HNL/NGAL were significantly increased in serum/plasma and urine after cardiac surgery and paralleled reduction in renal function (12,16,19). Several immunoassays have been developed for the measurement of HNL/NGAL. The assays are based on different formats and include RIA (20), Western blotting (21), ELISA (22,23), Triage device (24), and the Architect platform (16). Several research groups used one of these assays to determine the levels of HNL/NGAL in urine and drew the conclusion that HNL/NGAL is a biomarker of AKI (12,13,16–18). Our previous results indicated that the antibody configuration had an effect on the clinical performance of the assay (19,25). We also reported, for the first time, the existence of several molecular forms of HNL/NGAL in urine obtained from patients after cardiac surgery and that the presence of dimeric and monomeric forms and their ratios changed after operation (19). The source of the different molecular forms of HNL/NGAL and what they might reflect has not yet been elucidated. The aim of this report was therefore to study the possible cellular source of these different molecular forms and to investigate the possible effect of these different forms on the assay performances of HNL/NGAL assays using several different monoclonal and polyclonal antibodies with different epitope specificities.     

Incidence of Contrast-Induced Nephropathy after Contrast-Enhanced Computed Tomography in the Outpatient Setting

Background and objectives: No prospective study has reported the incidence of contrast-induced nephropathy (CIN) or the associated morbidity and mortality after contrast-enhanced computed tomography (CECT) in the outpatient setting.Design, setting, participants, & measurements: We enrolled and followed a prospective, consecutive cohort (June 2007 through January 2009) of patients who received intravenous contrast for CECT in the emergency department of a large, academic, tertiary care center. Outcomes measured were as follows (1) CIN: An increase in serum creatinine ≥0.5 mg/dl or ≥25% 2 to 7 d after contrast administration; (2) severe renal failure: An increase in serum creatinine to ≥3.0 mg/dl or the need for dialysis at 45 d; and (3) renal failure as a contributing cause of death (consensus of three independent physicians) at 45 d.Results: The incidence of CIN was 11% (70 of 633) among the 633 patients enrolled. Fifteen (2%) patients died within 45 d, including six deaths after study-defined CIN. Seven (1%) patients developed severe renal failure, six of whom had study-defined CIN. Of the six patients with CIN and severe renal failure, four died, and adjudicators determined that renal failure significantly contributed to all four deaths. Thus, CIN was associated with an increased risk for severe renal failure and death from renal failure.Conclusions: CIN occurs in >10% of patients who undergo CECT in the outpatient setting and is associated with a significant risk for severe renal failure and death.Contrast-induced nephropathy (CIN) is a known complication of intravenous, iodinated contrast; is a common cause of renal failure in the inpatient setting (1–5); and is associated with both short- and long-term adverse outcomes (6,7). Previous reports indicated that CIN occurs in 4 to 20% of patients after intra-arterial administration after coronary angiography (5–9). In the outpatient setting, the use of intravenous contrast to enhance (contrast-enhanced computed tomography [CECT]) imaging has increased sharply in recent years. Despite that >6% of all emergency department (ED) patients undergo CECT in the United States (10), no prospective data allow clinicians to estimate the rate of CIN or the associated morbidity and mortality after CECT in the outpatient setting in a heterogeneous population. Previous, retrospective work in outpatients who underwent CECT found the prevalence of CIN to be 5 to 13% (11–14) and indicates that patients without baseline renal insufficiency or chronic kidney disease may still be at risk for CIN in this population (11); however, these studies were limited by retrospective design and selection bias related to inclusion of inpatients with existing kidney disease (11–14). Thus, the absence of predicate literature required to estimate both the incidence and the clinical significance of CIN after CECT provided rationale for this work.In this study, we sought to define prospectively the incidence of CIN in an unselected, consecutive, heterogeneous population of ED patients who received low-osmolar, nonionic contrast for a CECT study of any body region. We tested the hypothesis that the incidence of CIN in the ED population exceeds 4% and that CIN is associated with a high rate of severe renal failure and death (5–9,11).     

Potent neutralization of anthrax edema toxin by a humanized monoclonal antibody that competes with calmodulin for edema factor binding

This study describes the isolation and characterization of a neutralizing monoclonal antibody (mAb) against anthrax edema factor, EF13D. EF13D neutralized edema toxin (ET)-mediated cyclic AMP (cAMP) responses in cells and protected mice from both ET-induced footpad edema and systemic ET-mediated lethality. The antibody epitope was mapped to domain IV of EF. The mAb was able to compete with calmodulin (CaM) for EF binding and displaced CaM from EF-CaM complexes. EF-mAb binding affinity (0.05–0.12 nM) was 50- to 130-fold higher than that reported for EF-CaM. This anti-EF neutralizing mAb could potentially be used alone or with an anti-PA mAb in the emergency prophylaxis and treatment of anthrax infection.Infection by inhalational anthrax is often fatal if treatment is delayed. Anthrax bacteria can be killed by vigorous treatment with antibiotics, but patients may still die because the lethality of anthrax is largely because of the action of toxins (1). Anti-toxin neutralizing monoclonal antibodies (mAbs) are the only viable choice for immediate neutralization of toxin and they could augment the effectiveness of antibiotics.Anthrax bacteria produce 3 toxin components: Protective antigen (PA), lethal factor (LF), and edema factor (EF) (2, 3). PA binds to cellular receptors and acts as a vehicle to deliver LF or EF into the cytosol where they exert their enzymatic activities (4–8). LF is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinases and causes lysis of macrophages (9, 10). EF is a calcium-calmodulin (CaM)-dependent adenylate cyclase and causes local inflammation and edema (11). The combination of PA with LF results in lethal toxin (LT). LT can replicate symptoms of anthrax disease when injected into animals (12). PA together with EF forms edema toxin (ET) and ET can produce a range of toxic effects in the host (11, 13).PA has been regarded as the most important target for prophylaxis and therapy of anthrax, because PA is common to both LTs and ETs, initiates the toxic process via receptor binding, and is highly immunogenic. In fact, PA is the major component in the current anthrax vaccine and the target for most of the available human or human-like neutralizing mAbs that have been shown to be very effective in protection against anthrax toxin or spore challenge (14–19). However, there is evidence that LF and EF may play important roles in providing protective immunity (20–22). Furthermore, concerns that PA could potentially be manipulated, such that it would no longer be neutralized by current anti-PA neutralizing mAbs have led to interest in therapeutics against the other 2 toxin components. A mixture of mAbs that recognize distinct epitopes on multiple toxin components (PA, LF, or EF) would not only enhance the protective efficacy but also broaden the spectrum of protection. Thus, in recent years, several anti-LF mAbs have also been reported (23–27). However, no anti-EF neutralizing mAbs have been reported to date. A previous report had indicated that immunization with the PA-binding N-terminal domain of EF (amino acids 1–254) resulted in polyclonal sera containing both EF and LF neutralizing activities (28).The purpose of this study was to determine (i) if anti-EF neutralizing mAbs could be isolated; (ii) the effectiveness of such antibodies against anthrax ET effects; and (iii) the neutralization mechanism of these antibodies. We have made a Fab combinatorial phage display library from chimpanzees that were immunized with anthrax toxins (17). From the library, 4 EF-specific Fabs were recovered, and 1 of them had potent neutralizing activity independent of the homologous PA-binding N-terminal (1–254) domain of LF. In this report, we describe the detailed characterization of these anti-EF clones.     

Recognition of the centromere-specific histone Cse4 by the chaperone Scm3

A specialized nucleosome is a component of all eukaryotic kinetochores. The core of this nucleosome contains a centromere-specific histone, CENP-A (the Cse4 gene product in budding yeast), instead of the usual H3. Assembly of a centromeric nucleosome depends on a specific chaperone, called Scm3 in yeast and HJURP in higher eukaryotes. We describe here the structure of a complex formed by an N-terminal fragment of Scm3 with the histone-fold domains of Cse4, and H4, all prepared as recombinant proteins derived from the budding yeast Kluyveromyces lactis. The contacts of Scm3 with Cse4 explain its selectivity for the centromere-specific histone; key residues at the interface are conserved in HJURP, indicating a common mechanism for centromeric-histone deposition. We also report the structure of a (Cse4 : H4)₂ heterotetramer; comparison with the structure of the Scm3:Cse4:H4 complex shows that tetramer formation and DNA-binding require displacement of Scm3 from the nucleosome core. The two structures together suggest that specific contacts between the chaperone and Cse4, rather than an altered overall structure of the nucleosome core, determine the selective presence of Cse4 at centromeres.Faithful transfer of genetic information from a mother cell is crucial for the survival of its daughters. During mitosis, an assembly of protein complexes, the kinetochore, connects each centromere with spindle microtubules and monitors bipolar attachment (1). A hallmark of kinetochores in all eukaryotes is a centromere-specific nucleosome, in which a centromere-specific H3 variant, CENP-A (sometimes designated CenH3 and known as Cse4 in budding yeast), replaces the canonical H3 (2–5). CENP-A/Cse4 is very well conserved, despite the divergence of centromeric DNA from budding yeast (which have short “point centromeres,” approximately 150–220 bp in length) to higher eukaryotes (with much longer, “regional centromeres”) (6).In point-centromere yeasts, a kinetochore-associated protein, Scm3, targets Cse4 nucleosomes to centromeres (7–9). Scm3, which associates specifically with Cse4 and not with H3, has orthologs in fission-yeast (Scm3^SP) and in higher eukaryotes (HJURP) (10–15). Centromeric localization of Scm3 is determined by Ndc10, a component of centromere-binding-factor 3 (CBF3) (8); elimination of CBF3 blocks deposition of the centromeric nucleosome (16). In organisms with regional centromeres, CENP-A deposition appears to be epigenetically directed. Localization of the Scm3 homolog depends on a set of proteins known as the Mis16–Mis18 complex (13, 17), as well as on the presence of CENP-A in neighboring nucleosomes and on defined H3 modifications in the interspersed chromatin (18).The so-called “CENP-A targeting domain” (CATD)—loop 1 and helix II of the CENP-A/Cse4 histone-fold domain (HFD)—is crucial for centromeric-histone function (19). Substitution of several CATD residues with their H3 counterparts disrupts CENP-A localization (20), and a chimeric H3-CATD histone functionally replaces CENP-A in vivo (19). Evidence that Scm3 and HJURP are assembly chaperones for Cse4 and CENP-A, respectively, therefore suggests that these proteins recognize features of the CATD (7–14).We report here the crystal structure (at 2.3-Å resolution) of a complex containing Scm3, Cse4, and H4, all from the budding yeast Kluyveromyces lactis. The structure shows that Scm3 interacts with Cse4 helix II and that its contacts explain selectivity for Cse4. Comparison of this structure with those of a (Cse4 : H4)₂ heterotetramer, also reported here, and of a conventional nucleosome (21) shows that tetramer formation and DNA-binding will displace Scm3. Conservation in HJURP and Scm3^SP of key residues at the Scm3:Cse4 interface indicates a common mechanism by which these chaperones recognize CENP-A/Cse4 and deposit it at centromeres. Our structure thus suggests that the principal difference between point and regional centromeres is in recruitment of the centromeric-histone–chaperone and that the structure and higher-order interactions of the centromere-specific nucleosome itself are essentially the same in all eukaryotes.     

Treatment Options for Sexual Dysfunction in Patients with Chronic Kidney Disease: A Systematic Review of Randomized Controlled Trials

Background and objectives: Sexual dysfunction is very common in patients with chronic kidney disease (CKD), but treatment options are limited. The benefits and harms of existing interventions for treatment of sexual dysfunction were assessed in patients with CKD.Design, setting, participants, & measurements: MEDLINE (1966 to December 2008), EMBASE (1980 to December 2008), and the Cochrane Trial Registry (Issue 4 2008) were searched for parallel and crossover randomized and quasi-randomized trials. Treatment effects were summarized as mean differences (MD) or standardized mean difference (SMD) with 95% confidence intervals (CI) using a random effects model.Results: Fourteen trials (328 patients) were included. Phosphodiesterase-5 inhibitors (PDE5i) compared with placebo significantly increased the overall International Index of Erectile Function-5 (IIEF-5) score (three trials, 101 patients, MD 1.81, 95% CI 1.51 to 2.10), all of its individual domains, and the complete 15-item IIEF-5 (two trials, 80 patients, MD 10.64, 95% CI 5.32 to 15.96). End-of-treatment testosterone levels were not significantly increased by addition of zinc to dialysate (two trials, 22 patients, SMD 0.19 ng/dl, 95% CI −2.12 to 2.50), but oral zinc improved end-of-treatment testosterone levels. There was no difference in plasma luteinizing and follicle-stimulating hormone level at the end of the study period with zinc therapy.Conclusions: PDE5i and zinc are promising interventions for treating sexual dysfunction in CKD. Evidence supporting their routine use in CKD patients is limited. There is an unmet need for studying interventions for male and female sexual dysfunction in CKD considering the significant disease burden.Sexual dysfunction is a set of disorders characterized by physical and psychologic changes that result in the inability to perform satisfactory sexual activities. The condition has been found to be significantly more common in men and women with chronic kidney disease (CKD) than in the general population (1). Men with CKD frequently suffer from reduced libido, erectile dysfunction, and difficulty reaching orgasm (2). Approximately 50% of male predialysis CKD patients and 80% of male dialysis patients have erectile dysfunction (3–6). Moreover, the prevalence of erectile dysfunction in male dialysis patients has been found to increase with age (63% <50 years versus 90% ≥50 years) (3). Similar results have been reported in women with CKD, with 55% of female dialysis patients reporting difficulty with sexual arousal (2). Dysmenorrhea, delayed sexual development, impaired vaginal lubrication, dyspareunia, and difficulties in reaching orgasm are also frequently observed (7,8).Multiple factors contribute to the frequent occurrence of sexual dysfunction in CKD patients, including hormonal disturbances (such as hyperprolactinemia, hypogonadism in males, and changes in hypothalamic-pituitary function in women) (9), anemia (10), CKD mineral and bone disorder (4), psychosocial factors (such as depression, anxiety, poor self-esteem, social withdrawal, marital discord, body image issues, fear of disability and death, loss of employment, and financial difficulties) (2,11,12), autonomic neuropathy (13), medications (including antihypertensives, antidepressant, and histamine receptor blockers) (2), and comorbid illness (such as diabetes mellitus, cardiovascular disease, and malnutrition) (2,14). Sexual dysfunction is inversely associated with GFR (7) and is improved after renal transplantation (15,16), suggesting that CKD per se may contribute to sexual dysfunction in these patients (15).Studies have also identified significant associations between sexual dysfunction in CKD patients and depression (8,17), impaired quality of life (8,17,18), and adverse cardiovascular outcomes (19). Effective treatment of sexual dysfunction in CKD patients may therefore potentially lead to improvement in these patient-level outcomes, although a causal link has not been definitively established (18).Therapies that have been used to treat sexual dysfunction include phosphodiesterase-5 inhibitors (PDE5i), intracavernosal injections, intraurethral suppositories, hormonal therapy, mechanical devices, and psychotherapy. Although many clinical trials and reviews have explored the role of these interventions for sexual dysfunction in nonuremic patients (20–24), the effectiveness and safety of these interventions in patients with CKD have not yet been studied thoroughly. Therefore, we aimed to evaluate the benefits and harms associated with various interventions for sexual dysfunction in patients with CKD.     

A Randomized Controlled Study Comparing Once-Weekly to Every-2-Week and Every-4-Week Dosing of Epoetin Alfa in CKD Patients with Anemia

Background and objectives: Extended-interval dosing of epoetin alfa (EPO) is commonly used to treat anemia in patients with chronic kidney disease (CKD). This study aimed to demonstrate that EPO dosed every 2 weeks (Q2W) and every 4 weeks (Q4W) was noninferior to once-weekly (QW) dosing.Design, setting, participants, & measurements: 430 anemic subjects with stage 3 to 4 CKD receiving a stable QW dose of EPO were randomized 1:1:2 to QW, Q2W, and Q4W dosing for 36 weeks. Hemoglobin (Hb) was measured weekly, and the dose of EPO was adjusted to maintain an Hb level of 11.0 to 11.9 g/dl. The primary endpoint was change in Hb from baseline to the average of the last 12 weeks of treatment.Results: Both the Q2W and Q4W dosing groups were noninferior to the QW group. The estimated difference of the mean change in Hb between Q2W and QW was −0.03 g/dl; and between Q4W and QW was −0.09 g/dl. From weeks 13 to 37, the mean percentage of weeks per subject with Hb 10.0 to 11.9 g/dl, inclusive, was 81% for QW, 81% for Q2W, and 75% for Q4W. Death occurred, respectively, in 4%, 3%, and 4%; thromboembolic vascular events occurred in 3%, 5%, and 3%; and serious adverse events occurred in 22%, 26%, and 26% of subjects.Conclusions: Q2W and Q4W EPO dosing maintained Hb levels in subjects with stage 3 to 4 CKD. Deaths, thromboembolic vascular events, and serious adverse events were comparable across the dosing groups.Chronic kidney disease (CKD) affects more than 25 million adults in the United States (1), with many developing anemia due to diminished erythropoietin production by the kidneys (2). Anemia of CKD is associated with decreased oxygen delivery and utilization, leading to weakness, fatigue, and frailty with increases in morbidity, mortality, and hospitalization rates (3,4). Overall, anemia in CKD patients leads to a poor quality of life, with a decrease in physical activity (5), congestive heart failure, and decreased cognition and mental acuity (6–10). Erythropoiesis-stimulating agents (ESAs) are considered the standard of care for the treatment of patients with anemia associated with CKD (11–13). Epoetin alfa (EPO) is an ESA with an amino acid sequence identical to endogenous human erythropoietin (14).Previous studies have suggested that extended-interval dosing of EPO could treat anemia in CKD patients not on dialysis (15–21). In a recent randomized, open-label, multicenter study (22), we demonstrated that once-weekly (QW) and every 2 weeks (Q2W) regimens of EPO could be used as potential alternatives to the approved three times weekly dosing for the treatment of anemia in ESA-naïve subjects with stage 3 to 4 CKD (22). The purpose of this study was to determine whether Q2W and every 4 weeks (Q4W) extended-interval dosing regimens would be as safe and effective as QW dosing for maintenance of hemoglobin (Hb) levels in subjects with stage 3 to 4 CKD.     

Antibody to Langerin/CD207 localizes large numbers of CD8α+ dendritic cells to the marginal zone of mouse spleen

Dendritic cells (DCs) are strategically positioned to take up antigens and initiate adaptive immunity. One DC subset expresses CD8αα in mice and is specialized to capture dying cells and process antigens for MHC class I “cross-presentation.” Because CD8⁺ DCs also express DEC205/CD205, which is localized to splenic T cell regions, it is thought that CD8⁺ DCs also are restricted to T zones. Here, we used a new antibody to Langerin/CD207, which colabels isolated CD8⁺ CD205⁺ DCs, to immunolabel spleen sections. The mAb labeled discrete cells with high levels of CD11c and CD8. Surprisingly most CD207⁺ profiles were in marginal zones surrounding splenic white pulp nodules, and only smaller numbers were in T cell areas, where CD205 colabeling was noted. Despite a marginal zone location, CD207⁺ DCs lacked identifying molecules for 3 different types of macrophages, localized in proximity and, in contrast to macrophages, marginal zone DCs were poor scavengers of soluble and particulate substrates. After stimulation with microbial agonists, Langerin expression disappeared from the marginal zone at 6–12 h, but was greatly expanded in the T cell areas, and by 24–48 h, Langerin expression disappeared. Therefore, anti-Langerin antibodies localize a majority of CD8⁺ DCs to non-T cell regions of mouse spleen, where they are distinct from adjacent macrophages.To induce adaptive immunity, a critical event is the uptake and presentation of antigen by dendritic cells (DCs) to naïve T cells. DCs initiate protective responses to infection and vaccination, and they also maintain self-tolerance (1, 2). Several subsets of DCs exist in the steady state, and these can have distinct functions (3, 4). In spleen, the main immune organ used for studies of immunity in mice, 2 main subsets of classical DCs are distinguished. Although both express high CD11c integrin, one subset expresses CD8αα, a marker of unknown function, and the other lacks CD8 but often expresses CD4 (5). CD8⁺ DCs are specialized to induce Th1 helper T cell development and to capture dying cells and cross-present antigens on MHC class I, whereas CD8⁻ DCs elicit IL-4 and more efficiently form peptide MHC II complexes (6–11).DC subsets differ in expression of antigen uptake receptors. CD8⁺ DCs express higher levels of DEC205/CD205 (5, 12), recognized by the mAb NLDC-145 (13, 14), whereas CD8⁻ DCs express DCIR2, recognized by 33D1 mAb (10). The capacity of DEC205 and DCIR2 to efficiently mediate antigen presentation in vivo can be demonstrated by injecting the corresponding anti-receptor mAbs engineered to deliver antigenic proteins (10, 15).The anatomy of mouse spleen is highly organized. The white pulp (WP) contains T cells, located in periarterial lymphoid sheaths (PALS), and B cells, found in discrete follicles. A marginal zone (MZ) rich in marginal zone macrophages (MZMs) and marginal metallophillic macrophages (MMMs) surrounds each WP nodule. Surrounding the MZ is the red pulp (RP), rich in red pulp macrophages (RPMs) (16, 17). CD11c-rich DCs are prevalent in the MZ and PALS (18). The location of both subsets of classic DCs (CD8⁺ and CD8⁻) is defined by using mAbs against DEC205 and DCIR2 in spleen sections. DEC205 staining in mouse spleen is restricted to PALS (13, 19), whereas DCIR2 staining is restricted to the bridging region of the MZ (10). Consequently, it is widely accepted that splenic CD8⁺ DCs are localized to DEC205-rich T cell areas (20–22).We recently developed an IgG mAb to the extracellular domain of mouse Langerin/CD207 (23). In the spleen, this mAb identifies CD207 selectively in CD8⁺ DCs, as also has been found with other approaches (12, 24). When anti-Langerin is engineered to deliver an antigen in vivo, it too mediates efficient presentation, both in peripheral lymph nodes and spleen (15). Here, we took advantage of the L31 anti-CD207 mAb to localize CD8⁺ DCs in spleen sections. Unexpectedly, CD207⁺ CD8⁺ DCs were mainly localized to the MZ, with fewer cells in the RP and PALS. Langerin⁺ cells lacked the major markers of 3 different groups of adjacent phagocytes, and despite their location, CD207⁺ CD8⁺ DCs only weakly cleared a variety of substrates from the blood.     

The Natural History of the Non-Nephrotic Membranous Nephropathy Patient

Background and objectives: Although early studies suggest that patients with idiopathic membranous nephropathy (MGN) and subnephrotic range proteinuria overall do well, these studies were small and follow-up was short or difficult to discern.Design, setting, participants, & measurements: Three hundred ninety-five cases of idiopathic MGN with at least 12 mo of follow-up from the Toronto Glomerulonephritis Registry were reviewed to determine the outcome of the subgroup of patients that presented with subnephrotic range proteinuria. Onset and follow-up data included mean arterial pressure (MAP) and creatinine clearance (CrCl) as determined by the Cockcroft-Gault equation. Outcome variables included the rate of progression (slope of CrCl), 50% reduction in initial CrCl, and end-stage renal disease (ESRD).Results: One hundred eight (27% of the total) patients presented with subnephrotic proteinuria and almost 40% (42 of 108) of this subgroup remained subnephrotic. Their long-term slope was −0.93 ml/min/yr. In contrast, those who subsequently developed nephrotic range proteinuria had a progression rate almost four times faster (−3.52 ml/min/yr). The majority who developed nephrotic syndrome did so within the first year of follow-up. The only distinguishing baseline feature between the two groups was a higher level of urine protein in the group that subsequently developed nephrotic syndrome (1.98 [0.3 to 3.4] versus 2.43 [0.5 to 3.4] g/d).Conclusions: Patients with MGN and sustained subnephrotic range proteinuria have an excellent prognosis. Conservative management with close monitoring is recommended given the difficulty predicting which patients will develop nephrotic range proteinuria and then progress more rapidly.Idiopathic membranous nephropathy (MGN) remains the most common cause of adult onset nephrotic syndrome. The natural history of the disease is stated to ascribe to the rule of thirds, wherein approximately one-third of the affected have a complete and spontaneous remission of their proteinuria, one-third enter a partial remission with subnephrotic range proteinuria, and a final third remain nephrotic and progress to end-stage renal disease (ESRD) (1–4). Studies utilizing multivariate analysis techniques have identified clinical predictors of poor renal survival. These include older age, male gender, and elevated serum creatinine at the time of diagnosis as well as the severity of proteinuria at the time of disease onset and during follow-up (5–10). However, despite these known predictors, the long-term outcome is variable with the 10 yr renal survival ranging from 60 to 80% (1–4). Thus, the optimal strategy for the management of patients with MGN has remained unclear with varying opinions emerging in the literature (11–13).This variation in opinion extends to the MGN patient with subnephrotic range proteinuria. Although there exists data to suggest these patients overall do well, previous natural history studies included only small numbers of this subset of patients, often with limited follow-up or inadequate details with respect to their clinical course. In particular, the time course for evolution to nephrotic range proteinuria was rarely defined (2,4,6,14–17). The largest natural history study was published in 1979 and included 116 untreated patients with MGN, of which 28 (24.2%) presented with subnephrotic range proteinuria. However, the outcome for this subgroup of patients was not described, and almost 25% of the patients were followed for less than 1 yr (2). In other studies, between 15 and 46% of patients presented with subnephrotic range proteinuria (6,14,17). In the largest of these reports, 19% entered a complete remission, 21% had persistent subnephrotic range proteinuria, and only 6% progressed to nephrotic syndrome (17). This study, along with smaller studies that included a total of only 42 patients presenting with subnephrotic range proteinuria, noted an excellent renal survival as long as proteinuria did not progress (4,14,15). Progression to nephrotic range proteinuria was noted to be rare, occurring in only four of 42 patients (10%) (4,15,16). In addition, the time course for the evolution to nephrotic range proteinuria could not be determined in these early studies, and all were published before the development of the classes of drugs that block the renin angiotensin system (RAS), which could potentially alter disease progression.We describe the long-term outcome of the largest cohort to date of patients with idiopathic MGN who present with subnephrotic proteinuria. This cohort was followed prospectively, allowing for the long-term assessment of these patients, including those that evolved to nephrotic range proteinuria, and compares these patients to the classic MGN patient who presents with nephrotic syndrome. Based on these data, a management strategy is suggested for this cohort.     

Phonon softening and crystallographic orientation of strained graphene studied by Raman spectroscopy

We present a systematic study of the Raman spectra of optical phonons in graphene monolayers under tunable uniaxial tensile stress. Both the G and 2D bands exhibit significant red shifts. The G band splits into 2 distinct subbands (G⁺, G⁻) because of the strain-induced symmetry breaking. Raman scattering from the G⁺ and G⁻ bands shows a distinctive polarization dependence that reflects the angle between the axis of the stress and the underlying graphene crystal axes. Polarized Raman spectroscopy therefore constitutes a purely optical method for the determination of the crystallographic orientation of graphene.Since the discovery of mechanical cleavage of graphene from graphite crystals (1), graphene has attracted intense interest because of properties that include high electron mobility (2, 3), novel quantum Hall physics (4, 5), superior thermal conductivity (6), and unusually high mechanical strength (7). Raman spectroscopy has emerged as a key diagnostic tool to identify single-layer graphene sheets (8) and probe their physical properties (9, 10). Because strain induces shifts in the vibrational frequencies, Raman spectroscopy can be applied to map built-in strain fields during synthesis (11) and device fabrication, as well as measure load transfer in composites. The rate of shift of the phonon frequencies with strain depends on the anharmonicity of the interatomic potentials of the atoms in the honeycomb lattice and thus can be used to verify theoretical models.Measurement of the strain dependence of the Raman active phonons is thus important for both applied and fundamental studies of this material system (12). By using graphene supported on a flexible substrate, we have been able to obtain precise information on the rate of frequency shift of the Raman G (zone-center optical) and 2D (two-phonon zone-edge optical) modes with strain. In addition, the polarization dependence of the Raman response in strained graphene can, as we demonstrate in this article, be used for an accurate determination of the crystallographic orientation. For unstrained graphene, such an orientation analysis is precluded by the high symmetry of the hexagonal lattice. A particularly important application of this capability lies in the study of nanopatterned graphene monolayers, such as nanoribbons (13) and quantum dots (14). Graphene nanoribbons possess electronic band gaps whose magnitude reflects both the ribbon width and crystallographic orientation (13, 15–17). The electronic states associated with graphene edges are also sensitive to the crystallographic orientation of the ribbon (18). It is thus crucial to be able to correlate the measured properties to the underlying crystallographic orientation of the sample. As we show here, polarized Raman spectroscopy provides a simple, but precise analytic tool that complements electron-spectroscopy techniques such as scanning tunneling microscopy (STM) (19), transmission electron microscopy (TEM) (20), and low-energy electron diffraction (LEED) (21), methods that typically require ultrahigh vacuum conditions and specialized equipment.     

Urinary Biomarkers in the Clinical Prognosis and Early Detection of Acute Kidney Injury

Background and objectives: Several novel urinary biomarkers have shown promise in the early detection and diagnostic evaluation of acute kidney injury (AKI). Clinicians have limited tools to determine which patients will progress to more severe forms of AKI at the time of serum creatinine increase. The diagnostic and prognostic utility of novel and traditional AKI biomarkers was evaluated during a prospective study of 123 adults undergoing cardiac surgery.Design, setting, participants, & measurements: Urinary neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CyC), kidney injury molecule-1 (KIM-1), hepatocyte growth factor (HGF), π-glutathione-S-transferase (π-GST), α-GST, and fractional excretions of sodium and urea were all measured at preoperative baseline, postoperatively, and at the time of the initial clinical diagnosis of AKI. Receiver operator characteristic curves were generated and the areas under the curve (AUCs) were compared.Results: Forty-six (37.4%) subjects developed AKI Network stage 1 AKI; 9 (7.3%) of whom progressed to stage 3. Preoperative KIM-1 and α-GST were able to predict the future development of stage 1 and stage 3 AKI. Urine CyC at intensive care unit (ICU) arrival best detected early stage 1 AKI (AUC = 0.70, P < 0.001); the 6-hour ICU NGAL (AUC = 0.88; P < 0.001) best detected early stage 3 AKI. π-GST best predicted the progression to stage 3 AKI at the time of creatinine increase (AUC = 0.86; P = 0.002).Conclusion: Urinary biomarkers may improve the ability to detect early AKI and determine the clinical prognosis of AKI at the time of diagnosis.Acute kidney injury (AKI) is a common and serious complication of cardiothoracic surgery (1); depending on the definition of AKI used, it may occur in over 40% of adults, with 1% to 5% requiring renal replacement therapy (RRT) (2–9). Recently, standardized clinical definitions of AKI have been implemented through the use of the RIFLE (Risk, Injury, Failure, Loss, and ESRD) and AKIN (Acute Kidney Injury Network) criteria (10,11). However, these criteria are still very much dependent on delayed serum creatinine elevations, the current gold standard for the diagnosis of AKI. Furthermore, as a functional marker of glomerular filtration, serum creatinine is not ideally suited to diagnose AKI caused by renal tubular injury, rather than reversible prerenal azotemia (10).In recent years, several novel human biomarkers have been demonstrated to detect acute tubular injury and have shown promise in their ability to precede and/or complement serum creatinine in the diagnosis of AKI (12–15). Cardiac surgery has long been used to study AKI because of the ability to prospectively follow patients before and after a well timed renal insult; for this reason, several urinary proteins have been shown to serve as biomarkers of AKI after cardiac surgery, including neutrophil gelatinase-associated lipocalin (NGAL) (16–20), cystatin C (CyC) (19,21), kidney injury molecule-1 (KIM-1) (18,21), interleukin-18 (IL-18) (22), and α-glutathione-S-transferase (α-GST) (23,24). Limited data are available comparing the ability of these markers to predict renal outcomes at the time of AKI diagnosis. In fact, nephrologists have limited tools in their arsenal to assess the presence and severity of renal tubular injury at the time of AKI diagnosis. Although urinalysis with microscopy has been shown to be of some utility in the differential diagnosis of AKI in a generalized hospital-based cohort (25), data supporting its use in the specific setting of cardiac surgery are lacking (24). Similarly, diagnostic mainstays of AKI evaluation such as the fractional excretion of sodium (FENa) have long been shown to be suboptimal tools in the complex setting of cardiac surgery AKI (24), in which volume status, fluid responsiveness, and diuretic use confound inferences regarding the relationship between tubular function and injury (26,27). Additionally, although recent data support the utility of the fractional excretion of urea (FEUrea) as a diagnostic tool in AKI (28), not all data support its use (29). Furthermore, very little is known about the utility of FENa or FEUrea compared with the novel urinary biomarkers discussed above for the differential diagnosis and prognostic evaluation of AKI.In this study, we assessed the diagnostic utility of urinary NGAL, CyC, KIM-1, hepatocyte growth factor (HGF), α-GST (a proximal tubular damage marker), π-GST (a marker specific to distal tubule damage), FENa, and FEUrea as biomarkers for the detection of early and severe AKI after adult cardiac surgery. These novel biomarkers can be thought of as falling into two categories: constitutive markers (proteins/enzymes that are normally present in renal tubular cells and not normally found in the urine in significant concentration but are released into the urine in direct response to cellular injury), and inducible biomarkers (proteins that are not normally found in high concentrations in renal tubular cells or urine until their production is directly upregulated in response to cellular injury). CyC, α-GST, and π-GST are constitutive proteins that are extruded into urine in the presence of site-specific renal tubular injury (CyC and α-GST are proximal and π-GST is distal); intracellular GSTs are released into urine by damaged tubular cells, whereas injured proximal tubules fail to reabsorb filtered CyC. In contrast, KIM-1 and NGAL are inducible biomarkers, gene products that are increased in direct response to nephron damage (30,31). We also evaluated the ability of these markers to predict the severity/stage of AKI at the time of clinical diagnosis by serum creatinine increase. We performed all of the above analyses for those subjects who developed AKI as defined by the AKIN (11). Recent data demonstrate that urine NGAL after cardiac surgery varies with baseline renal function (32); as such, a secondary analysis of baseline GFR was conducted for the aforementioned panel of biomarkers (32). Finally, we interpreted the data for all novel biomarker concentrations adjusted and unadjusted for dilution by indexing to urinary creatinine, but for brevity''s sake, we only report the indexed values unless otherwise noted.     

Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

Movement, cell division, protein biosynthesis, electron transfer against an electrochemical gradient, and many more processes depend on energy conversions coupled to the hydrolysis of ATP. The reduction of metal sites with low reduction potentials (E^0′ < -500 mV) is possible by connecting an energetical uphill electron transfer with the hydrolysis of ATP. The corrinoid-iron/sulfur protein (CoFeSP) operates within the reductive acetyl-CoA pathway by transferring a methyl group from methyltetrahydrofolate bound to a methyltransferase to the [Ni-Ni-Fe₄S₄] cluster of acetyl-CoA synthase. Methylation of CoFeSP only occurs in the low-potential Co(I) state, which can be sporadically oxidized to the inactive Co(II) state, making its reductive reactivation necessary. Here we show that an open-reading frame proximal to the structural genes of CoFeSP encodes an ATP-dependent reductive activator of CoFeSP. Our biochemical and structural analysis uncovers a unique type of reductive activator distinct from the electron-transferring ATPases found to reduce the MoFe-nitrogenase and 2-hydroxyacyl-CoA dehydratases. The CoFeSP activator contains an ASKHA domain (acetate and sugar kinases, Hsp70, and actin) harboring the ATP-binding site, which is also present in the activator of 2-hydroxyacyl-CoA dehydratases and a ferredoxin-like [2Fe-2S] cluster domain acting as electron donor. Complex formation between CoFeSP and its activator depends on the oxidation state of CoFeSP, which provides evidence for a unique strategy to achieve unidirectional electron transfer between two redox proteins.Energy transduction is fundamental for life. Aerobic and anaerobic organisms depend on coupling ATP hydrolysis to movement, activation of metabolites, or peptide bond formation, among others. Several metal-containing enzymes, such as nitrogenase, radical-dependent β,α-dehydratases, the related benzoyl-CoA reductases, and different cobalamin-dependent methyltransferases are able to convert unreactive molecules by acting in a low-potential regime. The highly energetic electrons required for these reactions (1–3) are injected by ATPases that enable the transfer of electrons against the redox potential gradient, driven by the hydrolysis of ATP. Three different types of reductive metallo-ATPase have been described so far.The enzyme nitrogenase is by reducing dinitrogen with six electrons to ammonia at the heart of the global nitrogen cycle (1, 4, 5). Nitrogenase consists of the dinitrogenase, also called MoFe protein for the predominant Mo-containing variant, and the dinitrogenase reductase, called Fe protein (1, 4–6). The Fe protein is a homodimer covalently linked through a [4Fe-4S] cluster bound within the dimer interface. Both monomers are able to bind and hydrolyze ATP in a cleft containing a P loop. For electron transfer (ET) between Fe and MoFe proteins to occur, the reduced Fe protein binds MgATP and forms a complex with the MoFe protein positioning the electron-donating [4Fe-4S] cluster and electron-accepting P cluster within the typical limits for physiological ET (< 15 Å) (1, 4, 7, 8). Hydrolysis of two ATP molecules initiates a one-electron transfer between both partners (9, 10). Conformational changes of the Fe protein induced by ATP hydrolysis are believed to act as switches for the association/dissociation of the Fe:MoFe protein complex and the delivery of electrons (8–11). The Fe protein is bifunctional and also acts as a molybdate/homocitrate insertase during the maturation of nitrogenase (5, 12).Benzoyl CoA reductases and 2-hydroxyacyl CoA dehydratases rely on homologous metallo-ATPases to catalyze the reduction of benzoyl-CoA or the β/α-dehydration of 2-hydroxyacyl-CoA compounds via formation of ketyl radicals (2). The structure of the homodimeric activator of 2-hydroxyglutaryl-CoA dehydratase revealed a [4Fe-4S] cluster covalently linking the two monomers, on a first glance resembling the Fe protein (13). The structure of the activator also showed it to be a member of the ASKHA (acetate and sugar kinases/heat shock protein 70/actin) superfamily. ASKHA proteins catalyze phosphoryl transfers or hydrolysis of ATP in a variety of biological contexts and are distinct from the P loop containing switch-type NTPases to which the Fe protein of nitrogenase belongs (13, 14). The binding of two MgATP molecules to the reduced activator is supposed to induce a conformational change and drives formation of the complex with the dehydratase. ATP hydrolysis likely increases the reducing power of the reduced [4Fe-4S] cluster of the activator, enabling the one-electron transfer to the low-potential [4Fe-4S] cluster of the dehydratase (2, 15). Unlike the 2-hydroxyacyl-CoA dehydratase system, the reduction of benzoyl-CoA is a two-step ET requiring a stoichiometric consumption of ATP (3).Recently, a third class of electron-transferring metallo-ATPases has been discovered (16–18). This enzyme class belongs to the COG3894 protein family and has been termed reductive activases for corrinoid enzymes (RACE) (17). The genome of several anaerobic microorganisms, which encode corrinoid-dependent methyltransferases and enzymes of the reductive acetyl-CoA pathway, also encode for proteins homologous to the two investigated RACE proteins with their characteristic binding motifs for one Fe/S cluster (17, 18). Bacterial RACE proteins typically show [2Fe-2S] cluster-binding-motifs, as in the veratrol O-demethylase system of Acetobacterium dehalogenans (16), whereas in archaea, as in the activator of the methylamine methyltransferase of the methanogenic archaeon Methanosarcina barkeri [4Fe-4S] cluster-binding motifs are more abundant (17, 18).The anaerobic hydrogenogenic bacterium Carboxydothermus hydrogenoformans is able to convert CO₂ into cellular carbon compounds via the reductive acetyl-CoA pathway (also known as the Wood–Ljungdahl pathway) (19–21). The corrinoid/iron-sulfur protein (CoFeSP) connects the methyl and carbonyl branch of this pathway by accepting a methyl group from methyltetrahydrofolate bound to a methyltransferase and donating it to the Ni,Fe-containing acetyl-CoA synthase (22, 23). Three redox states are known for the corrinoid cofactor of CoFeSP: The nucleophilic Co(I) acts as a methyl-acceptor, Co(II) is an oxidized inactive state, and CH₃ - Co(III) acts as the methyl donor of acetyl-CoA synthase (22, 23). The occasional oxidation of Co(I) to Co(II) inactivates CoFeSP, which has to be reactivated by a one-electron reduction (23, 24). The low midpoint potential needed to reduce Co²⁺ to Co¹⁺ (< -504 mV at pH 7.4) (25) can be achieved in vitro using either chemical reducing agents such as Na-dithionite (DT), Ti³⁺ citrate, via photoreduction with deazariboflavin as a catalyst or enzymatically with electrons generated by the oxidation of CO to CO₂ by carbon monoxide dehydrogenase (22, 26). An ATP-dependent reactivation of CoFeSP has not been reported so far.An open reading frame (orf7), situated between the structural genes coding for the CoFeSP subunits CfsA and CfsB of Moorella thermoacetica (27), codes for a member of the COG3894 protein family and contains the putative [2Fe-2S] cluster-binding motif CX₅CX₂CX_nC (17, 18). The genome of C. hydrogenoformans contains a similar arrangement of genes coding for enzymes of the reductive acetyl-CoA pathway as M. thermoacetica, including a homolog of Orf7 (CHY_1224 assigned as COG3894). To test whether an ATP-dependent reductive activator is operative in the reductive acetyl-CoA pathway, we established the heterologous production of the Orf7 homolog and investigated its activity, structure, and selective complex formation with CoFeSP. Furthermore, we reveal its relationship to known ATPases including the activator of 2-hydroxyacyl-CoA dehydratases and compare its strategy to achieve unidirectional electron transport with the other types of ATP-dependent activators/reductases.     

A Phase III,Randomized, Double-Blind,Placebo-Controlled Study of Tenecteplase for Improvement of Hemodialysis Catheter Function: TROPICS 3

Background and objectives: Despite widespread use of tunneled hemodialysis (HD) catheters, their utility is limited by the development of thrombotic complications. To address this problem, this study investigated whether the thrombolytic agent tenecteplase can restore blood flow rates (BFRs) in dysfunctional HD catheters.Design, setting, participants, & measurements: In this randomized, double-blind study, patients with dysfunctional tunneled HD catheters, defined as a BFR <300 ml/min at −250 mmHg pressure in the arterial line, received 1-hour intracatheter dwell with tenecteplase (2 mg) or placebo. The primary endpoint was the percentage of patients with BFR ≥300 ml/min and an increase of ≥25 ml/min above baseline 30 minutes before and at the end of HD. Safety endpoints included the incidence of hemorrhagic, thrombotic, and infectious complications.Results: Eligible patients (n = 149) were treated with tenecteplase (n = 74) or placebo (n = 75). Mean baseline BFR was similar for the tenecteplase and placebo groups at 151 and 137 ml/min, respectively. After a 1-hour dwell, 22% of patients in the tenecteplase group had functional catheters compared with 5% among placebo controls (P = 0.004). At the end of dialysis, mean change in BFR was 47 ml/min in the tenecteplase group versus 12 ml/min in the placebo group (P = 0.008). Four catheter-related bloodstream infections (one tenecteplase, three placebo) and one thrombosis (tenecteplase) were observed. There were no reports of intracranial hemorrhage, major bleeding, embolic events, or catheter-related complications.Conclusions: Tenecteplase improved HD catheter function and had a favorable safety profile compared with placebo.Effective hemodialysis (HD) requires reliable vascular access. Arteriovenous fistulas and grafts are preferred over catheters for their higher patency rates, prolonged survival, and lower complication rates (1–3). However, HD catheters are used by most dialysis patients to provide temporary access or to allow maturation of surgically placed fistulas (4). Tunneled catheters may also be used because of comorbidities or exhaustion of all graft and fistula sites (5,6).HD catheters must be carefully managed to mitigate a high complication rate. For example, catheter thrombosis is estimated to occur at a frequency of 0.5 to 3.0 events/1000 catheter-days (7–9). Among patients who experience access loss, catheter thrombosis is the precipitating event in 30% to 40% (1). Thrombotic obstruction of the catheter lumen reduces blood flow rate (BFR), frequently impeding the delivery of adequate HD. Treatment of partial occlusions of catheter lumens is often postponed by catheter line reversal, contributing to recirculation without addressing the underlying thrombus. The National Kidney Foundation''s Kidney Disease Outcomes Quality Initiative guidelines for HD vascular access advise against regular use of line reversal to manage low BFR (1).Maintenance of catheter patency is critical because many patients rely on catheter access for HD, and catheter insertion sites are limited. Administration of a thrombolytic directly into a dysfunctional HD catheter lumen may provide a way to salvage catheters with suboptimal BFRs while minimizing the risk of adverse events (AEs) associated with systemic delivery of these agents and avoid the need for catheter replacement. Previous studies evaluating the efficacy of alteplase or reteplase for clearance of HD catheters have yielded conflicting results (10–25). Limitations of prior studies include differences in trial design, sample size, thrombolytic dose, and definitions of treatment success. Until now there have been no large-scale, randomized, double-blind controlled trials using well defined efficacy and safety endpoints. As a result, many questions remain regarding the efficacy and safety of thrombolytics in the treatment of dysfunctional HD catheters.Tenecteplase is a recombinant serine protease that binds to fibrin and converts thrombus-bound plasminogen to plasmin, thereby stimulating local fibrinolysis. Tenecteplase has three engineered amino acid changes, resulting in greater fibrin specificity and an increased resistance to plasminogen activator inhibitor-1 compared with alteplase (26,27). When injected systemically, tenecteplase has a plasma half-life of approximately 22 minutes (28) and is primarily cleared by the liver (29). However, when tenecteplase is administered intraluminally for HD catheter dysfunction and subsequently withdrawn, circulating tenecteplase levels are not expected to reach detectable concentrations (30). Tenecteplase maintained clot lysis ability for 72 hours in a catheter in in vitro studies (Genentech data on file). In this study, we compared the efficacy of tenecteplase with placebo in improving BFR in dysfunctional HD catheters.     

Acute Decline in Renal Function,Inflammation, and Cardiovascular Risk after an Acute Coronary Syndrome

Background and objectives: Chronic kidney disease is associated with a higher risk of cardiovascular outcomes. The prognostic significance of worsening renal function has also been shown in various cohorts of cardiac disease; however, the predictors of worsening renal function and the contribution of inflammation remains to be established.Design, setting, participants, & measurements: Worsening renal function was defined as a 25% or more decrease in estimated GFR (eGFR) over a 1-mo period in patients after a non-ST or ST elevation acute coronary syndromes participating in the Aggrastat-to-Zocor Trial; this occurred in 5% of the 3795 participants.Results: A baseline C-reactive protein (CRP) in the fourth quartile was a significant predictor of developing worsening renal function (odds ratio, 2.48; 95% confidence interval, 1.49, 4.14). After adjusting for baseline CRP and eGFR, worsening renal function remained a strong multivariate predictor for the combined cardiovascular composite of CV death, recurrent myocardial infarction (MI), heart failure or stroke (hazard ratio, 1.6; 95% confidence interval, 1.1, 2.3).Conclusions: Patients with an early decline in renal function after an acute coronary syndrome are at a significant increased risk for recurrent cardiovascular events. CRP is an independent predictor for subsequent decline in renal function and reinforces the idea that inflammation may be related to the pathophysiology of progressive renal disease.Impaired renal function has consistently been shown to be an independent risk factor for cardiovascular outcomes across a broad spectrum of patients including population-based studies of patients with cardiovascular disease (1,2), acute coronary syndromes (3–6), chronic heart failure with either impaired or preserved ventricular systolic function (7), and after coronary artery bypass grafting (8). Worsening renal function (WRF), defined by small increases in creatinine or decreases in GFR, has also been independently associated with adverse cardiovascular outcomes and mortality in patients after an acute MI (9), cardiac surgery (10,11), and in patients with heart failure (12–14).Serum C-reactive protein (CRP), a marker of inflammation, has been associated with WRF in a population of nondiabetics (15), as well as in those after an MI (15,16). The predictors and prognostic significance of WRF in a cohort of patients after an acute coronary syndrome (ACS) is not well defined. Furthermore, the contribution of inflammatory markers to WRF in this cohort is unknown. We analyzed subjects from the phase Z of the A-Z trial (Early Intensive versus Delayed Conservative Simvastatin Strategy in Patients with Acute Coronary Syndromes), who had both measurements of serum creatinine and thus estimates of GFR (eGFR), at baseline and 1 mo. We have previously reported that both baseline CRP and eGFR were important prognostic markers after an ACS and that the increased cardiovascular hazard associated with a reduction in eGFR was independent and additive to baseline markers of inflammation (6). The objectives of this subsequent analysis was to determine clinical factors associated with an early decline in eGFR after an ACS and second to evaluate the cardiovascular prognostic importance of an early decline in eGFR in this patient population.     

Long-Term Outcome of Adults Who Undergo Transplantation with Single Pediatric Kidneys: How Young Is too Young?

Background and objectives: The optimal donor age for transplanting a single pediatric kidney in an adult recipient remains unknown. En block kidney transplantation is usually performed when the donor age is <5 yr.Design, setting, participants, & measurements: We compared the outcomes of adult patients who underwent transplantation with single pediatric kidneys from donors who were younger than 5 yr (group 1, n = 40) and from donors who were aged 5 to 10 yr of age (group 2, n = 39) in our center.Results: The donor kidney sizes were significantly smaller in group 1 than in group 2 (P < 0.001), and group 1 required more ureteral stents than group 2 (73 versus 38%). The surgical complications, delayed graft function, and development of proteinuria were similar in both groups. Group 1 had slightly higher rejection episodes than group 2 (25 versus 18%; P = 0.67), and graft function was comparable in both groups. There were no statistical differences between the two groups in patient (P = 0.73) or death-censored graft (P = 0.68) survivals over 5 yr.Conclusions: Single pediatric kidney transplants from donors who are younger than 5 yr can be used with acceptable complications and long-term outcomes as those from older donors.Transplantation of en block pediatric kidneys into an adult was first performed in 1972 (1). Giving both pediatric kidneys instead of one theoretically provides sufficient nephron mass to an adult body. Good long-term graft survival has been demonstrated (2,3). Splitting en block kidneys and transplanting a single pediatric kidney into each recipient could potentially increase kidney transplants. Mixed results have been reported (4–9). The technical concerns of vascular and ureteral complications (4,5,10,11) and the medical concerns of delayed graft function (DGF), rejection, and hyperfiltration injury have been raised (4,8,12).The minimum donor age or body weight that allows successfully splitting en block kidneys for adult recipients remains controversial. Registry data reported worse outcomes in single pediatric kidney transplants than en block transplants from donors who were younger than 5 yr or weighed <21 kg (2,3). As a result, en block transplantation has generally been considered the “preferred” method when donor age is <5 yr (2). In this study, we summarize our experience using single pediatric kidneys from donors who were younger than 5 yr. We compare the posttransplantation complications and the long-term outcomes of adult patients who underwent transplantation with single pediatric kidneys from donors who were younger than 5 yr with those who underwent transplantation with single kidneys from donors who were older than 5 but younger than 10 yr.     

Histopathologic Features Aid in Predicting Risk for Progression of IgA Nephropathy

Background and objectives: IgA nephropathy (IgAN) is the most common primary glomerular disease worldwide. Accurately identifying patients who are at risk for progressive disease is challenging. The extent to which histopathologic features improves prognostication is uncertain.Design, setting, participants, & measurements: We studied a retrospective cohort with biopsy-proven IgAN in Calgary, Canada. Renal biopsies were reviewed by a nephropathologist with histopathologic data abstracted using a standardized form. The primary outcome was the composite of doubling of serum creatinine, ESRD, or death. Spline models defined significant levels of interstitial fibrosis, glomerulosclerosis, hypertension, proteinuria, and creatinine. The prognostic significances of clinical and histopathologic parameters were determined using Cox proportional hazards models.Results: Data from 146 cases were available for analysis with a median follow-up of 5.8 years. Greater than 25% interstitial fibrosis, >40% glomerular sclerosis, and a systolic BP >150 mmHg were risk thresholds. In univariable analyses, baseline creatinine, proteinuria, systolic BP, glomerular sclerosis, interstitial fibrosis, and crescentic disease were predictors of the primary outcome. In multivariable models adjusted for clinical characteristics, interstitial fibrosis (hazard ratio [HR]2.7; 95% confidence interval [CI] 1.2 to 6.0), glomerular sclerosis (HR 2.6; 95% CI 1.2 to 4.5), and crescents (HR 2.4; 95% CI 1.2 to 5.1) remained independent predictors of the primary outcome and significantly improved model fit compared with clinical characteristics alone.Conclusions: Baseline histopathologic parameters are independent predictors of adverse outcomes in IgAN even after taking into consideration clinical characteristics. Relatively small degrees of interstitial fibrosis confer an increased risk for progressive IgAN.IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. Patients with IgAN have a variable clinical course with between 6 and 43% progressing to ESRD over 10 yr (1–5). Given this variability, identifying reliable prognostic factors is important to help stratify clinical monitoring and treatment regimens.Previous studies have identified clinical features including high-grade proteinuria, reduced kidney function, and hypertension at the time of diagnosis as predictors of adverse outcomes (4–7). Studies have identified interstitial fibrosis and glomerular sclerosis as poor prognostic features (8–10). This is not surprising considering that these features are a common final result of damage from glomerulonephritis; however, histopathologic features frequently correlate with serum creatinine, and whether they add prognostic values beyond the measurement of serum creatinine is uncertain. Histopathologic features are commonly categorized on the basis of arbitrary thresholds to denote significant degrees of damage (3,5,11), a factor that may contribute to poor performance in multivariable models of risk prediction in IgAN and result in an underestimation of their ability independently to predict outcomes. We performed a retrospective study using detailed baseline clinical data and quantitative analysis of renal biopsies, including the degree of interstitial fibrosis and glomerulosclerosis, to assess the factors that determine adverse outcomes including chronic kidney disease (CKD) progression and death.     

Reactogenicity to a live attenuated varicella vaccine in Canadian children

OBJECTIVE:

To assess the reactogenicity and safety of a thermostable, high titre, varicella vaccine in healthy infants and children.

DESIGN:

Open study of 505 children monitored for 42 days after vaccination.

SETTING:

Three urban Canadian centres (Halifax, Ottawa and Vancouver).

PARTICIPANTS:

505 healthy children one to 12 years of age were enrolled and 504 completed the study. All were susceptible to varicella by history.

INTERVENTIONS:

All participants received one dose of live attenuated varicella vaccine (1x10^4.5 plaque forming units/dose) subcutaneously.

MAIN OUTCOME MEASURES:

The children were monitored from the day of vaccine administration (day 0) until day 42. All local and general symptoms and signs were recorded on diary cards by the patients'' parents, who were encouraged to fill in the cards on days 2 to 3 and 18 to 24 via telephone calls from study personnel.

RESULTS:

Most of the symptoms noted after vaccine administration were mild and transient, and all resolved within the respective follow-up periods. Injection site symptoms included pain (17.5%, 13.9% and 30.4% in centres 1, 2 and 3 respectively), redness (21.1%, 32.1% and 48.8%) and swelling (7%, 10.3% and 29.2%). The general symptoms reported were fever 37.5°C or higher (3.5%, 4.8% and 3.0%) and varicella-like rashes (6.4%, 2.4% and 0%). Two subjects had severe symptoms (one with cervical lymphadenopathy, and one with a fever higher than 39°C) probably related to vaccine administration. No serious adverse events were reported during the entire study.

CONCLUSION:

The vaccine was well tolerated.Key Words: Chickenpox, Reactogenicity, Varicella vaccineInfection by varicella zoster virus (VZV) usually results in benign disease in children. However, complications such as pneumonia, encephalitis and bacterial superinfection of the skin lesions (1,2) occur in some patients, mainly in adolescents and adults, and in immunocompromised children (1,2). In addition, children born to nonimmune mothers who contract varicella during pregnancy can develop congenital varicella syndrome (1,3), with limb hypoplasia and central nervous system damage.A live varicella vaccine was developed in Japan in 1974 (4) using the OKA strain of the virus. The original wild type virus was isolated from a boy with natural varicella, and then attenuated by passages through human and guinea pig embryonic cells, and two human diploid cell lines, WI-38 and MRC-5 (4,5). The vaccine strain obtained after this treatment has different thermosensitivity and host range spectrum than the wild type virus (6). Additionally, it can be easily differentiated from wild type strains currently circulating in North America by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) testing (6,7).All OKA varicella vaccine production lots are derived directly from a working seed lot (8), following the World Health Organization guidelines (9). Many clinical trials have demonstrated that this strain is safe and immunogenic (2,5,10,11). The vaccine produced by SmithKline Beecham Biologicals (Rixensart, Belgium), VARILRIX, was first licensed in Europe in 1984 (10,11). An OKA-strain varicella vaccine, Oka/Biken (Biken, Osaka, Japan), has been licensed in Japan since 1987 (5). Vaccines using the same strain, were licensed in the United States in 1995 (Oka/Merck or VARIVAX, Merck and Company Inc, Whitehouse Station, New Jersey [12]) and in France in 1997 (Pasteur Merieux, Lyon, France).SmithKline Beecham Biologicals introduced recently a reformulation of the vaccine to increase its stability at 2 to 8°C and, therefore, facilitate its use for general vaccination (10). The vaccine will retain a titre of 1x10^3.3 plaque forming units (PFU) or more/dose after two years at 2 to 8°C. The safety and immunogenicity of this vaccine have been extensively tested (10,11).We conducted a multicentre study to assess the reactogenicity and safety of two lots of the reformulated varicella zoster vaccine produced by SmithKline Beecham Biologicals in children from one to 12 years of age. This vaccine has a higher titre (approximately 1x10^4.5 PFU per dose) of virus at release and is more thermostable than the vaccine presently licensed in Canada. The purpose of the study was to obtain daily information on local and general reactogenicity to the vaccine. Such information is required by licensing agencies before a vaccine is made available to the public.