John J. Moran

Genome-wide analysis of EGR2/SOX10 binding in myelinating peripheral nerve

Myelin is essential for the rapidity of saltatory nerve conduction, and also provides trophic support for axons to prevent axonal degeneration. Two critical determinants of myelination are SOX10 and EGR2/KROX20. SOX10 is required for specification of Schwann cells from neural crest, and is required at every stage of Schwann cell development. Egr2/Krox20 expression is activated by axonal signals in myelinating Schwann cells, and is required for cell cycle arrest and myelin formation. To elucidate the integrated function of these two transcription factors during peripheral nerve myelination, we performed in vivo ChIP-Seq analysis of myelinating peripheral nerve. Integration of these binding data with loss-of-function array data identified a range of genes regulated by these factors. In addition, although SOX10 itself regulates Egr2/Krox20 expression, leading to coordinate activation of several major myelin genes by the two factors, there is a large subset of genes that are activated independent of EGR2. Finally, the results identify a set of SOX10-dependent genes that are expressed in early Schwann cell development, but become subsequently repressed by EGR2/KROX20.

Xanthine oxidase inhibition attenuates endothelial dysfunction caused by chronic intermittent hypoxia in rats.

Background: Xanthine oxidase is a major source of superoxide in the vascular endothelium. Previous work in humans demonstrated improved conduit artery function following xanthine oxidase inhibition in patients with obstructive sleep apnea. Objectives: To determine whether impairments in endothelium-dependent vasodilation produced by exposure to chronic intermittent hypoxia are prevented by in vivo treatment with allopurinol, a xanthine oxidase inhibitor. Methods: Sprague-Dawley rats received allopurinol (65 mg/kg/day) or vehicle via oral gavage. Half of each group was exposed to intermittent hypoxia (FIO2 = 0.10 for 1 min, 15×/h, 12 h/day) and the other half to normoxia. After 14 days, gracilis arteries were isolated, cannulated with micropipettes, and perfused and superfused with physiological salt solution. Diameters were measured before and after exposure to acetylcholine (10–6M) and nitroprusside (10–4M). Results: In vehicle-treated rats, intermittent hypoxia impaired acetylcholine-induced vasodilation compared to normoxia (+4 ± 4 vs. +21 ± 6 µm, p = 0.01). Allopurinol attenuated this impairment (+26 ± 6 vs. +34 ± 9 µm for intermittent hypoxia and normoxia groups treated with allopurinol, p = 0.55). In contrast, nitroprusside-induced vasodilation was similar in all rats (p = 0.43). Neither allopurinol nor intermittent hypoxia affected vessel morphometry or systemic markers of oxidative stress. Urinary uric acid concentrations were reduced in allopurinol- versus vehicle-treated rats (p = 0.02). Conclusions: These data confirm previous findings that exposure to intermittent hypoxia impairs endothelium-dependent vasodilation in skeletal muscle resistance arteries and extend them by demonstrating that this impairment can be prevented with allopurinol. Thus, xanthine oxidase appears to play a key role in mediating intermittent hypoxia-induced vascular dysfunction.

Perioperative blood pressure control, delayed graft function, and acute rejection after renal transplantation.

Background. Blood pressure (BP) control immediately after renal transplantation is poorly understood, with patients experiencing both high and low BP levels. Donor kidneys lack the ability to autoregulate their blood flow, meaning high pressures are directly translated to the graft endothelium, whereas reduced perfusion may augment ischemic injury. We hypothesize that early BP control may therefore influence the early alloimmune response. Methods. A total of 276 patients undergoing primary cadaveric renal transplantation who received cyclosporine-based therapy were followed; standard transplant variables were identified. BP was serially recorded before, during, and after reperfusion until 50 hr after surgery. Variables predicting acute rejection and delayed graft function were identified using Cox and logistic regression models. Results. The mean (SD) BP after surgery was 161(19) mm Hg systolic and 73(12) mm Hg diastolic. Forty-two percent had perioperative hypertension defined by conventional parameters. Increasing postoperative systolic BP, measured as standardized area-under-the-curve, was associated with an increased risk for acute rejection (hazard ratio [per mm Hg]=1.008), independent of other covariables including the preoperative BP level. Diastolic BP was inversely associated with the risk of delayed graft function (odds ratio [per mm Hg]=0.956). Conclusions. Early hypertension is common after renal transplantation. Early BP control has the potential to influence the risk of allograft rejection and delayed graft function.

Concentration-dependent effects of antimicrobials on Staphylococcus aureus toxin-mediated cytokine production from peripheral blood mononuclear cells

BACKGROUND Toxins contribute to the pathogenicity of Staphylococcus aureus infections by inducing a dysregulated inflammatory response. This study evaluated the impact of anti-staphylococcal antibiotic exposures over an increasing concentration range on cytokine production from peripheral blood mononuclear cells (PBMCs) after S. aureus toxin exposures. METHODS Human PBMCs were suspended in complete Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum at 10(6) cells/mL with 100 ng/mL S. aureus toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxin A (SEA), α-toxin or Panton-Valentine leucocidin (PVL). Vancomycin, trimethoprim/sulfamethoxazole, tigecycline, daptomycin, linezolid, clindamycin and azithromycin were added at a concentration range of 0.5-100 mg/L. Cytokine [interleukin-1β (IL-1β), IL-6, IL-8, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α)] concentrations were measured in duplicate by ELISA following exposure and were compared with response with toxin alone. RESULTS At concentrations approximating serum C(max), tigecycline decreased IL-6 by 52%-57% and IFN-γ production by 43%-53% compared with toxin alone (P ≤ 0.05) and linezolid inhibited TNF-α by 12%-35% and IL-8 by 25%-42% (P ≤ 0.02). However, trimethoprim/sulfamethoxazole increased TNF-α and IL-8 production (P = 0.002). Clindamycin, daptomycin, vancomycin and azithromycin had no consistent significant effect at approximate serum C(max) concentrations. All antibiotics had a concentration-dependent effect on cytokine production, with tigecycline, clindamycin and trimethoprim/sulfamethoxazole being the most potent inhibitors of cytokine production at concentrations exceeding 25 mg/L. CONCLUSIONS S. aureus toxins stimulate production of inflammatory cytokines in PBMCs. Antimicrobials with high tissue penetration, including tigecycline, clindamycin, trimethoprim/sulfamethoxazole and linezolid, reduced cytokine production, which, along with their antimicrobial effects, may have importance in the therapeutic outcome of severe infections.

Added Value of Soluble Tumor Necrosis Factor-α Receptor 1 as a Biomarker of ESRD Risk in Patients With Type 1 Diabetes

OBJECTIVE Recent studies have suggested that circulating levels of the tumor necrosis factor-α receptor 1 (sTNFαR1) may be a useful predictor for the risk of end-stage renal disease (ESRD) in patients with diabetes. However, its potential utility as a biomarker has not been formally quantified. RESEARCH DESIGN AND METHODS Circulating levels of sTNFαR1 were assessed in 429 patients with type 1 diabetes and overt nephropathy from the Finnish Diabetic Nephropathy (FinnDiane) cohort study. Predictors of incident ESRD over a median of 9.4 years of follow-up were determined by Cox regression and Fine-Gray competing risk analyses. The added value of sTNFαR1 was estimated via time-dependent receiver operating characteristic curves, net reclassification index (NRI), and integrated discrimination improvement (IDI) for survival data. RESULTS A total of 130 individuals developed ESRD (28%; ESRD incidence rate of 3.4% per year). In cause-specific modeling, after adjusting for baseline renal status, predictors of increased incidence of ESRD in patients with overt nephropathy were an elevated HbA1c, shorter duration of diabetes, and circulating levels of sTNFαR1. Notably, sTNFαR1 outperformed estimated glomerular filtration rate in terms of R2. Circulating levels of the sTNFαR1 also remained associated with ESRD after adjusting for the competing risk of death. A prediction model including sTNFαR1 (as a −0.5 fractional polynomial) was superior to a model without it, as demonstrated by better global fit, an increment of R2, the C index, and area under the curve. Estimates of IDI and NRI(>0) were 0.22 (95% CI 0.16–0.28; P < 0.0001) and 0.98 (0.78–1.23; P < 0.0001), respectively. The median increment in the risk score after including sTNFαR1 in the prediction model was 0.18 (0.12–0.30; P < 0.0001). CONCLUSIONS Circulating levels of sTNFαR1 are independently associated with the cumulative incidence of ESRD. This association is both significant and biologically plausible and appears to provide added value as a biomarker, based on the absolute values of NRI and IDI.

Genome Editing-Enabled HTS Assays Expand Drug Target Pathways for Charcot–Marie–Tooth Disease

Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot–Marie–Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin.

Persistence of influenza vaccine‐induced antibodies in lung transplant patients between seasons

J.J.M. Moran, W.E. Rose, A.J. Darga, K.A. Rohde, M.S. Hayney. Persistence of influenza vaccine‐induced antibodies in lung transplant patients between seasons.
Transpl Infect Dis 2011: 13: 466–470. xx: 000–000. All rights reserved

Vancomycin Tolerance in Methicillin-Resistant Staphylococcus aureus: Influence of Vancomycin, Daptomycin, and Telavancin on Differential Resistance Gene Expression

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) isolates that are susceptible to vancomycin but are tolerant to its killing effect may present a potential challenge for effective treatment. This study compared the microbiologic characteristics of clinical vancomycin-tolerant (VT-MRSA) and vancomycin-susceptible (VS-MRSA) strains using phenotypic and gene regulation studies. MRSA isolates collected from vancomycin-treated patients with bacteremia over a 5-year period were analyzed for vancomycin, daptomycin, and telavancin susceptibility, as well as accessory gene regulator (agr) group and function. Vancomycin tolerance was defined by a minimum bactericidal concentration (MBC)/minimum inhibitor concentration (MIC) ratio of ≥32 mg/liter. VT-MRSA isolates were compared to VS-MRSA isolates for differences in antimicrobial susceptibility, time-kill activity, and gene expression of key cell envelope response genes vraSR, dltA, and mprF. All 115 isolates evaluated were susceptible to vancomycin, daptomycin, and telavancin. Seven isolates (6%) were VT-MRSA. agr group II was more prevalent in isolates with vancomycin MBC/MIC ratios of ≥8. In time-kill analyses, VT-MRSA had reduced vancomycin killing, but daptomycin and telavancin activities were maintained. Significantly greater gene expression was observed in VT-MRSA after 72 h of subinhibitory antibiotic exposures. Vancomycin most notably increased vraSR expression (P = 0.002 versus VS-MRSA strains). Daptomycin and telavancin increased expression of all genes studied, most significantly mprF expression (P < 0.001). Longer durations of antibiotic exposure (72 h versus 24 h) resulted in substantial increases in gene expression in VT-MRSA. Although the clinical impact of VT-MRSA is not fully recognized, these data suggest that VT-MRSA strains, while still susceptible, have altered gene regulation to adapt to the antimicrobial effects of glyco- and lipopeptides that may emerge during prolonged durations of exposure.

Decreased T‐Cell Responses to Influenza Vaccination in Patients with Heart Failure

Study Objective. To determine whether T‐cell immune responses to influenza vaccination in patients with chronic heart failure (CHF) are less vigorous than the responses of healthy control subjects.

Humoral immune responses to influenza vaccination in patients with obstructive sleep apnea.

Study Objective. To compare humoral immune responses to influenza vaccination in subjects with obstructive sleep apnea (OSA) and in healthy volunteers (control subjects).