Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that oxidative stress can regulate extracellular matrix in cardiac fibroblasts. Neonatal and adult rat cardiac fibroblasts in vitro were exposed to H(2)O(2) (0.05-5 microM) or the superoxide-generating system xanthine (500 microM) plus xanthine oxidase (0.00<em>1</em>-0.<em>1</em> mU/<em>ml</em>) (XXO) for 24 h. In-gel zymography demonstrated that H(2)O(2) and XXO each increased gelatinase activity corresponding to matrix metalloproteinases (MMP) MMP-<em>1</em>3, MMP-2, and MMP-9. H(2)O(2) and XXO decreased collagen synthesis (collagenase-sensitive [(3)H]proline incorporation) without affecting total protein synthesis ([(3)H]leucine incorporation). H(2)O(2) and XXO decreased the expression of procollagen alpha(<em>1</em>)(I), alpha(2)(I), and alpha(<em>1</em>)(III) mRNA but increased the expression of fibronectin mRNA, suggesting a selective transcriptional effect on collagen synthesis. H(2)O(2), but not XXO, also decreased the expression of nonfibrillar procollagen alpha(<em>1</em>)(IV) and alpha(2)(IV) mRNA. To determine the role of endogenous antioxidant systems, cells were treated with the superoxide dismutase (SOD) inhibitor diethyldithiocarbamic acid (DDC, <em>1</em>00 microM) to increase intracellular superoxide or with the glucose-6-phosphate dehydrogenase inhibitor dehydroisoandrosterone 3-acetate (DHEA; <em>1</em>0 microM) to increase intracellular H(2)O(2). DDC and DHEA decreased collagen synthesis and increased MMP activity, and both effects were inhibited by an SOD/catalase mimetic. Thus increased oxidative stress activates MMPs and decreases fibrillar collagen synthesis in cardiac fibroblasts. Oxidative stress may play a role in the pathogenesis of myocardial remodeling by regulating the quantity and quality of extracellular matrix.

OBJECTIVE

To report the short-term anatomic and visual acuity response after intravitreal injection of bevacizumab (Avastin, Genentech) in patients with proliferative diabetic retinopathy complicated by vitreous hemorrhage.

METHODS

Two patients with vitreous hemorrhage due to proliferative diabetic retinopathy were treated with at least one intravitreal injection of bevacizumab 1.25 mg in 0.05 mL. The patients underwent Snellen visual acuity testing, ophthalmoscopic examination, and fluorescein angiography at baseline and follow-up visits.

RESULTS

Both patients had proliferative diabetic retinopathy with vitreous hemorrhage extensive enough to preclude panretinal photocoagulation. Following intravitreal injection of bevacizumab both patients experienced improvement in visual acuity starting within the first week. At 1 month of follow-up one patient had 2 lines of improvement in visual acuity and the other 5 lines. Each patient had regression of retinal neovascularization at 1 month of follow-up. Repeat injection was given to one patient at the 1-month follow-up because of slight leakage from neovascularization on the nerve, and to the other patient at 3 months because the retinal neovascularization showed early signs of reperfusion. The vitreous hemorrhage in each patient showed partial resolution at 1 week and nearly complete regression at 1 month. No adverse events were observed in either patient.

CONCLUSIONS

Initial treatment results of patients with vitreous hemorrhage and proliferative diabetic retinopathy did not reveal any short-term safety concerns. Intravitreal bevacizumab resulted in marked regression of neovascularization and rapid resolution of vitreous hemorrhage. The favorable short-term results suggest further study is needed in a larger group of patients.

To infer possible mechanisms of acute airway inflammation and mucus hypersecretion in acute severe asthma, we performed cellular and biochemical analysis on sputum from <em>1</em>8 adults with acute severe asthma and compared the results with results of analysis of sputum from <em>1</em>2 adults with cystic fibrosis (CF). We found that in subjects with asthma neutrophils made up more than 75% of sputum cells in <em>1</em>0 samples whereas eosinophils made up more than 75% of cells in only three samples. Fifty percent of the subjects with asthma reported that their asthma exacerbation was precipitated by a respiratory tract infection, and these subjects had a significantly higher percentage of neutrophils in their sputum (85% +/- 6% vs 57% +/- <em>1</em>2%, p = 0.05). In the CF samples neutrophils made up more than 95% and eosinophils less than <em>1</em>% of cells in all samples analyzed. Analysis of fluid phase chemicals in asthmatic and CF sputum samples showed that despite overall lower mean values of neutrophil elastase (27 +/- <em>1</em><em>1</em> micrograms/<em>ml</em> vs 466 +/- <em>1</em>2<em>1</em> micrograms/<em>ml</em>, p = 0.000<em>1</em>) and interleukin-8 (IL-8) (55 +/- <em>1</em>5 ng/<em>ml</em> vs <em>1</em>86 +/- 24 ng/<em>ml</em>, p = 0.000<em>1</em>), some of the asthmatic samples had values for these variables that overlapped those in the CF samples. In addition, the asthmatic samples were distinguished by the presence of higher tryptase (<em>1</em>0 +/- 7 U/L vs 0.9 +/- 0.9 U/L, p = 0.000<em>1</em>) and interleukin-6 (<em>1</em><em>1</em>66 +/- 447 ng/<em>ml</em> vs <em>1</em>86 +/- 24 ng/<em>ml</em>; p = 0.000<em>1</em>) levels and by a higher ratio of albumin to mucin-like glycoprotein (0.8 +/- 0.5 vs 0.<em>1</em> +/- 0.002, p = 0.02). DNA levels were lower in the asthmatic samples (0.5 +/- 0.3 mg/<em>ml</em> vs 3.5 +/- <em>1</em>.2 mg/<em>ml</em>, p = 0.05). We conclude that neutrophils predominate more frequently than eosinophils as the major inflammatory cell in sputum from patients with asthma in acute exacerbation. We speculate that this may be because respiratory tract infections are a frequent precipitant of acute asthma. In addition, the high IL-8 levels and free neutrophil elastase activity observed in asthmatic sputum suggests that IL-8 may mediate airway neutrophilia in acute asthma and that neutrophil elastase may mediate mucin glycoprotein hypersecretion in acute asthma, as has been proposed for the mucin hypersecretion in CF.

BACKGROUND

Transcatheter, intramyocardial injections of bone marrow-derived cell therapy produces reverse remodeling in large animal models of ischemic cardiomyopathy.

OBJECTIVE

We used cardiac MRI (CMR) in patients with left ventricular (LV) dysfunction related to remote myocardial infarction (MI) to test the hypothesis that bone marrow progenitor cell injection causes functional recovery of scarred myocardium and reverse remodeling.

RESULTS

Eight patients (aged 57.2±<em>1</em>3.3 years) received transendocardial, intramyocardial injection of autologous bone marrow progenitor cells (mononuclear or mesenchymal stem cells) in LV scar and border zone. All patients tolerated the procedure with no serious adverse events. CMR at <em>1</em> year demonstrated a decrease in end diastolic volume (208.7±20.4 versus <em>1</em>67.4±7.32 <em>mL</em>; P=0.03), a trend toward decreased end systolic volume (<em>1</em>42.4±<em>1</em>6.5 versus <em>1</em>07.6±7.4 <em>mL</em>; P=0.06), decreased infarct size (P<0.05), and improved regional LV function by peak Eulerian circumferential strain in the treated infarct zone (-8.<em>1</em>±<em>1</em>.0 versus -<em>1</em><em>1</em>.4±<em>1</em>.3; P=0.04). Improvements in regional function were evident at 3 months, whereas the changes in chamber dimensions were not significant until 6 months. Improved regional function in the infarct zone strongly correlated with reduction of end diastolic volume (r(2)=0.69, P=0.04) and end systolic volume (r(2)=0.83, P=0.0<em>1</em>).

CONCLUSIONS

These data suggest that transcatheter, intramyocardial injections of autologous bone marrow progenitor cells improve regional contractility of a chronic myocardial scar, and these changes predict subsequent reverse remodeling. The findings support the potential clinical benefits of this new treatment strategy and ongoing randomized clinical trials.

We developed a mouse bone marrow culture system to examine the process of osteoclast-like multinucleated cell formation from its progenitors. When mouse marrow cells were cultured for 8 days with <em>1</em> alpha,25-dihydroxyvitamin D3 [<em>1</em> alpha,25-(OH)2D3, <em>1</em>0(-<em>1</em>0) to <em>1</em>0(-7) M] or human PTH (<em>1</em>-34) (25-<em>1</em>00 ng/<em>ml</em>), tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells formed. No TRACP-positive multinucleated cells appeared in the absence of these hormones. <em>1</em> alpha,25-(OH)2D3 and PTH also increased the number of the clusters of TRACP-positive mononuclear cells. Time course studies showed that these TRACP-positive mononuclear cell clusters appeared before the formation of TRACP-positive multinucleated cells, suggesting that the TRACP-positive mononuclear cells are precursors of the multinucleated cells. Salmon calcitonin markedly inhibited the formation of TRACP-positive multinucleated cells but not TRACP-positive mononuclear cell clusters induced by <em>1</em> alpha,25-(OH)2D3 or PTH. TRACP-positive mononuclear cells and multinucleated cells were rarely stained for nonspecific esterase, but some mononuclear cells were positively stained for both nonspecific esterase and TRACP. More that 90% of the TRACP-positive mononuclear cell clusters and multinucleated cells were found near colonies of alkaline phosphatase-positive mononuclear cells (possibly osteoblasts). When marrow mononuclear cells were cultured on sperm whale dentine slices in the presence of <em>1</em> alpha,25-(OH)2D3 or PTH, numerous resorption lacunae were formed. These results suggest that <em>1</em>) TRACP-positive multinucleated cells formed in response to osteotropic hormones in mouse marrow cultures satisfy most of the criteria of osteoclasts, and 2) osteoblasts may play an important role in osteoclast formation.

Microdialysis experiments in rodents indicate that ethanol promotes dopamine release predominantly in the nucleus accumbens, a phenomenon that is implicated in the reinforcing effects of drugs of abuse. The aim of the present study was to test the hypothesis in humans that an oral dose of ethanol would lead to dopamine release in the ventral striatum, including the nucleus accumbens. Six healthy subjects underwent two [(<em>1</em><em>1</em>)C]raclopride PET scans following either alcohol (<em>1</em> <em>ml</em>/kg) in orange juice or orange juice alone. Subjective mood changes, heart rate, and blood-alcohol levels were monitored throughout the procedure. Personality traits were evaluated using the tridimensional personality questionnaire. PET images were co-registered with MRI and transformed into stereotaxic space. Statistical parametric maps of [(<em>1</em><em>1</em>)C]raclopride binding potential change were generated. There was a significant reduction in [(<em>1</em><em>1</em>)C]raclopride binding potential bilaterally in the ventral striatum/nucleus accumbens in the alcohol condition compared to the orange juice condition, indicative of increased extracellular dopamine. Moreover, the magnitude of the change in [(<em>1</em><em>1</em>)C]raclopride binding correlated with the alcohol-induced increase in heart rate, which is thought to be a marker of the psychostimulant effects of the drug, and with the personality dimension of impulsiveness. The present study is the first report that, in humans, alcohol promotes dopamine release in the brain, with a preferential effect in the ventral striatum. These findings support the hypothesis that mesolimbic dopamine activation is a common property of abused substances, possibly mediating their reinforcing effects.

Interleukin <em>1</em> (IL-<em>1</em>) plays an important role in host defense mechanisms by increasing body temperature, inducing the synthesis of a variety of lymphokines and hepatic acute phase proteins and acting as a chemoattractant for lymphocytes. However, in some microenvironments such as injured tissue or joint spaces, elevated IL-<em>1</em> levels may contribute to pathologic processes, for example, proliferation and fibrosis of tissue involved in pannus formation as well as degradation of matrix and abnormal tissue architecture. To investigate potential mechanisms that may lead to excessive production of IL-<em>1</em>, we have examined the ability of IL-<em>1</em> to participate in an amplification event by inducing its own gene expression leading to synthesis of biologically active IL-<em>1</em>. When injected into rabbits, recombinant human IL-<em>1</em>-alpha induced biphasic fevers, and during the second temperature elevation 3 hr later, a circulating pyrogenic material was detected by passive transfer of plasma to other rabbits. Induction of the biphasic fever was not caused by endotoxin contamination of the recombinant IL-<em>1</em>. The 3-hr circulating pyrogen was heat-labile and was not residual injected IL-<em>1</em>-alpha. Chromatographic separation of this plasma and biologic assay suggested that it was new IL-<em>1</em> of rabbit origin. We next incubated human blood mononuclear cells with recombinant IL-<em>1</em>-alpha and measured the intracellular and extracellular levels of IL-<em>1</em> by bioassay using the D<em>1</em>0.G4.<em>1</em> murine T cell line. In order to control for the carryover of recombinant IL-<em>1</em>-alpha used to stimulate the mononuclear cells (MNC), we used neutralizing antibodies that were specific for IL-<em>1</em>-alpha or IL-<em>1</em>-beta. The results of these neutralizations showed that recombinant human IL-<em>1</em>-alpha induces the synthesis of IL-<em>1</em>-beta in human MNC in vitro. These results were verified with a radioimmunoassay specific for IL-<em>1</em>-beta. At concentrations of <em>1</em>00 ng/<em>ml</em>, IL-<em>1</em>-alpha induced prostaglandin E2 production in the MNC culture, and this was associated with decreased production of immunoreactive IL-<em>1</em>-beta. Adding indomethacin to the cultures prevented the decreased production of IL-<em>1</em>-beta induced by high concentrations of IL-<em>1</em>-alpha. Using nonadherent MNC, we observed an increase in IL-<em>1</em>-beta as well as IL-<em>1</em>-alpha mRNA after 4 hr of exposure to recombinant IL-<em>1</em>-alpha. These results demonstrate that IL-<em>1</em>-alpha induces biologically active and immunoreactive IL-<em>1</em>-beta from MNC in vitro and that the same concentrations of IL-<em>1</em>-alpha induce gene expression for both forms of IL-<em>1</em>.(ABSTRACT TRUNCATED AT 400 WORDS)

Oestrogen is considered to be the 'female' hormone, whereas testosterone is considered the 'male' hormone. However, both hormones are present in both sexes. Thus sexual distinctions are not qualitative differences, but rather result from quantitative divergence in hormone concentrations and differential expressions of steroid hormone receptors. In males, oestrogen is present in low concentrations in blood, but can be extraordinarily high in semen, and as high as 250 pg <em>ml</em>(-<em>1</em>) in rete testis fluids, which is higher than serum oestradiol in the female. It is well known that male reproductive tissues express oestrogen receptors, but the role of oestrogen in male reproduction has remained unclear. Here we provide evidence of a physiological role for oestrogen in male reproductive organs. We show that oestrogen regulates the reabsorption of luminal fluid in the head of the epididymis. Disruption of this essential function causes sperm to enter the epididymis diluted, rather than concentrated, resulting in infertility. This finding raises further concern over the potential direct effects of environmental oestrogens on male reproduction and reported declines in human sperm counts.

Antagonists of the human immunodeficiency virus type <em>1</em> (HIV-<em>1</em>) coreceptor, CCR5, are being developed as the first anti-HIV agents acting on a host cell target. We monitored the coreceptor tropism of circulating virus, screened at baseline for coreceptor tropism, in 64 HIV-<em>1</em>-infected patients who received maraviroc (MVC, UK-427,857) as monotherapy for <em>1</em>0 days. Sixty-two patients harbored CCR5-tropic virus at baseline and had a posttreatment phenotype result. Circulating virus remained CCR5 tropic in 60/62 patients, 5<em>1</em> of whom experienced an HIV RNA reduction from baseline of>><em>1</em> log(<em>1</em>0) copies/<em>ml</em>, indicating that CXCR4-using variants were not rapidly selected despite CCR5-specific drug pressure. In two patients, viral load declined during treatment and CXCR4-using virus was detected at day <em>1</em><em>1</em>. No pretreatment factor predicted the emergence of CXCR4-tropic virus during maraviroc therapy in these two patients. Phylogenetic analysis of envelope (Env) clones from pre- and posttreatment time points indicated that the CXCR4-using variants probably emerged by outgrowth of a pretreatment CXCR4-using reservoir, rather than via coreceptor switch of a CCR5-tropic clone under selection pressure from maraviroc. Phylogenetic analysis was also performed on Env clones from a third patient harboring CXCR4-using virus prior to treatment. This patient was enrolled due to a sample labeling error. Although this patient experienced no overall reduction in viral load in response to treatment, the CCR5-tropic components of the circulating virus did appear to be suppressed while receiving maraviroc as monotherapy. Importantly, in all three patients, circulating virus reverted to predominantly CCR5 tropic following cessation of maraviroc.

Cascade polymers also known as Starburst dendrimers are spheroidal polycations that can be synthesized with a well-defined diameter and a precise number of terminal amines per dendrimer. We show, using luciferase and beta-galactosidase containing plasmids, that dendrimers mediate high efficiency transfection of a variety of suspension and adherent cultured mammalian cells. Dendrimer-mediated transfection is a function both of the dendrimer/DNA ratio and the diameter of the dendrimer. Maximal transfection of luciferase are obtained using a diameter of 68 A and a dendrimer to DNA charge ratio of 6/<em>1</em> (terminal amine to phosphate). Expression is unaffected by lysomotrophic agents such as chloroquine and only modestly affected (2-fold decrease) by the presence of <em>1</em>0% serum in the medium. Cell viability, as assessed by dye reduction assays, decreases by only 30% at <em>1</em>50 micrograms dendrimer/<em>mL</em> in the absence of DNA and about 75% in the presence of DNA. Under similar conditions polylysine causes a complete loss of viability. Gene expression decreased by 3 orders of magnitude when the charge ratio is reduced to <em>1</em>:<em>1</em>. When GALA, a water soluble, membrane-destabilizing peptide, is covalently attached to the dendrimer via a disulfide linkage, transfection efficiency of the <em>1</em>:<em>1</em> complex is increased by 2-3 orders of magnitude. The high transfection efficiency of the dendrimers may not only be due to their diameter and shape but may also be caused by the pKa's (3.9 and 6.9) of the amines in the polymer. The low pKa's permit the dendrimer to buffer the pH change in the endosomal compartment. The characteristics of precise control of structure, favorable pKa's, and low toxicity make the dendrimers suitable for gene-transfer vehicles.

BACKGROUND

Limited data are available to assess whether endovascular repair of abdominal aortic aneurysm (AAA) improves short-term outcomes compared with traditional open repair.

OBJECTIVE

To compare postoperative outcomes up to 2 years after endovascular or open repair of AAA in a planned interim report of a 9-year trial.

METHODS

A randomized, multicenter clinical trial of 88<em>1</em> veterans (aged>> or = 49 years) from 42 Veterans Affairs Medical Centers with eligible AAA who were candidates for both elective endovascular repair and open repair of AAA. The trial is ongoing and this report describes the period between October <em>1</em>5, 2002, and October <em>1</em>5, 2008.

METHODS

Elective endovascular (n = 444) or open (n = 437) repair of AAA.

METHODS

Procedure failure, secondary therapeutic procedures, length of stay, quality of life, erectile dysfunction, major morbidity, and mortality.

RESULTS

Mean follow-up was <em>1</em>.8 years. Perioperative mortality (30 days or inpatient) was lower for endovascular repair (0.5% vs 3.0%; P = .004), but there was no significant difference in mortality at 2 years (7.0% vs 9.8%, P = .<em>1</em>3). Patients in the endovascular repair group had reduced median procedure time (2.9 vs 3.7 hours), blood loss (200 vs <em>1</em>000 <em>mL</em>), transfusion requirement (0 vs <em>1</em>.0 units), duration of mechanical ventilation (3.6 vs 5.0 hours), hospital stay (3 vs 7 days), and intensive care unit stay (<em>1</em> vs 4 days), but required substantial exposure to fluoroscopy and contrast. There were no differences between the 2 groups in major morbidity, procedure failure, secondary therapeutic procedures, aneurysm-related hospitalizations, health-related quality of life, or erectile function.

CONCLUSIONS

In this report of short-term outcomes after elective AAA repair, perioperative mortality was low for both procedures and lower for endovascular than open repair. The early advantage of endovascular repair was not offset by increased morbidity or mortality in the first 2 years after repair. Longer-term outcome data are needed to fully assess the relative merits of the 2 procedures.

BACKGROUND

clinicaltrials.gov Identifier: NCT00094575.

Angiogenesis is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals. ALK<em>1</em> (activin receptor like-kinase <em>1</em>) is an endothelial-specific type <em>1</em> receptor of the transforming growth factor-beta receptor family. Heterozygotes with mutations in the ALK<em>1</em> gene develop hereditary hemorrhagic telangiectasia type 2 (HHT2). Recently, we reported that bone morphogenetic protein (BMP)9 and BMP<em>1</em>0 are specific ligands for ALK<em>1</em> that potently inhibit microvascular endothelial cell migration and growth. These data lead us to suggest that these factors may play a role in the control of vascular quiescence. To test this hypothesis, we checked their presence in human serum. We found that human serum induced Smad<em>1</em>/5 phosphorylation. To identify the active factor, we tested neutralizing antibodies against BMP members and found that only the anti-BMP9 inhibited serum-induced Smad<em>1</em>/5 phosphorylation. The concentration of circulating BMP9 was found to vary between 2 and <em>1</em>2 ng/<em>mL</em> in sera and plasma from healthy humans, a value well above its EC(50) (50 pg/<em>mL</em>). These data indicated that BMP9 is circulating at a biologically active concentration. We then tested the effects of BMP9 in 2 in vivo angiogenic assays. We found that BMP9 strongly inhibited sprouting angiogenesis in the mouse sponge angiogenesis assay and that BMP9 could inhibit blood circulation in the chicken chorioallantoic membrane assay. Taken together, our results demonstrate that BMP9, circulating under a biologically active form, is a potent antiangiogenic factor that is likely to play a physiological role in the control of adult blood vessel quiescence.

We tested the hypothesis that the inflammatory cytokines can regulate fibroblast extracellular matrix metabolism. Neonatal and adult rat cardiac fibroblasts cultures in vitro were exposed to interleukin (IL)-<em>1</em>beta (4 ng/<em>mL</em>), tumor necrosis factor-alpha (TNF-alpha; <em>1</em>00 ng/<em>mL</em>), IL-6 (<em>1</em>0 ng/<em>mL</em>), or interferon-gamma (IFN-gamma; 500 U/<em>mL</em>) for 24 hours. IL-<em>1</em>beta, and to a lesser extent TNF-alpha, decreased collagen synthesis, which was measured as collagenase-sensitive [(3)H]proline incorporation, but had no effect on cell number or total protein synthesis. IL-<em>1</em>beta decreased the expression of procollagen alpha(<em>1</em>)(I), alpha(2)(I), and alpha<em>1</em>(III) mRNA, but increased the expression of procollagen alpha(<em>1</em>)(IV), alpha(2)(IV), and fibronectin mRNA, indicating a selective transcriptional downregulation of fibrillar collagen synthesis. IL-<em>1</em>beta and TNF-alpha each increased total matrix metalloproteinase (MMP) activity as measured by in-gel zymography, causing specific increases in the bands corresponding to MMP-<em>1</em>3, MMP-2, and MMP-9. IL-<em>1</em>beta increased the expression of proMMP-2 and proMMP-3 mRNA, suggesting that increased metalloproteinase activity is due, at least in part, to increased transcription. The effects of IL-<em>1</em>beta were not dependent on NO production. Thus, IL-<em>1</em>beta and TNF-alpha decrease collagen synthesis and activate MMPs that degrade collagen. These observations suggest that IL-<em>1</em>beta and TNF-alpha may contribute to ventricular dilation and myocardial failure by promoting the remodeling of interstitial collagen.

<em>1</em>. This study was performed to test the hypothesis that inflammatory cytokines are produced in skeletal muscle in response to prolonged intense exercise. Muscle biopsies and blood samples were collected from runners before, immediately after, and 2 h after a marathon race. 2. The concentration of interleukin (IL)-6 protein in plasma increased from <em>1</em>.5 +/- 0.7 to 94.4 +/- <em>1</em>2.6 pg <em>ml</em>-<em>1</em> immediately post-exercise and to 22.<em>1</em> +/- 3.8 pg <em>ml</em>-<em>1</em> 2 h post-exercise. IL-<em>1</em> receptor antagonist (IL-<em>1</em>ra) protein in plasma increased from <em>1</em>23 +/- 23 to 2795 +/- 55<em>1</em> pg <em>ml</em>-<em>1</em>, and increased further to 4<em>1</em><em>1</em>9 +/- 527 pg <em>ml</em>-<em>1</em> 2 h post-exercise. 3. The comparative polymerase chain reaction technique was used to evaluate mRNA for IL-6, IL-<em>1</em>ra, IL-<em>1</em>beta and tumour necrosis factor (TNF)-alpha in skeletal muscle and blood mononuclear cells (BMNC) (n = 8). Before exercise, mRNA for IL-6 could not be detected either in muscle or in BMNC, and was only detectable in muscle biopsies (5 out of 8) after exercise. Increased amounts of mRNA for IL-<em>1</em>ra were found in two muscle biopsies and five BMNC samples, and increased amounts of IL-<em>1</em>beta mRNA were found in one muscle and four BMNC samples after exercise. TNF-alpha mRNA was not detected in any samples. 4. This study suggests that exercise-induced destruction of muscle fibres in skeletal muscles may trigger local production of IL-6, which stimulates the production of IL-<em>1</em>ra from circulating BMNC.

BACKGROUND

The arteriovenous fistula is the preferred type of vascular access for hemodialysis because of lower thrombosis and infection rates and lower health care expenditures compared with synthetic grafts or central venous catheters. Early failure of fistulas due to thrombosis or inadequate maturation is a barrier to increasing the prevalence of fistulas among patients treated with hemodialysis. Small, inconclusive trials have suggested that antiplatelet agents may reduce thrombosis of new fistulas.

OBJECTIVE

To determine whether clopidogrel reduces early failure of hemodialysis fistulas.

METHODS

Randomized, double-blind, placebo-controlled trial conducted at 9 US centers composed of academic and community nephrology practices in 2003-2007. Eight hundred seventy-seven participants with end-stage renal disease or advanced chronic kidney disease were followed up until <em>1</em>50 to <em>1</em>80 days after fistula creation or 30 days after initiation of dialysis, whichever occurred later.

METHODS

Participants were randomly assigned to receive clopidogrel (300-mg loading dose followed by daily dose of 75 mg; n = 44<em>1</em>) or placebo (n = 436) for 6 weeks starting within <em>1</em> day after fistula creation.

METHODS

The primary outcome was fistula thrombosis, determined by physical examination at 6 weeks. The secondary outcome was failure of the fistula to become suitable for dialysis. Suitability was defined as use of the fistula at a dialysis machine blood pump rate of 300 mL/min or more during 8 of <em>1</em>2 dialysis sessions.

RESULTS

Enrollment was stopped after 877 participants were randomized based on a stopping rule for intervention efficacy. Fistula thrombosis occurred in 53 (<em>1</em>2.2%) participants assigned to clopidogrel compared with 84 (<em>1</em>9.5%) participants assigned to placebo (relative risk, 0.63; 95% confidence interval, 0.46-0.97; P = .0<em>1</em>8). Failure to attain suitability for dialysis did not differ between the clopidogrel and placebo groups (6<em>1</em>.8% vs 59.5%, respectively; relative risk, <em>1</em>.05; 95% confidence interval, 0.94-<em>1</em>.<em>1</em>7; P = .40).

CONCLUSIONS

Clopidogrel reduces the frequency of early thrombosis of new arteriovenous fistulas but does not increase the proportion of fistulas that become suitable for dialysis. Trial Registration clinicaltrials.gov Identifier: NCT00067<em>1</em><em>1</em>9.

Hypertension is associated with insulin-resistant states such as diabetes and obesity. Nitric oxide (NO) contributes to regulation of blood pressure. To gain insight into potential mechanisms linking hypertension with insulin resistance we directly measured and characterized NO production from human umbilical vein endothelial cells (HUVEC) in response to insulin using an amperometric NO-selective electrode. Insulin stimulation of HUVEC resulted in rapid, dose-dependent production of NO with a maximal response of approximately <em>1</em>00 nM NO (200,000 cells in 2 <em>ml</em> media; ED50 approximately 500 nM insulin). Although HUVEC have many more IGF-<em>1</em> receptors than insulin receptors (approximately 400,000, and approximately 40,000 per cell respectively), a maximally stimulating dose of IGF-<em>1</em> generated a smaller response than insulin (40 nM NO; ED50 approximately <em>1</em>00 nM IGF-<em>1</em>). Stimulation of HUVEC with PDGF did not result in measurable NO production. The effects of insulin and IGF-<em>1</em> were completely blocked by inhibitors of either tyrosine kinase (genestein) or nitric oxide synthase (L-NAME). Wortmannin (an inhibitor of phosphatidylinositol 3-kinase [PI 3-kinase]) inhibited insulin-stimulated production of NO by approximately 50%. Since PI 3-kinase activity is required for insulin-stimulated glucose transport, our data suggest that NO is a novel effector of insulin signaling pathways that are also involved with glucose metabolism.

OBJECTIVE

To explore a more effective treatment for newly diagnosed stage IV, relapsed, or refractory extranodal natural killer/T-cell lymphoma, nasal type (ENKL), we conducted a phase II study of the steroid (dexamethasone), methotrexate, ifosfamide, L-asparaginase, and etoposide (SMILE) regimen.

METHODS

Patients with newly diagnosed stage IV, relapsed, or refractory disease and a performance status of 0 to 2 were eligible. Two cycles of SMILE chemotherapy were administered as the protocol treatment. The primary end point was the overall response rate (ORR) after the protocol treatment.

RESULTS

A total of 38 eligible patients were enrolled. The median age was 47 years (range, <em>1</em>6 to 67 years), and the male:female ratio was 2<em>1</em>:<em>1</em>7. The disease status was newly diagnosed stage IV in 20 patients, first relapse in <em>1</em>4 patients, and primary refractory in four patients. The eligibility was revised to include lymphocyte counts of 500/<em>μL</em> or more because the first two patients died from infections. No treatment-related deaths were observed after the revision. The ORR and complete response rate after two cycles of SMILE chemotherapy were 79% (90% CI, 65% to 89%) and 45%, respectively. In the 28 patients who completed the protocol treatment, <em>1</em>9 underwent hematopoietic stem-cell transplantation. The <em>1</em>-year overall survival rate was 55% (95% CI, 38% to 69%). Grade 4 neutropenia was observed in 92% of the patients. The most common grade 3 or 4 nonhematologic complication was infection (6<em>1</em>%).

CONCLUSIONS

SMILE chemotherapy is an effective treatment for newly diagnosed stage IV, relapsed or refractory ENKL. Myelosuppression and infection during the treatment should be carefully managed.

Recombinant adenoviruses containing the canine factor IX (FIX) cDNA were directly introduced in the hind leg muscle of mice. We show that (i) in nude mice, high expression (<em>1</em>-5 micrograms/<em>ml</em> in plasma) of FIX protein can be detected for>> 300 days; (ii) in contrast, expression of FIX protein was transient (7-<em>1</em>0 days) in normal mice; (iii) CD8+ lymphocytes could be detected within 3 days in the infected muscle tissue; (iv) use of beta 2-microglobulin and immunoglobulin M heavy chain "knockout" mice showed that lack of sustained expression of FIX protein is due to cell-mediated and humoral immune responses; (v) normal mice, once infected with recombinant adenovirus, could not be reinfected efficiently for at least 30 days due to neutralizing viral antibodies; and, finally, (vi) using immunosuppressive drugs, some normal mice can be tolerized to produce and secrete FIX protein for>> 5 months. We conclude that currently available adenoviral vectors have serious limitations for use for long-term gene therapy.

OBJECTIVE

The aim of this study was to determine whether fetal exposure to intra-amniotic inflammation and a systemic fetal inflammatory response (funisitis) are associated with the development of cerebral palsy at the age of 3 years.

METHODS

This cohort study included <em>1</em>23 preterm singleton newborns (gestational age at birth, </=35 weeks) born to mothers who underwent amniocentesis and were followed up for>>/=3 years. The presence of intra-amniotic inflammation was determined by elevated amniotic fluid concentrations of proinflammatory cytokines such as interleukins 6 and 8 and by amniotic fluid white blood cell count. Cytokine concentrations were measured with sensitive and specific immunoassays. Funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton jelly. Cerebral palsy was diagnosed by neurologic examination at the age of 3 years.

RESULTS

Newborns with subsequent development of cerebral palsy had a higher rate of funisitis and were born to mothers with higher median concentrations of interleukins 6 and 8 and higher white blood cell counts in the amniotic fluid compared with newborns without subsequent development of cerebral palsy (funisitis: 75% [9/<em>1</em>2] vs 23% [24/<em>1</em>05]; interleukin 6: median, <em>1</em>8.9 ng/<em>mL</em>; range, 0. 02-92.5 ng/<em>mL</em>; vs median, <em>1</em>.0 ng/<em>mL</em>; range, 0.0<em>1</em>-<em>1</em><em>1</em>5.2 ng/<em>mL</em>; interleukin 8: median, <em>1</em>3.0 ng/<em>mL</em>; range, 0.<em>1</em>-294.5 ng/<em>mL</em>; vs median, <em>1</em>.2 ng/<em>mL</em>; range, 0.05-285.0 ng/<em>mL</em>; white blood cell count: median, <em>1</em>98 cells/mm(3); range, 0>><em>1</em>000 cells/mm(3); vs median, 3 cells/mm(3); range, 0-<em>1</em>9,764 cells/mm(3); P <.0<em>1</em> for each). After adjustment for the gestational age at birth, the presence of funisitis and elevated concentrations of interleukins 6 and 8 in amniotic fluid significantly increased the odds of development of cerebral palsy (funisitis: odds ratio, 5.5; 95% confidence interval, <em>1</em>.2-24.5; interleukin 6: odds ratio, 6.4; 95% confidence interval, <em>1</em>. 3-33.0; interleukin 8: odds ratio, 5.9; 95% confidence interval, <em>1</em>. <em>1</em>-30.7; P <.05 for each).

CONCLUSIONS

Antenatal exposure to intra-amniotic inflammation and evidence of a systemic fetal inflammatory response (funisitis) are strong and independent risk factors for the subsequent development of cerebral palsy at the age of 3 years.

Several high molecular weight polypeptides have been shown to quantitatively copurify with brain tubulin during cycles of in vitro assembly-disassembly. These microtubule-associated proteins (MAPs) have been shown to influence the rate and extent of microtubule assembly in vitro. We report here that a heat-stable fraction highly enriched for one of the MAPs, MAP2 (mol wt approximately 300,000 daltons), devoid of MAP<em>1</em> (mol wt approximately 350,000 daltons), has been purified from calf neurotubules. This MAP2 fraction stoichiometrically promotes microtubule assembly, lowering the critical concentration for tubulin assembly to 0.05 mg/<em>ml</em>. Microtubules saturated with MAP2 contain MAP2 and tubulin in a molar ratio of approximately <em>1</em> mole of MAP2 to 9 moles of tubulin dimer. Electron microscopy of thin sections of the MAP2-saturated microtubules fixed in the presence of tannic acid demonstrates a striking axial periodicity of 32 +/- 8 nm.

Amino acid sequences, carbohydrate compositions and residue volumes are used to compare critically calculations of partial specific volumes v, neutron scattering matchpoints and 280-nm absorption coefficients with experimental v values for proteins and glycoproteins. The v values that are obtained from amino acid densitometry underestimate experimental v values by 0.0<em>1</em>-0.02 <em>ml</em>/g while the v values from crystallographic volumes overestimate the experimental v values by 0.04-0.05 <em>ml</em>/g. An intermediate consensus volume set of amino-acid-residue volumes is proposed in order to predict experimental v values using sequence information. The method is extended to carbohydrates and glycoproteins. Neutron scattering matchpoints can be calculated from crystallographic residue volumes on the basis of the non-exchange of <em>1</em>0% of the main-chain NH protons. Crystallographic results on protein-bound water are used to account for the experimental values of v and matchpoints. Finally, 280-nm absorption coefficients, A<em>1</em>%, <em>1</em> cm 280, of 5-27 are found to be well predicted by the Wetlaufer procedure based on the totals of Trp, Tyr and Cys residues. Average errors are +/- 0.7, and the experimental A(<em>1</em>%,<em>1</em>cm)280 values can be larger than the predicted values by 3%.

We sought to determine whether intramuscular injection of a recombinant adeno-associated virus (rAAV) vector expressing human factor IX (hF.IX) could direct expression of therapeutic levels of the transgene in experimental animals. High titer (<em>1</em>0(<em>1</em>2)-<em>1</em>0(<em>1</em>3) vector genomes/<em>ml</em>) rAAV expressing hF.IX was prepared, purified, and injected into hindlimb muscles of C57BL/6 mice and Rag <em>1</em> mice. In the immunocompetent C57BL/6 mice, immunofluorescence staining of muscle harvested 3 months after injection demonstrated the presence of hF.IX protein, and PCR analysis of muscle DNA was positive for AAV DNA, but no hF.IX was detected in mouse plasma. Further studies showed that these mice had developed circulating antibodies to hF.IX. In follow-up experiments in Rag <em>1</em> mice, which carry a mutation in the recombinase activating gene-<em>1</em> and thus lack functional B and T cells, similar results were seen on DNA analysis of muscle, but these mice also demonstrated therapeutic levels (200-350 ng/<em>ml</em>) of F. IX in the plasma. The time course of F.IX expression demonstrates that levels gradually increase over a period of several weeks before reaching a plateau that is stable 6 months after injection. In other experiments we demonstrate colocalization of hF.IX and collagen IV in intersitial spaces between muscle fibers. Collagen IV has recently been identified as a F.IX-binding protein; this finding explains the unusual pattern of immunofluorescent staining for F.IX shown in these experiments. Thus rAAV can be used to direct stable expression of therapeutic levels of F.IX after intramuscular injection and is a feasible strategy for treatment of patients with hemophilia B.

The present studies were performed to explore potentially selective mechanisms of leukocyte adhesion in an attempt to understand how preferential recruitment of eosinophils and basophils might occur during allergic and other inflammatory reactions. Stimulation of human vascular endothelial cells for 24 h with IL-4 (30 to <em>1</em>,000 U/<em>ml</em>) induced adhesion for eosinophils (up to approximately four-fold of control) and basophils (up to approximately twofold of control) but not neutrophils (less than <em>1</em>25% of control). Analysis of endothelial expression of adhesion molecules by flow cytometry revealed that IL-4 treatment induced vascular cell adhesion molecule-<em>1</em> (VCAM-<em>1</em>) expression without significantly affecting the expression of other adhesion molecules, namely endothelial-leukocyte adhesion molecule-<em>1</em> (ELAM-<em>1</em>) or intercellular adhesion molecule-<em>1</em> (ICAM-<em>1</em>). The concentration-response curve for IL-4-induced VCAM-<em>1</em> expression paralleled that for adhesion. Endothelial cells stimulated with IL-4 expressed adhesive properties for eosinophils by 3 h; the response increased steadily during a 24-h time course study. Eosinophils and basophils adhered to plates coated with a recombinant form of VCAM-<em>1</em>. This adhesion was blocked with antibodies to VCAM-<em>1</em> but not ELAM-<em>1</em>. mAb directed against either VCAM-<em>1</em> or VLA-4 inhibited (by approximately 75%) the binding of eosinophils and basophils to IL-4-stimulated endothelial cells. Because VLA-4 and VCAM-<em>1</em> have been demonstrated to bind to each other in other adhesion systems, these results suggest that IL-4 stimulates eosinophil and basophil adhesion by inducing endothelial cell expression of VCAM-<em>1</em> which binds to eosinophil and basophil VLA-4. The lack of expression of VLA-4 on neutrophils and the failure of IL-4 to stimulate neutrophil adherence support this conclusion. It is proposed that local release of IL-4 in vivo in allergic diseases or after experimental allergen challenge may partly explain the enrichment of eosinophils and basophils (vs neutrophils) observed in these situations.

OBJECTIVE

This study sought to examine whether exenatide is capable of reducing myocardial infarct size.

BACKGROUND

Exenatide is a glucagon-like peptide (GLP)-<em>1</em> analogue with insulinotropic and insulinomimetic properties. Because insulin and GLP-<em>1</em> have been described as reducing apoptosis, exenatide might confer cardioprotection after acute myocardial infarction (MI).

METHODS

Pigs were randomized to exenatide or phosphate-buffered saline (PBS) treatment after 75 min of coronary artery ligation and subsequent reperfusion. Infarct size was assessed with Evans Blue (Sigma-Aldrich, St. Louis, Missouri) and triphenyltetrazolium chloride. Cardiac function was measured with epicardial ultrasound and conductance catheter-based pressure-volume loops. Western blotting, histology, and activity assays were performed to determine markers of apoptosis/survival and oxidative stress.

RESULTS

Exenatide reduced myocardial infarct size (32.7 +/- 6.4% vs. 53.6 +/- 3.9%; p = 0.03<em>1</em>) and prevented deterioration of systolic and diastolic cardiac function (systolic wall thickening: 47.3 +/- 6.3% vs. 8.<em>1</em> +/- <em>1</em>.9%, p < 0.00<em>1</em>; myocardial stiffness: 0.<em>1</em>2 +/- 0.06 mm Hg/ml vs. 0.22 +/- 0.07 mm Hg/ml; p = 0.004). After exenatide treatment, myocardial phosphorylated Akt and Bcl-2 expression levels were higher compared with those after PBS treatment, and active caspase 3 expression was lower. In addition, fewer cells were terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling-positive. In addition, nuclear oxidative stress as assessed with an 8-hydroxydeoxyguanosine staining was reduced in the exenatide treatment arm, and superoxide dismutase activity and catalase activity were increased. Serum insulin levels increased after exenatide treatment, without affecting glucose levels.

CONCLUSIONS

These data identify exenatide as a potentially effective compound to reduce infarct size in adjunction to reperfusion therapy in patients with acute MI.