New graphic AUC-based method to estimate overall survival benefit: pomalidomide reanalysis.

WHAT IS KNOWN AND OBJECTIVE: Difference in median survival is an erratic measure and sometimes does not provide a good assessment of survival benefit. The aim of this study was to reanalyse the overall survival benefit of pomalidomide from pivotal clinical trial using a new area under curve (AUC)-based method. COMMENT: In the pivotal trial, pomalidomide plus low-dose dexamethasone showed a significant survival benefit over high-dose dexamethasone, with a difference between medians of 4.6 months. The new AUC method applied to the survival curves, obtained an overall survival benefit of 2.6 months for the pomalidomide treatment. This average difference in OS was calculated for the 61.5% of patients for whom the time to event is reliable enough. WHAT IS NEW AND CONCLUSION: This 2-month differential would have major clinical and pharmacoeconomic implications, on both cost-effectiveness studies and on the willingness of the healthcare systems to pay for this treatment.

Long-term type 1 diabetes influences haematopoietic stem cells by reducing vascular repair potential and increasing inflammatory monocyte generation in a murine model.

AIMS/HYPOTHESIS: We sought to determine the impact of long-standing type 1 diabetes on haematopoietic stem/progenitor cell (HSC) number and function and to examine the impact of modulating glycoprotein (GP)130 receptor in these cells. METHODS: Wild-type, gp130(-/-) and GFP chimeric mice were treated with streptozotocin to induce type 1 diabetes. Bone marrow (BM)-derived cells were used for colony-formation assay, quantification of side population (SP) cells, examination of gene expression, nitric oxide measurement and migration studies. Endothelial progenitor cells (EPCs), a population of vascular precursors derived from HSCs, were compared in diabetic and control mice. Cytokines were measured in BM supernatant fractions by ELISA and protein array. Flow cytometry was performed on enzymatically dissociated retina from gfp(+) chimeric mice and used to assess BM cell recruitment to the retina, kidney and blood. RESULTS: BM cells from the 12-month-diabetic mice showed reduced colony-forming ability, depletion of SP-HSCs with a proportional increase in SP-HSCs residing in hypoxic regions of BM, decreased EPC numbers, and reduced eNos (also known as Nos3) but increased iNos (also known as Nos2) and oxidative stress-related genes. BM supernatant fraction showed increased cytokines, GP130 ligands and monocyte/macrophage stimulating factor. Retina, kidney and peripheral blood showed increased numbers of CD11b(+)/CD45(hi)/ CCR2(+)/Ly6C(hi) inflammatory monocytes. Diabetic gp130(-/-) mice were protected from development of diabetes-induced changes in their HSCs. CONCLUSIONS/INTERPRETATION: The BM microenvironment of type 1 diabetic mice can lead to changes in haematopoiesis, with generation of more monocytes and fewer EPCs contributing to development of microvascular complications. Inhibition of GP130 activation may serve as a therapeutic strategy to improve the key aspects of this dysfunction.

Markers of airway inflammation in the exhaled breath condensate of preschool wheezers.

BACKGROUND: Leukotrienes (LT), isoprostanes, and nitrites/nitrates are biomarkers of airway inflammation and oxidative stress that can be detected in exhaled breath condensate (EBC). The aim of this study was to evaluate LTB4, LTE4, 8-isoprostane, and nitrite/nitrate levels in the EBC of healthy and wheezing preschool children. METHODS: We included 21 healthy nonatopic children and 25 patients with recurrent wheezing episodes in a cross-sectional study. LTB4, LTE4, and 8-isoprostane concentrations were measured directly in EBC using a specific enzyme immunoassay; nitrite/nitrate concentrations were measured using a colorimetric assay. RESULTS: LTB4 concentrations were higher in children with wheezing episodes than in healthy controls (76 pg/mL vs 20 pg/mL, P < .001). LTE4 was increased in children with wheezing episodes than in healthy controls (68 pg/mL vs 35 pg/mL, P < .001). Nitrite concentrations were higher in children with wheezing episodes than in healthy controls (14 pg/mL vs 9.7 pg/mL, P < .03). We found no differences in 8-isoprostane and nitrate concentrations between the patients and the healthy controls. CONCLUSIONS: Our findings suggest that EBC is a suitable noninvasive method for the assessment of airway inflammation and oxidative stress in preschool children. Levels of LTB4, LTE4, and nitrites were higher in children with recurrent wheezing episodes than in healthy controls.

Direct and indirect comparison of the efficacy and safety of adalimumab, etanercept, infliximab and golimumab in psoriatic arthritis.

WHAT IS KNOWN AND OBJECTIVE: Psoriatic arthritis is an autoimmune disease characterized by chronic inflammation of the skin and joints. Anti-TNF drugs reduce the severity of the disease in the long term. This study compares the efficacy and safety of adalimumab, etanercept, infliximab and golimumab in patients with psoriatic arthritis. METHODS: Direct comparison was based on a literature search of drug comparison studies, whereas indirect treatment comparison was based on phase III clinical trials with biological agents, involving similar populations and durations, and with the same outcome. ACR50 was taken as primary outcome for comparison, whereas ACR20 and ACR70 were used as secondary outcomes. Indirect comparisons were made using infliximab as the reference drug and the Bucher method. In calculating δ (the maximum acceptable difference as a clinical criterion of equivalence), use was made of half of the absolute risk reduction obtained in the meta-analysis of the clinical trials included in the indirect comparison (ARR 32%; δ: 16%). The four anti-TNF drugs were also compared in relation to the secondary outcomes and adverse effects. RESULTS AND DISCUSSION: Reported direct and indirect comparisons of the four drugs did not include golimumab, and did not yield conclusive results. Four clinical trials - one for each drug studied - were identified. The estimated differences for the primary outcome, ACR50, between infliximab and the other drugs were adalimumab (ARR 4%, 95% CI -9·5 to 17·5), etanercept (ARR 4%, 95% CI -10·5 to 18·5) and golimumab (ARR 9%, 95% CI -5·4 to 23·4). Likewise, there were no relevant differences between the drugs in relation to the secondary efficacy outcomes, except for etanercept, which was less effective in ACR70 response. For adverse reactions, there were also no significant differences except for injection site, reactions which were more frequent with etanercept, with a mean difference of 26% relative to infliximab. WHAT IS NEW AND CONCLUSION: No significant differences were found in ACR50 responses to the four drugs after 24 weeks. Injection-site reactions were more common with etanercept, but this was insufficient to invalidate the inference that clinically the four drugs can be regarded as clinically equivalent for the treatment of psoriatic arthritis.

A pleural ultrasound image of a collapsed lung surrounded by pleural fluid ("jellyfish sign") may correspond to an intrapericardial mass.

Lung ultrasound has been shown to be a valuable diagnostic tool. It has become the main way to get to the diagnosis of pleural effusion with much more specificity and sensibility than the x-ray. The diagnosis of pleural effusion with ultrasound is easily obtained after the visualization of hypoechoic fluid surrounding the lung. Sometimes it appears as an image of a collapsed lung moving with the surrounded pleural fluid ("jellyfish sign"). Until now this sign was almost pathognomonic of pleural effusion, but we explore a case in which this sign could have led to a misleading diagnosis. We present the case of a child admitted to intensive care with respiratory distress. In the point of care lung ultrasound we believed to see a pleural effusion with a collapsed lung moving into the effusion. Due to the enlargement of the pericardial sac, we did not realize that what we thought to be the pleural space was in fact the pericardial space. Unfortunately, there was a more echogenic area inside the pericardial effusion which led to a misleading fake lung atelectasis with pleural effusion ("jellyfish sign"). The correct diagnosis was properly obtained after assessing a cardiac point of care ultrasound using a four chambers view. The left side of the thorax is more difficult to be sonographed than the right due to the presence of the heart fossa that occupies a significant part of that side. Obtaining the diagnosis of pleural effusion on that side is more difficult for this reason and can sometimes be misleading with a pericardial effusion. The presence of the "jellyfish sign" is not pathognomonic and may lead to an error if we are guided only by the presence of that sign. To avoid such a misleading diagnosis, we highly recommend performing a point of care cardiac ultrasound if a pleural effusion is primarily seen in the lung ultrasound.

Development of a constitutive model for the compaction of recovered polyethylene terephthalate packages.

To date, PET (polyethylene terephthalate) is the most widely used plastic in packaging and also one of the most recycled polymers worldwide. However, the high transport costs and stagnated prices of recycled PET undermine recycling process profits. Transport costs can lower through compaction, which is still not a completely well-known process. Due to heterogeneous designs, the output density of the compaction process varies. This poses problems during equipment design, selection or operation processes as recovery costs sharply increase if the required density is not met. In this manuscript, the authors develop a constitutive model for the compaction of recovered PET packaging. This experimentally validated model, based on the elasto-plastic behaviour of PET packages, allows the output density range to be predicted according to the compression pressure during PET compaction. Unlike other generic compaction models that need more than two parameters, this model uses only one and better correlates with the experimental results. Unlike existing generic models, the model parameters have a physical meaning, which allows the influence of different factors on the compaction process to be assessed. Finally as a result of the model analysis, we provide some tips to enhance compaction equipment efficiency.

Captive-born intergeneric hybrid of a Guiana and bottlenose dolphin: Sotalia guianensis x Tursiops truncatus.

Bottlenose dolphins (Tursiops truncatus) and Guiana dolphin (Sotalia guianensis) live in sympatry along the Caribbean Coast of Central and South America and social interactions between these species have been described in the Caribbean Coast of Costa Rica, including sexual encounters. Here we examine and document the only known hybridization event between a male Guiana dolphin and a female bottlenose dolphin, in captivity at Oceanario Islas del Rosario (Colombian Caribbean), using photographic and genetic evidence from mitochondrial DNA markers and nuclear autosomal introns.

Elimination capacity of a TSE-model agent in the manufacturing process of Alphanate/Fanhdi, a human factor VIII/VWF complex concentrate.

The variant Creutzfeldt-Jakob disease (vCJD) is a transmissible spongiform encephalopathy (TSE), mainly present in the UK and is associated with the ingestion of bovine products affected with bovine spongiform encephalopathy. Manufacturers of biological products must investigate the ability of their production processes to remove TSE agents. We studied the purification steps in the manufacturing process of two FVIII/VWF concentrates (Alphanate) and Fanhdi in their ability to eliminate an experimental TSE-model agent. Hamster scrapie strain 263K brain-derived materials were spiked into samples of the solutions taken before various stages during its production: 3.5% polyethylene glycol (PEG) precipitation, heparin affinity chromatography and saline precipitation/final filtrations. PEG precipitation and affinity chromatography were studied both as isolated and combined steps. TSE agent removal was determined using a laboratory scale model representative of the industrial manufacturing process. The prion protein (PrP(Sc)) was measured with Western blot and TSE infectivity was measured with bioassay. Western blot results were in agreement with those obtained by bioassay, showing a significant removal capacity in the production process: 3.21-3.43 log(10) for the PEG precipitation; about 3.45 log(10) for the affinity chromatography; and around 2.0 log(10) for the saline precipitation plus final filtrations. PEG precipitation and heparin affinity chromatography were demonstrated to be two complementary TSE-model agent removal mechanisms with total removal being the sum of the two. An overall reduction factor of around 8 log(10) can be deduced. The tests from the production process of FVIII/VWF complex concentrates have demonstrated their potential for eliminating TSE agents.

Electroencephalographic evaluation of gold wire implants inserted in acupuncture points in dogs with epileptic seizures.

The purpose of this study was to evaluate both, clinically and with electroencephalographic (EEG) recordings, the effect of gold wire implants in acupuncture points in dogs with uncontrolled idiopathic epileptic seizures. Fifteen dogs with such diagnosis were enrolled in the study. A first EEG recording was performed in all dogs under anaesthesia with xylazine (1mg/kg) and propofol (6 mg/kg) before the treatment protocol, and a second EEG was performed 15 weeks later. Relative frequency power, intrahemispheric coherence available through EEG, number of seizures and seizure severity were compared before and after treatment using a Wilcoxon signed-rank test. There were no significant statistical differences before and after treatment in relative power or in intrahemispheric coherence in the EEG recording. However, there was a significant mean difference in seizure frequency and seizure severity between control and treatment periods. After treatment, nine of the 15 dogs (60%) had at least a 50% reduction in seizures frequency during the 15 weeks established as follow-up of this treatment.

[Invasive mechanical ventilation in cystic fibrosis: Influence in lung transplant]. / Ventilación mecánica invasiva en fibrosis quística: influencia en el trasplante pulmonar.

INTRODUCTION: Invasive mechanical ventilation (IMV) in patients with advanced cystic fibrosis (CF) is a relative contraindication for lung transplant (LT) in adults, although there is currently no data on children. PATIENTS AND METHODS: An 8-year retrospective study on 21 children with CF who underwent LT was performed, analysing their results as they were receiving (n = 8) or not (n = 13) IMV pretransplant. Demographic and surgical data, postoperative course, lung function and survival (immediate and 1-year) were compared between both groups. The role of the IMV pretransplant as a postoperative risk factor was estimated (odds ratio) and Kaplan Meier survival study was performed in both groups. RESULTS: No differences in patient age, sex and nutritional parameters were observed between both groups. Those on IMV who received LT required more frequent and longer bypass, more need for tracheotomy, a higher number of rejection episodes per patient and multiorgan failure, longer PICU stay and longer time on IMV than those who were not on IMV when LT was received. Nevertheless, no differences could be found regarding graft function and immediate and 1-year survivals (62.5% vs. 92.3% with and without IMV respectively). On the other hand, long-term survival was significantly lower than in patients on IMV. CONCLUSIONS: In our experience, children with CF on IMV who receive LT have more complicated surgery and immediate postoperative course. Though immediate and 1-year results and survivals may be encouraging, medium and long-term ones are significantly lower.

Selective laser fiber welding on woven polymer fabrics for biomedical applications.

Localized cartilage damage is a common problem for younger patients. This can heal, but often results in a painful condition that requires intervention. A welded-woven three-dimensional polymer fabric has been suggested as a suitable cartilage replacement because such materials closely match the mechanical properties of cartilage. However, such materials fare poorly when evaluated with respect to wear. A microscopic investigation of wear mechanisms showed that it is critical that the fibers not deflect laterally under a normal load. This observation led to the use of a new process for selective laser welding of the surface layers of three-dimensional fabrics in order to improve their wear resistance. Experimental evaluations were made in a pin-on-disc arrangement with a biomimetic loading. All materials used in the studies have previously been used in orthopedic devices or meet the requirements for United States Pharmacopeial Convention (USP) Class VI biocompatibility approval. The wear rates were significantly reduced and the lifespan of the fabrics was markedly improved due to surface welding, making this a viable option for cartilage replacement in vivo.

Evaluation of the vasoactive-inotropic score, mid-regional pro-adrenomedullin and cardiac troponin I as predictors of low cardiac output syndrome in children after congenital heart disease surgery.

OBJECTIVE: To determine the predictive value of the inotropic score (IS) and vasoactive-inotropic score (VIS) in low cardiac output syndrome (LCOS) in children after congenital heart disease surgery involving cardiopulmonary bypass (CPB), and to establish whether mid-regional pro-adrenomedullin (MR-proADM) and cardiac troponin I (cTn-I), associated to the IS and VIS scores, increases the predictive capacity in LCOS. DESIGN: A prospective observational study was carried out. SETTING: A Paediatric Intensive Care Unit. PATIENTS: A total of 117children with congenital heart disease underwent CPB. Patients were divided into two groups: LCOS and non-LCOS. INTERVENTIONS: The clinical and analytical data were recorded at 2, 12, 24 and 48h post-CPB. Logistic regression was used to develop a risk prediction model using LCOS as dependent variable. MAIN OUTCOME MEASURES: LCOS, IS, VIS, MR-proADM, cTn-I, age, sex, CPB time, PIM-2, Aristotle score. RESULTS: While statistical significance was not recorded for IS in the multivariate analysis, VIS was seen to be independently associated to LCOS. On the other hand, VIS>15.5 at 2h post-CPB, adjusted for age and CPB timepoints, showed high specificity (92.87%; 95%CI: 86.75-98.96) and increased negative predictive value (75.59%, 95%CI: 71.1-88.08) for the diagnosis of LCOS at 48h post-CPB. The predictive power for LCOS did not increase when VIS was combined with cTn-I >14ng/ml at 2h and MR-proADM >1.5nmol/l at 24h post-CPB. CONCLUSIONS: The VIS score at 2h post-CPB was identified as an independent early predictor of LCOS. This predictive value was not increased when associated with LCOS cardiac biomarkers. The VIS score was more useful than IS post-CPB in making early therapeutic decisions in clinical practice post-CPB.

[Hypobaric spinal anesthesia in a large ventral hernia]. / Anestesia espinal hipobara en hernia supraumbilical de gran tamaño.

High abdominal wall surgery may require general anesthesia but, in patients with high risk of difficult airway and respiratory complications, local or regional anesthesia is the choice whenever possible. Spinal anesthesia usually used (both isobaric and hyperbaric) could compromise the respiratory function due to blockade of the T6 metamere or higher. Hypobaric spinal anesthesia (HSA) at low doses (3.6 cc of 0.1% hypobaric bupivacaine plus 0.2 cc of 0.005% fentanyl) achieves sufficient analgesia with minimal motor blockade. We present the case of a patient with a large supraumbilical hernia with high risk of difficult airway and respiratory complications, who went through HSA. The patient did not report pain or dyspnea during the surgical procedure thus, HSA at low doses is an option to be taken into account in high abdominal wall surgery despite not having been described for this use.

[Care of the critically ill child with heart disease in Spain]. / La asistencia al niño crítico con cardiopatía en España.

INTRODUCTION: The objective of the present study is to present the organization of the resources of paediatric cardiac critical care in Spain. PATIENTS AND METHODS: Data were collected through questionnaires sent by e-mail to Spanish PICUs. RESULTS: 22 PICUs were enrolled. The median number of beds were 9.5 (4-18 beds). Total cardiac admissions represented a 20 % of total PICUs admissions per year, firstly for congenital heart defects, and secondly for respiratory problems. Cardiac surgical activities were carried out in 16 centres, centralized in PICU in 10 cases. Mechanical support of the myocardium was performed in 7 PICUs. A total of 10 participating PICUs considered echocardiograph training necessary and also an increase in the amount of activity for better results. CONCLUSIONS: Paediatric cardiac critical care involves a significant use of resources, including PICUs with no surgical activity. This study is useful for detecting common problems and for improving clinical care.

Species Richness and Abundance of Cerambycidae (Coleoptera) in Huatulco, Oaxaca, Mexico; Relationships with Phenological Changes in the Tropical Dry Forest.

Cerambycidae have an important ecological role in initiating the degradation process of dead wood, but few studies have evaluated Cerambycidae community attributes in relation to ecosystem phenology. We surveyed the cerambicid fauna of the tropical dry forest in Huatulco, Oaxaca, Mexico, and explored the relationship of Cerambycidae species richness and abundance with phenological changes in vegetation. We applied three collecting methods of light traps, direct collection, and Malaise traps to survey Cerambycidae throughout 2005. To determine seasonal variations, we collected samples in the dry season month of February in the rainy season of May-July and August-September, and in the transition months of October and November through. We collected and identified 145 species, 88 genera, 37 tribes, and four subfamilies. The subfamily with the highest number of species was Cerambycinae (100 species), and the tribe with the highest number of genera and species was Elaphidiini with 13 genera and 33 species. The ICE non-parametric estimator determined an overall expected richness of 373 species, while the overall Shannon Diversity Index was 4.1. Both species richness and abundance varied seasonally, with the highest values recorded in the rainy season and the lowest in the dry season. Overall species abundance was not significantly correlated to monthly rainfall or EVI neither, only for "direct collecting" the EVI vs Richness and EVI vs Shannon Diversity Index were significantly correlated. We propose that the seemingly contradictory relationships between seasonal richness patterns of Cerambycidae and the greening/senescence of vegetation (EVI) may be explained by the seasonal availability of dead organic matter, flowers, or leafy vegetation that may be synchronized with the behavior of different cerambycid species.

Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis?

Apoptosis is a phenomenon fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other proapoptotic factors. Mitochondrial apoptosis-induced channel (MAC) is the mitochondrial apoptosis-induced channel and is responsible for cytochrome c release early in apoptosis. MAC activity is detected by patch clamping mitochondria at the time of cytochrome c release. The Bcl-2 family proteins regulate apoptosis by controlling the formation of MAC. Depending on cell type and apoptotic inducer, Bax and/or Bak are structural component(s) of MAC. Overexpression of the antiapoptotic protein Bcl-2 eliminates MAC activity. The focus of this review is a biophysical characterization of MAC activity and its regulation by Bcl-2 family proteins, and ends with some discussion of therapeutic targets.

Effect of pulsed laser light in patients with dry eye syndrome. / Efecto del láser de luz pulsada en pacientes con síndrome de ojo seco.

OBJECTIVES: The objective of this study was to determine the clinical benefits of pulsed light therapy for the treatment of Dry Eye Syndrome (DES) due to the decrease in aqueous tear production (aqueous deficient DES) and/or excessive tear evaporation (evaporative DES) due to Meibomian Gland Dysfunction (MGD). METHODS: A study was conducted on 72 eyes corresponding to 36 patients with DES. Out of these 72 eyes, 60 underwent refractive surgery (48 with femtosecond laser, 6 were operated with a mechanical microkeratome, and 6 with refractive photo-keratectomy[RPK], 6 treated with phacoemulsification, and 6 with no previous surgical treatment. Pulsed laser light (Intense Pulsed Light Regulated [IRPL®]) was use to stimulate the secretion of the Meibomian glands during 4 sessions, one every 15 days. RESULTS: Patients with aqueous deficient DES did not show any improvement. Eyes with no previous surgery and those treated with phacoemulsification and PRK had a favourable outcome. On the other hand, less conclusive results were observed in the eyes treated with excimer laser. CONCLUSIONS: This treatment could be very helpful to treat evaporative DES produced by MGD. On the other hand, it is not helpful for those cases related to an isolated damage in the aqueous phase, or the mucin phase.

Surface depression with double-angle geometry during the discharge of grains from a silo.

When rough grains in loose packing conditions are discharged from a silo, a conical depression with a single slope is formed at the surface. We observed that the increase of volume fraction generates a more complex depression, characterized by two angles of discharge: one at the bottom similar to the angle of repose and a considerably larger upper angle. The change in slope appears at the boundary between a dense stagnant region at the periphery and the central flowing channel formed over the aperture. Since the material in the latter zone is always fluidized, the flow rate is unaffected by the initial packing of the bed. On the other hand, the contrast between both angles is markedly smaller when smooth particles of the same size and density are used, which reveals that high packing fraction and friction must combine to produce the observed geometry. Our results show that the surface profile helps to identify by simple visual inspection the packing conditions of a granular bed, being useful to prevent undesirable collapses during silo discharge in industry.

Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells.

Adenosine, released in increased amounts by hypoxic tissues, is thought to be an angiogenic factor that links altered cellular metabolism caused by oxygen deprivation to compensatory angiogenesis. Adenosine interacts with 4 subtypes of G protein-coupled receptors, termed A(1), A(2A), A(2B), and A(3). We investigated whether adenosine causes proliferation of human retinal endothelial cells (HRECs) and synthesis of vascular endothelial growth factor (VEGF) and, if so, which adenosine receptor subtype mediates these effects. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), in a concentration-dependent manner, increased both VEGF mRNA and protein expression by HRECs, as well as proliferation. This proliferative effect of NECA was inhibited by the addition of anti-human VEGF antibody. NECA also increased insulin-like growth factor-I and basic fibroblast growth factor mRNA expression in a time-dependent manner and cAMP accumulation in these cells. In contrast, neither the A(1) agonist N(6)-cyclopentyladenosine nor the A(2A) agonist 2-p-(2-carboxyethyl) phenethylamino-NECA caused any of the above effects of NECA. The effects of NECA were not significantly attenuated by either the A(2A) antagonist SCH58261 or the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine. However, the nonselective adenosine receptor antagonist xanthine amine congener completely inhibited the effects of NECA. Addition of antisense oligonucleotide complementary to A(2B) adenosine receptor mRNA inhibited VEGF protein production by HRECs after NECA stimulation. Thus, the A(2B) adenosine receptor subtype appears to mediate the actions of adenosine to increase growth factor production, cAMP content, and cell proliferation of HRECs. Adenosine activates the A(2B) adenosine receptor in HRECs, which may lead to neovascularization by a mechanism involving increased angiogenic growth factor expression.

Reduction in preretinal neovascularization by ribozymes that cleave the A2B adenosine receptor mRNA.

Adenosine modulates a variety of cellular functions by interacting with specific cell surface G protein-coupled receptors (A1, A2A, A2B, and A3) and is a potential mediator of angiogenesis through the A2B receptor. The lack of a potent, selective A2B receptor inhibitor has hampered its characterization. Our goal was to design a hammerhead ribozyme that would specifically cleave the A2B receptor mRNA and examine its effect on retinal angiogenesis. Ribozymes specific for the mouse and human A2B receptor mRNAs were designed and cloned in expression plasmids. Human embryonic kidney (HEK) 293 cells were transfected with these plasmids and A2B receptor mRNA levels were determined by quantitative real-time RT-PCR. Human retinal endothelial cells (HRECs) were also transfected and cell migration was examined. The effects of these ribozymes on the levels of preretinal neovascularization were determined using a neonatal mouse model of oxygen-induced retinopathy (OIR). We produced a ribozyme with a Vmax of 515+/-125 pmol/min and a Kcat of 36.1+/-8.3 min(-1) (P< or =1x10(-5)). Transfection of HEK293 cells with the plasmid expressing the ribozyme reduced A2B receptor mRNA levels by 45+/-4.8% (P=5.1x10(-5)). Transfection of HRECs reduced NECA-stimulated migration of cells by 47.3+/-1.2% (P=7x10(-4)). Intraocular injection of the constructs into the mouse model reduced preretinal neovascularization by 53.5+/-8.2% (P=4.5x10(-5)). Our results suggest that the A2B receptor ribozyme will provide a tool for the selective inhibition of this receptor and provide further support for the role of A2B receptor in retinal angiogenesis.