Stent-based delivery of adeno-associated viral vectors with sustained vascular transduction and iNOS-mediated inhibition of in-stent restenosis.

In-stent restenosis remains an important clinical problem in the era of drug eluting stents. Development of clinical gene therapy protocols for the prevention and treatment of in-stent restenosis is hampered by the lack of adequate local delivery systems. Herein we describe a novel stent-based gene delivery platform capable of providing local arterial gene transfer with adeno-associated viral (AAV) vectors. This system exploits the natural affinity of protein G (PrG) to bind to the Fc region of mammalian IgG, making PrG a universal adaptor for surface immobilization of vector-capturing antibodies (Ab). Our results: 1) demonstrate the feasibility of reversible immobilization of AAV2 vectors using vector tethering by AAV2-specific Ab appended to the stent surface through covalently attached PrG, 2) show sustained release kinetics of PrG/Ab-immobilized AAV2 vector particles into simulated physiological medium in vitro and site-specific transduction of cultured cells, 3) provide evidence of long-term (12 weeks) arterial expression of luciferase with PrG/Ab-tethered AAV2Luc, and 4) show anti-proliferative activity and anti-restenotic efficacy of stent-immobilized AAV2iNOS in the rat carotid artery model of stent angioplasty.

Deletion of Rapgef6, a candidate schizophrenia susceptibility gene, disrupts amygdala function in mice.

In human genetic studies of schizophrenia, we uncovered copy-number variants in RAPGEF6 and RAPGEF2 genes. To discern the effects of RAPGEF6 deletion in humans, we investigated the behavior and neural functions of a mouse lacking Rapgef6. Rapgef6 deletion resulted in impaired amygdala function measured as reduced fear conditioning and anxiolysis. Hippocampal-dependent spatial memory and prefrontal cortex-dependent working memory tasks were intact. Neural activation measured by cFOS phosphorylation demonstrated a reduction in hippocampal and amygdala activation after fear conditioning, while neural morphology assessment uncovered reduced spine density and primary dendrite number in pyramidal neurons of the CA3 hippocampal region of knockout mice. Electrophysiological analysis showed enhanced long-term potentiation at cortico-amygdala synapses. Rapgef6 deletion mice were most impaired in hippocampal and amygdalar function, brain regions implicated in schizophrenia pathophysiology. The results provide a deeper understanding of the role of the amygdala in schizophrenia and suggest that RAPGEF6 may be a novel therapeutic target in schizophrenia.

C1 esterase inhibitor concentrate in 1085 Hereditary Angioedema attacks--final results of the I.M.P.A.C.T.2 study.

BACKGROUND: The placebo-controlled study International Multicentre Prospective Angioedema C1-INH Trial 1 (I.M.P.A.C.T.1) demonstrated that 20 U/kg C1 esterase inhibitor (C1-INH) concentrate (Berinert®; CSL Behring, Marburg, Germany) is effective in treating acute abdominal and facial Hereditary Angioedema (HAE) attacks. METHODS: I.M.P.A.C.T.2 was an open-label extension study of I.M.P.A.C.T.1 to evaluate the safety and efficacy of long-term treatment with 20 U/kg C1-INH for successive HAE attacks at any body location. Efficacy outcomes included patient-reported time to onset of symptom relief (primary) and time to complete resolution of all symptoms (secondary), analysed on a per-patient and per-attack basis. Safety assessments included adverse events, vital signs, viral safety and anti-C1-INH antibodies. RESULTS: During a median study duration of 24 months, 1085 attacks were treated in 57 patients (10-53 years of age). In the per-patient analysis, the median time to onset of symptom relief was 0.46 h and was similar for all types of attacks (0.39-0.48 h); the median time to complete resolution of symptoms was 15.5 h (shortest for laryngeal attacks: 5.8 h; 12.8-26.6 h for abdominal, peripheral and facial attacks). Demographic factors, type of HAE, intensity of attacks, time to treatment, use of androgens and presence of anti-C1-INH antibodies had no clinically relevant effect on the efficacy outcomes. There were no treatment-related safety concerns. No inhibitory anti-C1-INH antibodies were detected in any patient. CONCLUSIONS: A single dose of 20 U/kg C1-INH concentrate is safe and provides reliable efficacy in the long-term treatment of successive HAE attacks at any body location.

Efficacy and safety of a new immunoglobulin G product, Gammaplex(®), in primary immunodeficiency diseases.

This open-label multi-centre study evaluated a new intravenous immunoglobulin, Gammaplex®, in the treatment of 50 patients with primary immunodeficiency and significant hypogammglobulinaemia. Patients treated previously with other intravenous immunoglobulins received Gammaplex® on their same infusion schedule for 1 year; 22 were on a 21-day and 28 on a 28-day regimen (300-800 mg/kg/infusion). There were no serious, acute bacterial infections, whereas six subjects (12·0%) had at least one such infection in the 6 months before enrollment. Forty subjects (80·0%) had at least one non-serious infection; the median number of infective episodes per subject per year was 3·07. Antibiotics were taken by 38 subjects therapeutically and prophylactically by 16 at some time. Fewer than half (46·0%) missed any time off work or school because of infection or other illness. Trough immunoglobulin (Ig)G levels were above 6·00 g/l in all subjects at all assessments after 15 weeks with two exceptions. Overall, 21·2% of infusions were associated with an adverse event up to 72 h after infusion. The frequency of adverse events increased with infusion rate. Headache was the most common product-related adverse event (7·5% of 703 infusions). In conclusion, Gammaplex® is effective in primary immunodeficiency and is well tolerated.

Insights into serotonin signaling mechanisms associated with canine degenerative mitral valve disease.

Little is known about the molecular abnormalities associated with canine degenerative mitral valve disease (DMVD). The pathology of DMVD involves the differentiation and activation of the normally quiescent mitral valvular interstitial cell (VIC) into a more active myofibroblast phenotype, which mediates many of the histological and molecular changes in affected the valve tissue. In both humans and experimental animal models, increased serotonin (5-hydroxytryptamine, 5HT) signaling can induce VIC differentiation and myxomatous valve damage. In canine DMVD, numerous lines of evidence suggest that 5HT and related molecules such as transforming growth factor-beta play a critical role in the pathogenesis of this disease. A variety of investigative techniques, including gene expression, immunohistochemistry, protein blotting, and cell culture, shed light on the potential role of 5HT in the differentiation of VIC, elaboration of myxomatous extracellular matrix components, and activation of mitogen-activated protein kinase pathways. These studies help support a hypothesis that 5HT and its related pathways serve as an important stimulus in canine DMVD. This review describes the pathological characteristics of canine DMVD, the organization and role of the 5HT pathway in valve tissue, involvement of 5HT in human and experimental models of valve disease, avenues of evidence that suggest a role for 5HT in naturally occurring DMVD, and finally, a overarching hypothesis describing a potential role for 5HT in canine DMVD.

Serum serotonin concentrations in dogs with degenerative mitral valve disease.

BACKGROUND: Increased serotonin (5HT) signaling has been implicated in valvular disease of humans and animals, including canine degenerative mitral valve disease (DMVD). High circulating 5HT concentration is a potential source of increased signaling, and serum 5HT concentrations have not been previously reported in dogs with DMVD. HYPOTHESIS: Dogs with DMVD and small breed dogs predisposed to DMVD have higher serum 5HT concentrations than large breed controls. ANIMALS: Fifty dogs affected with DMVD, 34 dogs predisposed to DMVD but without cardiac murmur or echocardiographic evidence of DMVD, and 36 healthy large breed control dogs. METHODS: Prospective analysis. Serum 5HT concentration was measured by an ELISA test. RESULTS: Median serum 5HT concentration was significantly higher in dogs with DMVD and in dogs predisposed to DMVD as compared with controls (DMVD, 765.5 ng/mL [interquartile range, 561.3-944.4]; predisposed, 774.9 ng/mL [528.3-1,026]; control, 509.8 ng/mL [320.8-708.8]; P= .0001). Subgroup analysis of predisposed dogs indicated significantly higher serum 5HT concentrations in Cavalier King Charles Spaniel (CKCS) dogs than in other breeds (CKCS, 855.0 ng/mL [635.8-1,088]; non-CKCS, 554.2 ng/mL [380.6-648.4]; P= .0023). Age, platelet count, and platelet morphology were not correlated with 5HT concentration in any group. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with DMVD had significantly higher serum 5HT concentrations when compared with large breed control dogs. Healthy CKCS dogs had significantly higher serum 5HT concentrations than other healthy dogs predisposed to DMVD. Additional investigation into a possible role of 5HT in the pathogenesis of DMVD is warranted.

Immobilization of gene vectors on polyurethane surfaces using a monoclonal antibody for localized gene delivery.

BACKGROUND: Conventional strategies of gene therapy using viral vectors result in suboptimal localization and potentially dangerous distal spread of vector. We hypothesized that localized delivery of adenoviral gene vectors could be achieved from a polyurethane (PU) film through a mechanism involving anti-viral antibody tethering. METHODS: PU films were formulated with a collagen coating. Anti-adenoviral monoclonal antibodies were covalently bound to the collagen surface. These antibodies enabled tethering of replication-defective adenoviruses [Ad-GFP (encoding green fluorescent protein)] through highly specific antigen-antibody affinity. The binding stability and in vitro delivery of virus bound on PU films were investigated. Cell culture studies with rat arterial smooth muscle cells (A10) assessed transduction on or near the PU matrix. In vivo experiments with collagen-coated PU films investigated atrial epicardial implant and subdermal implant models in Yorkshire swine. RESULTS: We report for the first time successful PU film-based gene delivery using antibody-tethered adenovirus encoding the green fluorescent protein (GFP), demonstrating efficient and highly localized gene delivery to arterial smooth muscle cells in cell culture and pig implant. In comparison, direct injections of viral vectors into subcutaneous sites gave sparse, needle-track-oriented GFP expression patterns. CONCLUSION: We conclude that PU film is a suitable platform for a localizable viral vector delivery system that also prevents systemic spread of vector. Gene delivery using PU film-based anti-viral antibody tethering of vectors should be suitable for a wide array of single or multiple therapeutic gene strategies, and for further device-based gene delivery therapeutic strategies.

Immobilization of gene vector on polyurethane surface using monoclonal antibody for site-specific gene therapy.

Conventional strategies of gene therapy using viral vectors result in suboptimal localization and potentially dangerous distal spread of vector. We hypothesized that site-specific delivery of adenoviral gene vectors could be achieved from a polyurethane (PU) film through a mechanism involving anti-viral antibody tethering. PU films were formulated with a collagen coating. Anti-adenoviral monoclonal antibodies were covalently bound to the collagen surface. These antibodies enabled tethering of replication defective adenoviruses through highly specific antigen-antibody affinity. We report for the first time successful PU filmbased gene delivery using antibody-tethered adenovirus encoding green fluorescent protein (GFP), demonstrating efficient and highly localized gene delivery to arterial smooth muscle cells in cell culture. We conclude that PU film is a suitable platform for a localizable viral vector delivery system that also prevents systemic spread of vector. Gene delivery using PU film-based anti-viral antibody tethering of vectors should be suitable for a wide array of single or multiple therapeutic gene strategies, and for further stent-based gene delivery therapeutic strategies.

Localized gene delivery using antibody tethered adenovirus from polyurethane heart valve cusps and intra-aortic implants.

The present study investigated a novel approach for gene therapy of heart valve disease and vascular disorders. We formulated and characterized implantable polyurethane films that could also function as gene delivery systems through the surface attachment of replication defective adenoviruses using an anti-adenovirus antibody tethering mechanism. Our hypothesis was that we could achieve site-specific gene delivery to cells interacting with these polyurethane implants, and thereby demonstrate the potential for intravascular devices that could also function as gene delivery platforms for therapeutic vectors. Previous research by our group has demonstrated that polyurethane elastomers can be derivatized post-polymerization through a series of chemical reactions activating the hard segment amide groups with alkyl bromine residues, which can enable a wide variety of subsequent chemical modifications. Furthermore, prior research by our group investigating gene delivery intravascular stents has shown that collagen-coated balloon expandable stents can be configured with anti-adenovirus antibodies via thiol-based chemistry, and can then tether adenoviral vectors at doses that lead to high levels of localized arterial neointima expression, but with virtually no distal spread of vector. Thus, we sought to create two-device configurations for our investigations building on this previous research. (1) Polyurethane films coated with Type I collagen were thiol activated to permit covalent attachment of anti-adenovirus antibodies to enable gene delivery via vector tethering. (2) We also formulated polyurethane films with direct covalent attachment of anti-adenovirus antibodies to polyurethane hard segments derivatized with alkyl-thiol groups, thereby also enabling tethering of replication-defective adenoviruses. Both formulations demonstrated highly localized and efficient transduction in cell culture studies with rat arterial smooth muscle cells. In vivo experiments with collagen-coated polyurethane films investigated an abdominal aorta implant model in pigs using a button configuration that simulated the blood contacting environment of a vascular graft. One week explants of the collagen-coated polyurethane films demonstrated 14.3+/-2.5% of neointimal cells on the surface of the implant transduced with green fluorescent protein - adenovirus (AdGFP) vector loadings of 1 x 10(8) PFU. PCR studies demonstrated no detectable vector DNA in blood or distal organs. Similarly, polyurethane films with direct attachment of antivector antibodies to the surface were used in sheep pulmonary valve leaflet replacement studies, simulating the blood contacting environment of a prosthetic heart valve cusp. Polyurethane films with antibody tethered AdGFP vector (10(8) PFU) demonstrated 25.1+/-5.7% of attached cells transduced in these 1 week studies, with no detectable vector DNA in blood or distal organs. In vivo GFP expression was confirmed with immunohistochemistry. It is concluded that site-specific intravascular delivery of adenoviral vectors for gene therapy can be achieved with polyurethane implants utilizing the antivector antibody tethering mechanism.

DNA delivery from an intravascular stent with a denatured collagen-polylactic-polyglycolic acid-controlled release coating: mechanisms of enhanced transfection.

We previously demonstrated that DNA-polylactic-polyglycolic acid (PLGA)-coated stents can deliver genes to the arterial wall with reporter expression involving 1% of neointimal cells. The present study investigated a novel formulation utilizing denatured collagen in DNA-stent coatings; denatured collagen was hypothesized to enhance gene transfer due to adhesion molecule interactions and actin-related mechanisms. Arterial smooth muscle cells (SMCs) cultivated on denatured collagen had significantly greater plasmid DNA (beta-galactosidase) transfection than SMC grown on native collagen (18.3+/-1.2 vs 1.0+/-0.1%, P<0.001). The denatured-collagen effect was completely blocked with anti-alpha(v)beta(3) integrin antibody. SMCs cultivated on native collagen supplemented with tenascin-C (TN-C), a protein recognized by alpha(v)beta(3) integrins, showed a 33-fold increase in transfection compared to control (P<0.001); this effect was also blocked with anti-alpha(v)beta(3) antibody. We observed that cells grown on denatured collagen had marked F-actin-enriched stress fibers and intense perinuclear G actin, compared to those grown on native collagen, which demonstrated F-actin-enriched focal adhesions without perinuclear G-actin localization. Cytochalasin-D, an F actin depolymerizing agent, caused significantly increased SMC transfection in cells cultivated on native collagen compared to control cells (18.0+/-1.8 vs 3.02+/-0.9%, P<0.001) further supporting the view that actin-related cytoskeletal changes influence transfection. A denatured-collagen-PLGA composite vascular stent coating similarly resulted in increased plasmid DNA green fluorescent protein (GFP) expression compared to controls (P<0.001) in SMC cultures; the increased transfection was blocked by anti-alpha(v)beta(3) antibody. Pig coronary studies comparing denatured-collagen-PLGA-coated stents containing plasmid DNA (encoding GFP) to coated stents without DNA demonstrated 10.8% of neointimal cells transfected; this level of expression was almost an order of magnitude greater than previously reported with a DNA delivery stent. It is concluded that denatured collagen incorporated into plasmid DNA-stent coating formulations may increase the level of gene expression in vitro and in vivo because of integrin-related mechanisms and associated changes in the arterial smooth muscle cell actin cytoskeleton.

Enhanced immune response with a combination of alum and biodegradable nanoparticles containing tetanus toxoid.

The purpose of the study was to determine the synergistic adjuvant effect of sustained release biodegradable nanoparticles in combination with alum. Nanoparticles containing tetanus toxoid (TT) were formulated using a biodegradable polymer, polylactic polyglycolic acid co-polymer (50:50, molecular weight 100000). The immunization studies were carried out subcutaneously in rats. The results were expressed as mean serum anti-TT IgG levels. I'he nanoparticles demonstrated a TT loading of 4% w/w with mean particle diameter of 238 +/- 31 nm. The TT encapsulated in nanoparticles was released slowly under in vitro conditions, with 67.5% cumulative release occurring in 20 days. A single injection of TT-nanoparticles (TT dose = 10 Lf) mixed with TT-Alum (TT dose= 5 Lf) induced a four-fold greater mean serum anti-TT IgG response than a single injection of TT nanoparticles alone (TT dose=15Lf) (2235 +/- 310 vs. 539 +/- 49 microg/ml, mean +/- sem, p = >0.001). In addition, the mean immune response induced with the single injection of combination of nanoparticles and alum was comparable to the two injections of TT-alum alone (5 Lf each dose) given at 3 week intervals IgG = 1998 +/- 333 microg/ ml). Furthermore, the combination induced a peak immune response (IgG = 4215 +/- 546 microg/ml) as early as the first time point at 3 weeks post-immunization. In the case of a TT-alum alone, the animals showed a weaker immune response at 3 weeks and required a second dose of TT-alum to enhance the antibody response. The data thus suggest that the combination of TT-nanoparticles and TT-alum acts as a much better adjuvant than nanoparticles or alum alone. A rapid induction of immune response is useful to curb the spread of communicable diseases in the case of an outbreak.

High reactivity of alkyl sulfides towards epoxides under conditions of collagen fixation--a convenient approach to 2-amino-4-butyrolactones.

Epoxy crosslinking agents have been investigated for use in the fabrication of bioprosthetic devices, such as heterograft heart valve prostheses. It has been generally assumed that epoxy crosslinking takes place via amino-epoxy reactions. The present study investigated the hypothesis that the reactions of methionine residues with epoxides also can occur in biomaterial crosslinking. A series of model reactions were studied in which a mono-epoxide was combined with individual alkyl sulfides. In the present studies epoxides rapidly alkylate aliphatic sulfides, including methionine derivatives, in buffered aqueous solutions at room temperature and pH close to neutral, forming sulfonium compounds, which are stable at pH 5-7 at temperatures up to 50 degrees C, except for cases in which methionine derivatives with non-protected carboxy groups are used. The rate of reaction remains practically unchanged within the range of pH from 5 to 12, whereas in strongly alkaline media the reverse reaction occurs. This discovery can provide a better understanding of processes occurring in the fixation of bioprosthetic tissues with polyepoxides. It can also develop into a site-specific method to label methionine residues in proteins. The carboxy group-containing sulfonium betaines derived from N-protected methionines undergo cyclization in unexpectedly mild conditions, which can be used as an efficient method for preparation of N-protected 2-amino-4-butyrolactones with sensitive protective groups.

Matrix metalloproteinase-2 is associated with tenascin-C in calcific aortic stenosis.

We previously showed that the expression of tenascin (TN-C), an extracellular matrix glycoprotein found in developing bone and atherosclerotic plaque, and matrix metalloproteinase-2 (MMP-2) are coordinated and interdependent in cultured vascular smooth muscle cells. In this study, we hypothesized that TN-C and MMP-2 are mechanistically involved in the pathobiology of calcific aortic stenosis. Human calcific aortic stenosis cusps demonstrated immunohistochemically prominent deposition of TN-C, MMP-2, and alkaline phosphatase activity, as well as MMP-2 gelatinolytic activity. Although far lesser amounts of TN-C were noted in several of the grossly non-calcified valve cusps, MMP-2 and AP were never detected. Further, when aortic valve interstitial cells (both sheep and human) were cultivated on collagen supplemented with TN-C, both MMP-2 mRNA expression and MMP-2 gelatinolytic activity (both pro and active forms), were up-regulated compared to control. These observations support the view that accumulation of first TN-C and then MMP-2 are associated with progression of calcification. The residual presence of these proteins in severe calcifications is indicative of their involvement in the pathogenesis.

Localized adenovirus gene delivery using antiviral IgG complexation.

Gene therapy with viral vectors has progressed to clinical trials. However, the localization of viral vector delivery to diseased target sites remains a challenge. We tested the hypothesis that an adenoviral vector could be successfully delivered by complexation with a specific antibody that is bound to a biodegradable matrix designed for achieving localized gene transduction. We report the first successful delivery system based upon antibody immobilization of virions in a type I collagen-avidin gel using a polyclonal biotinylated IgG specific for the adenovirus hexon. In vitro stability studies demonstrated retention of viral vector activity with antibody-complexed adenovirus collagen gel preparations, in comparison to loss of vector activity from collagen gels prepared with nonspecific biotinylated IgG. Cell culture investigations using this antibody-controlled release system for adenoviral vector transduction of rat aortic smooth muscle cells (A10) demonstrated a significantly more localized reporter expression (beta-galactosidase) compared with non-antibody-complexed controls. Herpes simplex thymidine kinase (HSVtk) adenoviral vectors were immobilized on avidin-collagen gels via this antibody-complexation approach, and ganciclovir was added to rat smooth muscle cells (A10) in culture with the gels. With complexed HSVtk adenovirus, only cells either in contact with the virus-containing gel or within 50 microm were killed. By comparison, at the same adenovirus and ganciclovir dose, non-antibody-complexed HSVtk adenoviral delivery with ganciclovir resulted in the death of virtually all cells. Myocardial gene transfer studies in pigs demonstrated significantly more efficient right ventricular adenoviral GFP expression with anti-hexon antibody-complexed matrix injections, compared with direct vector injections. Thus, our results show that matrix formulations based on antibody-complexation delivery of adenovirus resulted in site-specific localization of transgene expression that enhances the efficiency of therapeutic vector strategies and provides a potent means for localization, to avoid distal side-effects. This approach has therapeutic potential as an implantable preparation that through the means of antibody-complexation, can localize and optimize viral vector gene therapy.

Enhanced plasmid DNA transfection with lysosomotropic agents in cultured fibroblasts.

Transfer of plasmid DNA into mammalian cells has posed major challenges for gene therapy. Most non-viral vectors are known to internalize in the cells by endocytosis. Therefore, low transfection efficiency of non-viral vectors may be due to intracellular degradation of input DNA in the endosomes and/or lysosomes. DNA degradation can be inhibited either by inactivating the lysosomal enzymes or obliterating endosome fusion to lysosomes using lysosomotropic agents. We report here the effects of individual lysosomotropic agents such as chloroquine, polyvinylpyrolidone (PVP) and sucrose on beta-gal expression in cultured fibroblasts COS, 293 and CHO. Cell viability was influenced by type, exposure time and concentration of lysosomotropic agents. Exposure to chloroquine at high concentration (1000 microM) or more than 4 h at any concentration (10-1000 microM) caused extensive cell death, however, cytotoxicity due to sucrose (5-500 mM) and PVP (0.01-1 mg/ml) was minimal in the cell lines tested. All the agents utilized in this study enhanced the gene expression and the transfection efficiency followed the order of sucrose>chloroquine>PVP at the concentrations used in all cell lines. Results suggest that lysosomotropic agents can enhance transfection efficiency but the degree of transgene expression may be cell- and agent-specific. Of the agents studied, sucrose appears to be an attractive agent in improving gene expression without toxic effect in the cultured fibroblasts. Thus, it can be used as an excipient in the formulation of new gene delivery systems.

Gene delivery from a DNA controlled-release stent in porcine coronary arteries.

Expandable intra-arterial stents are widely used for treating coronary disease. We hypothesized that local gene delivery could be achieved with the controlled release of DNA from a polymer coating on an expandable stent. Our paper reports the first successful transfection in vivo using a DNA controlled-release stent. Green fluorescent protein (GFP) plasmid DNA within emulsion-coated stents was efficiently expressed in cell cultures (7.9% +/- 0.7% vs. 0.6% +/- 0.2% control, p < 0.001) of rat aortic smooth muscle cells. In a series of pig stent-angioplasty studies, GFP expression was observed in all coronary arteries (normal, nondiseased) in the DNA-treated group, but not in control arteries. GFP plasmid DNA in the arterial wall was confirmed by PCR, and GFP presence in the pig coronaries was confirmed by immunohistochemistry. Thus, DNA-eluting stents are capable of arterial transfection, and could be useful as delivery systems for candidate vectors for gene therapy of cardiovascular diseases.

Inhibition of matrix metalloproteinase activity attenuates tenascin-C production and calcification of implanted purified elastin in rats.

Elastin, a major extracellular matrix protein present in arterial walls provides elastic recoil and resilience to arteries. Elastin is prone to calcification in a number of cardiovascular diseases including atherosclerosis and bioprosthetic heart valve mineralization. We have recently shown that purified elastin when implanted subdermally in rats undergoes severe calcification. In the present study, we used this elastin implant model to investigate the molecular mechanisms underlying elastin calcification. Intense matrix metalloproteinase (MMP-2) and tenascin-C (TN-C) expression were seen in the proximity of the initial cal-cific deposits at 7 days. Gelatin zymography studies showed both MMP-2 (latent and active form) and MMP-9 expression within the implants. To investigate the role of MMPs in calcification, rats were administered a MMP inhibitor, (2S:-allyl-N:-hydroxy-3R:-isobutyl-N:-(1S:-methylcarbamoyl-2-ph enylet hyl)-succinamide (BB-1101) by daily injection, either systemically or at the implant site. The site-specific BB-1101 administration almost completely suppressed TN-C expression, as shown by immunohistochemical staining, within the implants. The systemic BB-1101 injections also significantly reduced TN-C expression within the elastin implants. Moreover, calcification of elastin implants was significantly reduced in the site-specific administration group (5.43 +/- 1.03 microg/mg Ca for BB-1101 group versus 21.71 +/- 1.19 for control group, P: < 0.001). Alizarin Red staining clearly showed that the elastin fibers were heavily calcified in the control group, whereas in BB-1101 group the calcification was scarce with few fibers showing initial calcification deposits. The systemic administration of BB-1101 also significantly reduced elastin calcification (28.07 +/- 5.81 control versus 16.92 +/- 2.56 in the BB-1101 group, P: < 0.05), although less than the site-specific administration. Thus, the present studies indicate that MMPs and TN-C play a role in elastin-oriented calcification.

Pediatric airway issues.

Airway management in the pediatric patient requires an understanding and knowledge of the differences and characteristics unique to the child and infant. New and exciting techniques are currently being explored and developed for management of the pediatric airway. Technology in the area of imaging has allowed clinicians to better visualize the airway and aberrations of it. Presently, there are many different modes and routes of ventilation and oxygenation that are being applied to the pediatric patient for different disease states. Work continues to probe for methods and ways that will allow us to take care of infants and children better and to provide the safest and most effective means of delivering that care. No doubt, there will be more advances and exciting ideas to come that lead to better management of the pediatric airway.

Prevention of glutaraldehyde-fixed bioprosthetic heart valve calcification by alcohol pretreatment: further mechanistic studies.

BACKGROUND AND AIM OF THE STUDY: Calcification is a major cause of failure of bioprosthetic heart valves derived from glutaraldehyde-crosslinked bovine pericardium or porcine aortic valve (PAV) cusps. Recently, we have shown that ethanol pretreatment of PAV cusps prevents calcification in animal models. METHODS AND RESULTS: In this study we showed that ethanol pretreatment was equally effective in preventing calcification of glutaraldehyde-crosslinked bovine pericardium (control Ca2+ = 121.16+/-7.49 microg/mg tissue; ethanol-pretreated Ca2+ = 2.95+/-0.78 microg/mg). Furthermore, other low-molecular weight alcohols such as methanol and isopropanol were also effective in mitigating calcification of PAV cusps. Storage of ethanol-pretreated cusps in glutaraldehyde before implantation allowed partial return of calcification, suggesting a role for ethanol-glutaraldehyde interactions in preventing calcification. However, when ethanol-pretreated cusps were stored in ethanolic glutaraldehyde up to one month, the anti-calcification effect of ethanol persisted. The conditions whereby PAV cusps were crosslinked in pure, non-aqueous, alcoholic glutaraldehyde solutions were also examined. The crosslinking was equivalent to the standard aqueous glutaraldehyde crosslinking as indicated by thermal denaturation temperatures (Td) obtained by differential scanning calorimetry (DSC) and resistance to collagenase digestion. However, these cusps had lower water content and showed a marked decrease in spin-lattice relaxation times (T1) obtained by solid-state proton nuclear magnetic resonance (NMR). Moreover, these cusps calcified heavily in the 21-day rat subdermal implants. Thus, alcohol treatment during glutaraldehyde crosslinking was not useful. CONCLUSION: Glutaraldehyde storage after ethanol pretreatment aggravates calcification; moreover, alcoholic-glutaraldehyde crosslinking solutions are not beneficial for anti-calcification. Ethanol pretreatment of glutaraldehyde-pretreated bovine pericardium prevents its calcification.

Immune response with biodegradable nanospheres and alum: studies in rabbits using staphylococcal enterotoxin B-toxoid.

In this study, the adjuvant effect of the sustained release biodegradable nanospheres (100-150 nm in diameter) has been compared with alum. Nanospheres were formulated using a biodegradable polylactic polyglycolic acid copolymer (PLGA, 50:50) containing Staphylococcal Enterotoxin B (SEB) toxoid as a model vaccine antigen. Systemic immune response of the nanospheres containing toxoid was studied in rabbits by subcutaneous immunization. The data demonstrated that approximately 30% of the toxoid activity was lost following its encapsulation into nanospheres. Under in vitro conditions, nanospheres demonstrated sustained release of the toxoid. However, only 20% of the antigenic toxoid was released over the first 2 weeks of the release study. Immunization of animals with equal doses of toxoid, either using nanospheres or alum induced a comparable systemic immune response (IgG, IgM and IgA). The immune response reached a maximum level at 7 weeks post-immunization, which then gradually declined with time. The booster dose of toxoid at 19 weeks, either using alum or nanospheres induced similar immune response in both the groups, but was greater than the primary immune response. The studies, thus, suggest that biodegradable nanospheres could be used as a vaccine adjuvant.