Study of novel coating strategy for coronary stents: simutaneous coating of VEGF and anti- CD34 antibody.

INTRODUCTION: Intravascular coronary stenting has been used in the treatment of coronary artery disease (CAD), with a major limitation of in-stent restenosis (ISR). The 316 stainless steel has been widely used for coronary stents. In this study, we developed a novel coating method to reduce ISR by simultaneously coating vascular endothelial growth factor (VEGF) and anti-CD34 antibody on 316L stainless steel. METHODS: Round 316L stainless steel sheets in the D-H group were polymerized with compounds generated from condensation reaction of dopamine and heparin using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). Sixteen sheets from the D-H group were further immersed into 1ug/ml VEGF165 and 3mg/ml heparin sodium one after another for 10 times, and named as the D-(H-V)10 group. Eight sheets from the D-(H-V)10 group were coated with anti-CD34 antibody and termed as the D-(H-V)10-A group. Immunofluorescence assay and ELISA were used to evaluate whether the 316L stainless steel disks were successfully coated with VEGF and anti-CD34 antibody. RESULTS: The results of immunofluorescence assay and ELISA showed that VEGF could be detected in the D-(H-V)10 and D-(H-V)10-A group, suggesting the steel sheets were successfully covered with VEGF. Anti-CD34 antibody could only be observed in the D-(H-V)10-A group, which was the only group coated with CD34 antibody. Both results suggested that the 316L stainless steel sheets were successfully coated with VEGF and anti-CD34 antibody. CONCLUSION: Our study developed a method to simultaneously coat VEGF and anti-CD34 antibody to stainless metal steel. This research serves as a fundamental role for a novel coating strategy.

Study of novel coating strategy for coronary stents: simutaneous coating of VEGF and anti- CD34 antibody / Estudo da nova estratégia de revestimento para stents coronários: revestimento simultâneo de VEGF e anticorpo anti-CD34

Abstract Introduction: Intravascular coronary stenting has been used in the treatment of coronary artery disease (CAD), with a major limitation of in-stent restenosis (ISR). The 316 stainless steel has been widely used for coronary stents. In this study, we developed a novel coating method to reduce ISR by simultaneously coating vascular endothelial growth factor (VEGF) and anti-CD34 antibody on 316L stainless steel. Methods: Round 316L stainless steel sheets in the D-H group were polymerized with compounds generated from condensation reaction of dopamine and heparin using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). Sixteen sheets from the D-H group were further immersed into 1ug/ml VEGF165 and 3mg/ml heparin sodium one after another for 10 times, and named as the D-(H-V)10 group. Eight sheets from the D-(H-V)10 group were coated with anti-CD34 antibody and termed as the D-(H-V)10-A group. Immunofluorescence assay and ELISA were used to evaluate whether the 316L stainless steel disks were successfully coated with VEGF and anti-CD34 antibody. Results: The results of immunofluorescence assay and ELISA showed that VEGF could be detected in the D-(H-V)10 and D-(H-V)10-A group, suggesting the steel sheets were successfully covered with VEGF. Anti-CD34 antibody could only be observed in the D-(H-V)10-A group, which was the only group coated with CD34 antibody. Both results suggested that the 316L stainless steel sheets were successfully coated with VEGF and anti-CD34 antibody. Conclusion: Our study developed a method to simultaneously coat VEGF and anti-CD34 antibody to stainless metal steel. This research serves as a fundamental role for a novel coating strategy. .

Resumo Introdução: O stent coronário intravascular tem sido utilizado no tratamento de doença arterial coronária, com uma maior limitação de restenose intra-stent (RIS). O aço inoxidável 316 tem sido amplamente utilizado para stents. Neste estudo, foi desenvolvido um novo método de revestimento para reduzir a RIS para revestir simultaneamente o fator de crescimento endotelial vascular (VEGF) e anti-CD34 em aço inoxidável 316L. Métodos: Placas de aço inoxidável 316L redondas no grupo DH foram polimerizadas com compostos gerados a partir da reacção de condensação de dopamina e heparina utilizando N- (3-dimetilaminopropil) -N'-etilcarbodiimida (EDC) e N-hidroxissuccinimida (NHS). Dezesseis folhas a partir do grupo DH foram ainda imersas em 1 ug/ml de VEGF 165 e 3 mg/ml de heparina sódica, um após outro por 10 vezes, sendo denominado como o grupo D-(HV)10. Oito folhas de D-(HV)10 foram revestidas com anticorpo anti-CD34 e denominado como grupo D-(HV)10-A. Testes de imunofluorescência e ELISA foram usados para avaliar se os discos de aço inoxidável 316L foram revestidos com sucesso com VEGF e anticorpo anti-CD34. Resultados: Os resultados dos testes de imunofluorescência e ELISA mostraram que o VEGF pôde ser detectado nos grupos D-(HV)10 e D-(HV)10-A, evidenciando que as chapas de aço foram cobertas com VEGF com sucesso. O anticorpo anti-CD34 podia apenas ser observado no grupo D-(HV)10-A, o único grupo revestido com anticorpo CD34. Ambos os resultados sugerem que as chapas de aço inoxidável 316L foram revestidas com sucesso com VEGF e anticorpo anti-CD34. Conclusão: Nosso estudo desenvolveu um método para revestir simultaneamente VEGF e anti-CD34 de aço inoxidável. Esta pesquisa tem um papel fundamental para a nova estratégia de revestimento. .

Atividade antimicrobiana da carbodiimida (EDC) sobre microorganismos presentes em lesões cariosas / Antimicrobial activity of carbodiimide (EDC) against caries-related microorganisms

Tem sido demonstrado que a carbodiimida (EDC) apresenta notável potencial inibidor de proteases (MMPs) e de melhorar as propriedades mecânicas do colágeno quando aplicada sobre a dentina desmineralizada. Entretanto, não existem informações a respeito de sua ação antimicrobiana sobre microrganismos comumente encontrados em lesões de cárie ou mesmo após a sua remoção. Objetivo: Investigar a atividade antimicrobiana do EDC em diferentes concentrações sobre microrganismos presentes em cavidades cariosas. Métodos: Soluções de EDC foram preparadas e testadas contra S. mutans e sobrinus, L. acidophilus e Candida albicans. Inicialmente, foi utilizado o teste de difusão em ágar, no qual discos de papel filtro foram impregnados com EDC 2, 1, 0,5, 0,3 ou 0,1 mol/L, clorexidina 0,12%, nistatina 1% ou tampão Sorensen pH 6,2 (n=6). Em seguida, foi determinada a concentração inibitória mínima (CIM) e bactericida mínima (CBM) do EDC sobre L. acidophilus em suspensão planctônica (n=9), por meio de turvamento. Por fim, a atividade do EDC (de 0,01 à 2 mol/L) sobre L. acidophilus em biofilme monoespécie foi definida por meio do ensaio de XTT (n=6). Os dados foram submetidos aos testes estatísticos de ANOVA e Tukey ou Mann-Whitney (p<0,05). Resultados: No teste de difusão em ágar, nenhuma atividade antimicrobiana foi observada para EDC nas concentrações de 0,1 e 0,3 mol/L, assim como para o grupo controle. EDC 0,5, 1 e 2 mol/L exerceu efeito antimicrobiano apenas sobre L. acidophilus. A CIM do EDC foi de 0,01 mol/L e a CBM foi de 0,03 mol/L. Todas as concentrações de EDC igual ou superiores a 0,05 mol/L foram capazes de reduzir significantemente o metabolismo do biofilme formado por L. acidophilus. Essa redução variou de 84,2 para 0,05 mol/L até 93,4% para 2 mol/L. Conclusão: O EDC apresentou atividade antimicrobiana apenas contra L. acidophilus reduzindo significantemente o crescimento deste microrganismo quando em suspensão planctônica e o seu metabolismo quando em biofilme monoespécie a partir de 0,05 mol/L

It has been demonstrated that carbodiimide (EDC) is a potent protease inhibitor (MMPs) and is able to improve the mechanical properties of collagen when applied on the demineralized dentin. However, there is no information about its antimicrobial effect on microorganisms commonly found in caries lesions or even after its removal. Objective: To investigate the antimicrobial activity of different concentrations of EDC against microorganisms present in caries lesions. Methods: EDC solutions were prepared and tested against S. mutans and sobrinus, L. acidophilus and Candida albicans. Initially, the agar diffusion test was used, where paper discs were impregnated with 2, 1, 0.5, 0.3 or 0.1 mol/L EDC, 0.12% chlorhexidine, nistatin 1% or Sorensen's buffer pH 6.2 (control) (n=6). Then, the minimum inhibitory (MIC) and bactericide concentrations (MBC) of EDC were determined against L. acidophilus using turbidity. Finally, the growth inhibitory activity of EDC (from 0.01 to 2 mol/L) against L. acidophilus in monoespecies biofilm was defined using the XTT assay (n=6). Data were submitted to ANOVA and Tukey tests or Mann-Whitney (p<0.05). Results: For the agar diffusion test, lack of antimicrobial activity was seen for EDC at 0.1 and 0.3 mol/L, as well as for the control group. 0.5, 1 and 2 mol/L EDC exerted a growth inhibitory effect only against L. acidophilus. The MIC for EDC was set as 0.01 mol/L and the MBC as 0.03 mol/L. Concentrations equal to or greater than 0.05 mol/L were capable of significantly reducing the metabolism of L. acidophilus when in monospecies biofilm. This reduction ranged from 84.2% for 0.05 mol/L to 93.4% for 2 mol/L. Conclusion: EDC exerted antibacterial activity only against L. acidophilus significantly reducing its growth in planktonic suspension and its metabolism in biofilms in the concentration of 0.05 mol/L or higher

The interplay of processivity, substrate inhibition and a secondary substrate binding site of an insect exo-beta-1,3-glucanase.

Abracris flavolineata midgut contains a processive exo-beta-glucanase (ALAM) with lytic activity against Saccharomyces cerevisiae, which was purified (yield, 18%; enrichment, 37 fold; specific activity, 1.89 U/mg). ALAM hydrolyses fungal cells or callose from the diet. ALAM (45 kDa; pI 5.5; pH optimum 6) major products with 0.6 mM laminarin as substrate are beta-glucose (61%) and laminaribiose (39%). Kinetic data obtained with laminaridextrins and methylumbelliferyl glucoside suggest that ALAM has an active site with at least six subsites. The best fitting of kinetic data to theoretical curves is obtained using a model where one laminarin molecule binds first to a high-affinity accessory site, causing active site exposure, followed by the transference of the substrate to the active site. The two-binding-site model is supported by results from chemical modifications of amino acid residues and by ALAM action in MUbetaGlu plus laminarin. Low laminarin concentrations increase the modification of His, Tyr and Asp or Glu residues and MUbetaGlu hydrolysis, whereas high concentrations abolish modification and inhibit MUbetaGlu hydrolysis. Our data indicate that processivity results from consecutive transferences of substrate between accessory and active site and that substrate inhibition arises when both sites are occupied by substrate molecules abolishing processivity.

Optimization of the conjugation method for a serogroup B/C meningococcal vaccine.

A conjugate meningococcal vaccine against serogroup B/C consisting of capsular PS (polysaccharide) from serogroup C conjugated to OMV (outer membrane vesicle) from serogroup B would be a very useful vaccine in regions where there is a prevalence of both serogroups, for example in Brazil. For this purpose, the conjugation method that uses ADHy (adipic acid dihydrazide) as spacer and a carbodi-imide derivative, EDAC [1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide], as catalyser was optimized looking for synthesis yield and maintenance of the antigenicity of both components. The best synthesis conditions preserving the vaccine immunogenicity resulted in a final yield of approx. 17%. Immunogenicity of the vaccine was highest when 10% of the sialic acid residues of the PS were occupied by the ADHy spacer. Sterilization of the conjugate by filtration through a 0.22-microm-pore-size membrane resulted in a low recovery of protein and PS (approximately 50%), although the vaccine immunogenicity was maintained. Using gamma irradiation on freeze-dried sample, it was possible to maintain the integrity of OMV structure and, consequently, its ability to induce bactericidal antibodies.

Identification of functionally important acidic residues in transducin by group-specific labeling.

Transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells, is a heterotrimer arranged as two units, the alpha-subunit (T alpha) and the beta gamma-complex (T beta gamma). The role of the carboxyl groups in T was evaluated by labeling with N,N'-dicyclohexylcarbodiimide (DCCD) and 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide (EDC). Only a minor effect on the binding of beta, gamma-imido guanosine 5'-triphosphate (GMPpNp) to T was observed in the presence of the hydrophobic carbodiimide, DCCD. Similarly, the GMPpNp binding activity of the reconstituted holoenzyme was not significantly affected when T alpha was combined with DCCD-treated T beta gamma. However, the binding of guanine nucleotides to the reconstituted T was approximately 50% inhibited when DCCD-labeled T alpha was incubated with T beta gamma. In contrast, treatment of T with the hydrophilic carbodiimide, EDC, completely impaired its GMPpNp-binding ability. EDC-modified T was incapable of interacting with illuminated rhodopsin, as determined by sedimentation experiments. However, rhodopsin only partially protected against the inactivation of T. Additionally, analyses of trypsin digestion patterns showed that fluoroaluminate was not capable of activating the EDC-labeled T sample. The function of the reconstituted holoenzyme was also disrupted when EDC-modified T alpha was combined with T beta gamma, and when EDC-treated T beta gamma was incubated with T alpha.