RESUMEN
BACKGROUND AND OBJECTIVE: Batroxobin, a snake venom thrombin-like enzyme, converts fibrinogen into fibrin by cleaving fibrinopeptide A. It is used for hemostasis; however, the supply of native batroxobin is limited. Therefore, we developed a recombinant batroxobin (r-batroxobin) from Pichia pastoris and evaluated its pharmacodynamics and safety in humans. METHODS: A randomized, double-blind, placebo-controlled, single ascending-dose study was performed. Eight healthy subjects were enrolled in each r-batroxobin dose group (2.5, 5.0, or 10.0 BU/2.0 mL administered intravenously), and randomized to receive r-batroxobin (n = 6) or matching placebo (n = 2). Safety was evaluated during the study, and pharmacodynamics was assessed using prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen level. RESULTS: All subjects in each cohort completed the study. No significant changes in PT or aPTT occurred after intravenous r-batroxobin administration. Compared with the placebo group, the fibrinogen level in all r-batroxobin dose groups decreased significantly to 8.68-33.57% from the baseline within 12 h (p ≤ 0.05). The TT in the 5.0 and 10.0 BU/2.0 mL groups significantly increased to 7.53-18.48% from baseline within 12 h compared with that of the placebo group (p ≤ 0.05), whereas that of the 2.5 BU/2.0 mL group exhibited non-significant changes compared with the placebo group. No serious adverse events occurred. CONCLUSIONS: A single intravenous injection of r-batroxobin within a dose range of 2.5-10.0 BU/2.0 mL was well tolerated and resulted in a significant decrease in fibrinogen and prolongation of TT. REGISTRATION: This study is registered at the Clinical Research Information Service (CRIS, http://cris.nih.go.kr ), number KCT0002518.
Asunto(s)
Batroxobina/administración & dosificación , Batroxobina/sangre , Coagulación Sanguínea/efectos de los fármacos , Hemostáticos/administración & dosificación , Hemostáticos/sangre , Tiempo de Protrombina , Adulto , Coagulación Sanguínea/fisiología , Estudios de Cohortes , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Voluntarios Sanos , Humanos , Inyecciones Intravenosas , Masculino , Persona de Mediana Edad , Tiempo de Protrombina/métodos , Proteínas Recombinantes/administración & dosificación , Trombina/metabolismo , Adulto JovenRESUMEN
The choice of hemostat is determined by the situation and the degree of hemorrhage. One common hemostat, the nonwoven dressing, is easy to handled and controls severe bleeding on wider wounds. In this study, chitosan-based nonwoven dressings with recombinant batroxobin (rBat) were used as efficacious hemostatic dressing agents. Hemostatic agents need to absorb blood quickly in the early stages of blood coagulation cascade to rapidly and effectively control of excessive hemorrhages. To date, most studies of hemostatic agents focused on a single material and hemostats composed of multiple materials have not been studied sufficiently. Thus, we made a chitosan dressing coated with rBat and investigated the microstructure, mechanical properties, hemostatic efficacy, and clotting properties of the coated dressing. Our results showed that the rBat had a synergetic effect on chitosan that improved blood coagulation. Furthermore, the dressing had excellent bleeding control in an Sprague-Dawley (SD) rat femoral artery hemorrhage model. In conclusion, hemostasis can be improved by combining a chitosan-based nonwoven dressing with other agents, and rBat-coated chitosan-based nonwoven dressings have enormous potential to improve blood coagulation.
Asunto(s)
Vendajes , Batroxobina/química , Quitosano/química , Quitosano/farmacología , Hemorragia/tratamiento farmacológico , Hemostasis/efectos de los fármacos , Proteínas Recombinantes/química , Animales , Coagulación Sanguínea/efectos de los fármacos , Quitosano/uso terapéutico , Arteria Femoral/efectos de los fármacos , Arteria Femoral/fisiopatología , Hemorragia/fisiopatología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Infections of Toxoplasma gondii and Babesia microti are reported in many wild animals worldwide, but information on their incidence and molecular detection in Korean wild fields is limited. In this study, the prevalence of T. gondii and B. microti infection in blood samples of 5 animal species (37 Chinese water deer, 23 raccoon dogs, 6 roe deer, 1 wild boar, and 3 Eurasian badgers) was examined during 2008-2009 in Gangwon-do (Province), the Republic of Korea (=Korea) by using serological and molecular tests. The overall seropositivity of T. gondii was 8.6% (6/70); 10.8% in Chinese water deer, 4.3% in raccoon dogs, and 16.7% in roe deer. PCR revealed only 1 case of T. gondii infection in Chinese water deer, and phylogenic analysis showed that the positive isolate was practically identical to the highly pathogenetic strain type I. In B. microti PCR, the positive rate was 5.7% (4/70), including 2 Chinese water deer and 2 Eurasian badgers. Phylogenetic analysis results of 18S rRNA and the ß-tubulin gene showed that all positive isolates were US-type B. microti. To our knowledge, this is the first report of B. microti detected in Chinese water deer and Eurasian badger from Korea. These results indicate a potentially high prevalence of T. gondii and B. microti in wild animals of Gangwon-do, Korea. Furthermore, Chinese water deer might act as a reservoir for parasite infections of domestic animals.
Asunto(s)
Animales Salvajes/sangre , Animales Salvajes/parasitología , Babesia microti/aislamiento & purificación , Toxoplasma/aislamiento & purificación , Toxoplasmosis Animal/epidemiología , Toxoplasmosis Animal/parasitología , Animales , Anticuerpos Antiprotozoarios/sangre , Babesia microti/genética , Babesia microti/inmunología , Babesia microti/patogenicidad , Reservorios de Enfermedades/parasitología , Reservorios de Enfermedades/veterinaria , Filogenia , Reacción en Cadena de la Polimerasa , Prevalencia , ARN Ribosómico 18S/genética , República de Corea/epidemiología , Toxoplasma/genética , Toxoplasma/inmunología , Toxoplasma/patogenicidad , Tubulina (Proteína)/genéticaRESUMEN
Recombinant batroxobin is a thrombin-like enzyme of Bothrops atrox moojeni venom. To evaluate its toxicological effect, it was highly expressed in Pichia pastorisand successfully purified to homogeneity from culture broth supernatant following Good Manufacturing Practice (GMP). The maximum tolerated dose of the recombinant batroxobin was examined in Sprague-Dawley (SD) rat and Beagle dogs following Good Laboratory Practice (GLP) regulations. The approximate lethal dose of recombinant batroxobin was 10 National Institute of Health (NIH) u/kg in male and female rats. Slight test substance-related effects were clearly in male and female dogs at more than 10 NIH u/kg. The maximum tolerated dose (MTD) was considered to be greater than 30 NIH u/kg in dogs. To investigate the repeated dose toxicity of batroxobin, the test item was intravenously administered to groups of SD rat and Beagle dog every day for 4 weeks. We observed that all animals survived the duration of the study without any effects on their mortality. There were no effects in both rats and dogs regarding their clinical signs, body weight, food consumption, ophthalmological examination, urinalysis, hematology, clinical chemistry, organ weightand gross post mortem examinations. The no adverse effect level (NOAEL) of recombinant batroxobin for both males and females is considered to be greater than 2.5 NIH u/kgin rats and 1 NIH u/kg in dogs, respectively. No toxic effects were noted in target organs. In conclusion, these results show a favorable preclinical profile and may contribute clinical development of recombinant batroxobin.
Asunto(s)
Batroxobina/toxicidad , Venenos de Serpiente/química , Pruebas de Toxicidad Aguda , Animales , Peso Corporal , Perros , Relación Dosis-Respuesta a Droga , Femenino , Fermentación , Dosificación Letal Mediana , Masculino , Nivel sin Efectos Adversos Observados , Tamaño de los Órganos/efectos de los fármacos , Pichia/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/toxicidad , TrombinaRESUMEN
Although a number of natural materials have been used as hemostatic agents, many substances do not act quickly enough. Here, we created a novel dressings using collagen and chitosan with recombinant batroxobin (r-Bat) to promote faster and more effective hemostasis. We hypothesized that r-Bat would promote synergetic blood coagulation because it contains a blood coagulation active site different than those of collagen and chitosan. Our results suggest that each substances can maintain hemostatic properties while in the mixed dressings and that our novel hemostatic dressings promotes potent control of bleeding, as demonstrated by a whole blood assay and rat hemorrhage model. In a rat femoral artery model, the scaffold with a high r-Bat concentration more rapidly controlled excessive bleeding. This novel dressings has enormous possible for rapidly controlling bleeding and it improves upon the effect of collagen and chitosan used alone. Our novel r-Bat dressings is a possible candidate for improving preoperative care and displays promising properties as an absorbable agent in hemostasis. STATEMENT OF SIGNIFICANCE: Despite the excellent hemostatic properties of collagen and chitosan pads, they reported to brittle behavior and lack sufficient hemostatic effect within relevant time. Therefore, we created a novel pad using collagen and chitosan with recombinant batroxobin (r-Bat). r-Bat acts as a thrombin-like enzyme in the coagulation cascade. Specifically, r-Bat, in contrast to thrombin, only splits fibrinopeptide A off and does not influence other hemostatic factors or cells, which makes it clinically useful as a stable hemostatic agent. Also the materials in the pad have synergetic effect because they have different hemostatic mechanisms in the coagulation cascade. This report propose the novel hemostatic pad isreasonable that a great potential for excessive bleeding injury and improve effects of natural substance hemostatic pad.
Asunto(s)
Vendajes , Batroxobina/farmacología , Hemostáticos/farmacología , Proteínas Recombinantes/farmacología , Animales , Coagulación Sanguínea/efectos de los fármacos , Bovinos , Modelos Animales de Enfermedad , Arteria Femoral/efectos de los fármacos , Arteria Femoral/patología , Fibrinógeno/metabolismo , Hemorragia/patología , Hígado/efectos de los fármacos , Hígado/patología , Microscopía Electrónica de Rastreo , Nefelometría y Turbidimetría , Activación Plaquetaria/efectos de los fármacos , Ratas Sprague-DawleyRESUMEN
Many types of hemostatic agents have been studied for the effective control of bleeding. In this study, a powdery medical adhesive composed of aldehyded dextran and ε-poly (L-lysine) was used with the recombinant batroxobin. Batroxobin is a venomous component from the snake Bothrops atrox moojeni and catalyzes fibrinogen conversion to form soluble fibrin clots. This research aims to examine the performance of the batroxobin-containing adhesive for hemostasis, and evaluate its potential as a novel hemostatic adhesive. The fibrinogen conversion ability of batroxobin was evaluated by a fibrinogen clotting assay and a whole blood clotting assay. Both experiments demonstrated the effectiveness of the batroxobin-containing adhesive for blood clot formation. Animal experiments were also conducted. After a pricking wound was made in an ICR (imprinting control region) mouse liver, the adhesive and various concentrations of batroxobin were applied. The total amount of blood loss was reduced with increasing concentrations of batroxobin. For excessive bleeding conditions, the femoral artery wound model of SD (Sprague-Dawley) rats was adopted. With higher concentrations of batroxobin, hemostasis was more rapidly achieved. Histological analysis of the liver model also supports the hemostatic effects through fibrin clot formation. In conclusion, batroxobin and medical adhesive effectively facilitate blood coagulation, and could be developed for clinical use.
Asunto(s)
Vendajes , Batroxobina/administración & dosificación , Coagulación Sanguínea/efectos de los fármacos , Hemorragia/tratamiento farmacológico , Hemostáticos/administración & dosificación , Adhesivos , Aldehídos , Animales , Bothrops , Dextranos/química , Arteria Femoral/patología , Fibrina/química , Fibrinógeno/química , Hemostasis , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Polilisina/química , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/químicaRESUMEN
Terminalia chebula Retz. has been used in India for a long time to treat many diseases, and its extract was reported to have antidiabetic activity in vivo. In this study, T. chebula methanolic extract (TCE) containing 2.7 % chebulic acid was evaluated for its preventive effects against the formation of advanced glycation end products (AGEs) and endothelial cell dysfunction. When the effects of TCE on AGE formation and on protein crossing-linking by glycation with D-threose and lens crystallines were examined, TCE showed inhibitory activity in a dose-dependent manner, and the concentration of 1000 µg/mL presented an activity similar to that of 5 mM aminoguanidine as a positive control. Upon investigating the protective activity of TCE against AGE-induced vascular endothelium dysfunction, human umbilical vein endothelial cells (HUVEC) incubated with 100 µg/mL of AGEs had significantly enhanced reactive oxygen species (ROS) formation, whereas the treatment of T. chebula reduced AGE-induced ROS generation. The incubation of HUVEC with 100 µg/mL of AGEs caused a considerable increase in THP-1 monocytic cell adhesion, but this adhesion was reduced by the treatment of TCE. These results suggest that TCE is a potential agent for alleviating diabetic complications.
Asunto(s)
Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Extractos Vegetales/farmacología , Terminalia , Antioxidantes/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Humanos , Cristalino/efectos de los fármacos , Monocitos/citología , Monocitos/efectos de los fármacos , Plantas Medicinales , Venas Umbilicales/citologíaRESUMEN
The importance of expression of matrix metalloproteinase (MMP) in keratinocyte migration is well established, but its role remains unclear. Here we investigated the function of MMP-14 in TGF-beta1-induced keratinocyte migration. TGF-beta1 stimulated cell migration and the expression of MMP-2, -9 in HaCaT human keratinocyte cells. When we lowered MMP-14 mRNA with siRNA, cell migration, and MMP-9 expression decreased. Furthermore, the MMP-14 siRNA also reduced activation of JNK in response to TGF-beta1, and a JNK-specific inhibitor decreased both cell migration and MMP-9 expression. Taken together, these results suggest that TGF-beta1-induced HaCaT cell migration is mediated by MMP-14, which regulates MMP-9 expression via JNK signaling.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Queratinocitos/citología , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Comunicación Celular , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular , Humanos , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas , Modelos Biológicos , Fosforilación , Transducción de SeñalRESUMEN
PURPOSE: Oxidative damage is one of the major factors associated with the formation of age-related cataract and with senescence of various cell types. Although the effects of oxidative stress are complex, we focused on whether oxidative damage affects control of the cell cycle in lens epithelial cells. METHODS: BrdU labeling and FACS analysis were used to investigate the effect of H2O2 on the cell cycle of HLE B-3 cells. In addition, western and Northern blot analysis were performed to assess the expression of cell cycle regulatory proteins and transfection with siRNA was used to knock out expression of p21Cip1. The activation of MAPK family members by oxidative stress was assessed using antibodies to detect the activated forms. To confirm the effect of H2O2 on an ex vivo model, its effect on cultures of the lenses of 3-week-old SD rats were examined. The localization and expression of PCNA and p21Cip1 in the rat lenses were analyzed by immunohistochemistry. RESULTS: FACS analysis showed that H2O2 treatment induced G2/M phase arrest of HLE B-3 cells. p21Cip1 was strongly induced by H2O2, whereas expression of other cell cycle genes was unchanged. Attenuation of p21Cip1 expression using siRNA reduced the H2O2 induced G2/M arrest. Furthermore, JNK and ERK were activated by H2O2 and their specific inhibitors SP600125 (for JNK) and U0126 (for ERK1/2) prevented p21Cip1 expression and blocked cell cycle arrest. H2O2 treatment of a rat lens organ culture also caused an increase in p21Cip1. However, H2O2 treatment lowered the levels of p27Kip1, cdc2, and PCNA in the rat lens culture, unlike in the HLE B-3 cells. CONCLUSIONS: The accumulation of p21Cip1 in lenses exposed to oxidative stress may play a role as a defensive mediator of oxidative damage, an indicator for senescence or aging, or an inducer for the formation of cataract. This finding links oxidative stress with p21Cip1-mediated control of the cell cycle in lens epithelial cells.
Asunto(s)
División Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Fase G2/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Cristalino/efectos de los fármacos , Animales , Northern Blotting , Western Blotting , Proteína Quinasa CDC2/metabolismo , Catarata/inducido químicamente , Catarata/metabolismo , Técnicas de Cultivo de Célula , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Citometría de Flujo , Humanos , Inmunohistoquímica , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Cristalino/citología , Cristalino/metabolismo , Masculino , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Técnicas de Cultivo de Órganos , Estrés Oxidativo/efectos de los fármacos , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
An after-cataract is caused by the proliferation of residual cells over the equator of the lens. These cells subsequently migrate to the posterior lens capsule, where they undergo aberrant differentiation into fiber-like cells or transdifferentiation into fibroblast-like cells. To study the precise molecular mechanisms of transdifferentiation, an attempt was made to establish an in vitro system, in which the lens epithelial cells (LECs) of the pre-equatorial zone could be transdifferentiated into fibroblast-like cells. The required conditions for culturing the LECs were identified as consisting of four phases; intact bovine explants, explant-cultured, serum-modulated and additionally modulated LECs. The LECs of each phase were compared by examining changes in the expression of the epithelial-mesenchymal transition (EMT)-related genes and changes in cellular morphology and adhesion. The explants that were cultured in a medium containing 10% fetal bovine serum (FBS) for 2 weeks, showed changes in the expression of the EMT-related genes, although the other explant-cultured cells maintained an epithelial morphology. To introduce a transition into mesenchymal cells, the explant cultures were subcultured in a medium containing 20% FBS for six passages. These cells displayed an elongated morphology and were able to grow and migrate in a similar way to fibroblast cells. The expression of the EMT-related genes, such as, extracellular matrix proteins and integrins, was altered. This was similar to the alteration of the 3-dimensional collagen gels model previously reported. During a further process of EMT by additional serum modulation, the inhibitory effect of disintegrin on cell adhesion was gradually decreased, integrin expression was differentially regulated and alpha-smooth muscle actin was post-translationally modified from the point of passage number six. Overall, it can be concluded that terminal transdifferentiation accompanies changes in the cytoskeletal proteins and cell surface molecules. These are modulated in systematic patterns of post-transcriptional and post-translational regulation and patterns of gene regulation, by the synergic effects of several transforming factors contained in serum. Therefore, posterior capsular opacification may also be accompanied by this molecular mechanism.
Asunto(s)
Proteínas Sanguíneas/farmacología , Células Epiteliales/citología , Fibroblastos/citología , Cristalino/citología , Animales , Bovinos , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Células Epiteliales/fisiología , Fibroblastos/fisiología , Expresión Génica/efectos de los fármacos , Cadena A de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/genéticaRESUMEN
The proliferation, migration and transdifferentiation of the remaining lens epithelial cells (LECs) after cataract surgery are a major cause of posterior capsular opacification (PCO). It has previously been reported that salmosin, a novel disintegrin, significantly inhibits solid tumor growth in mice by perturbation of tumor-specific angiogenesis via blocking alpha v beta 3 integrin expressed on vascular endothelial cells. In this study, the inhibitory function of salmosin in PCO was investigated and was found that salmosin inhibits the attachment of bovine LECs and rabbit lens cells (N/N1003A) to extracellular matrix-coated plates. The anti-adhesive activity of salmosin was approximately 1000 times higher than that of synthetic Arg-Gly-Asp peptide. In addition, the cell proliferation and migration of bovine LECs and N/N1003A were strongly inhibited by salmosin, whereas the proliferation of corneal endothelial cells was less affected. LEC migration and proliferation were also decreased by salmosin treatment in rabbit eyes without any toxic effect in the cornea, iris and retina. In this study, salmosin was shown to specifically inhibit LEC migration and proliferation in an animal model. Therefore, the authors suggest that further investigation may show salmosin to be a good candidate for inhibiting PCO development.
Asunto(s)
Catarata/prevención & control , Venenos de Crotálidos/uso terapéutico , Cápsula del Cristalino/patología , Animales , Catarata/patología , Extracción de Catarata , Bovinos , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Venenos de Crotálidos/farmacología , Endotelio Corneal/efectos de los fármacos , Proteínas de la Matriz Extracelular/metabolismo , Cápsula del Cristalino/efectos de los fármacos , Cápsula del Cristalino/metabolismo , Conejos , RecurrenciaRESUMEN
Transforming growth factor-beta (TGF-beta) regulates a wide range of physiological and pathological cellular processes, including cell migration, mesenchymal transition, extracellular matrix synthesis, and cell death. Cas (Crk-associated substrate, 130 kDa), an adaptor protein localized at focal adhesions and stress fibers, is also known to have important functions in cell migration and the induction of immediate-early gene expression. Here, we report that a rapid and transient tyrosine phosphorylation of Cas is induced by TGF-beta 1 and that E-cadherin-mediated cell-cell interaction and the Src kinase pathway are involved in this early TGF-beta signaling. The addition of TGF-beta 1 to epithelial cells rapidly induced tyrosine phosphorylation of Cas and promoted the formation of complexes between focal adhesion molecules. Cas phosphorylation required the integrity of the actin cytoskeleton but was not dependent on cell adhesion, implying that Cas-dependent signaling may be distinct from integrin signaling. TGF-beta 1 also stimulated Src kinase activity, and specific inhibitors of Src completely blocked the induction of Cas phosphorylation by TGF-beta 1. The Cas phosphorylation and Src kinase activation seen in our results were induced in an epithelial phenotype-specific manner. Stable transfection of E-cadherin to L929 cells and L cells as well as E-cadherin blocking assay revealed that E-cadherin-mediated cell-cell interactions were essential for both Cas phosphorylation and Src kinase activation. Taken together, our data suggest that rapid Cas phosphorylation and Src kinase activation may play a novel role in TGF-beta signal transduction.