Vo Hoai Bac

Colon ascendens stent peritonitis--a model of sepsis adopted to the rat: physiological, microcirculatory and laboratory changes.

The colon ascendens stent peritonitis (CASP) procedure creates an intestinal leakage of feces, resulting in diffuse peritonitis and polymicrobial sepsis. Mouse models of CASP have been used to study sepsis experimentally. The aim of the present study was to establish CASP sepsis in rats and to provide basic functional characteristics of this model. In analogy to the mouse model, 3 degrees of severity of CASP sepsis, 2 sublethal and 1 lethal, were established depending on the stent diameter. Radio-telemetric recordings in a sublethal model showed that the nonsurvivors remained hemodynamically stable until approximately 1 h before death, when heart rate and blood pressure fell rapidly. Intestinal microcirculatory changes were analyzed 3, 6, 12, and 18 h after CASP surgery using intravital microscopy in a sublethal model. After 18 h, the numbers of the leukocytes firmly adhering to the endothelium and of the ones temporarily interacting were significantly increased. The levels of IL-6 and IL-1&bgr; increased continuously during the CASP experiments while remaining unchanged in the sham group. TNF-&agr; and IL-10 levels of CASP animals reached a maximum after 12 h. In conclusion, a rat model of CASP sepsis has been established and characterized with regard to alterations in cardiovascular and microcirculatory function as well as plasma cytokine levels. In experimental settings where genetically engineered animals are not required, it will facilitate detailed examination of dynamic changes in integrated organ function during the course of sepsis and the investigation of treatment strategies.

Thermostatic tissue platform for intravital microscopy: 'the hanging drop' model

Intravital microscopy imposes the particular problem of the combined control of the body temperature of the animal and the local temperature of the observed organ or tissues. We constructed and tested, in the rat ileum microcirculation preparation, a new organ‐support platform. The platform consisted of an organ bath filled with physiological solution, and contained a suction tube, a superfusion tube, an intestine‐support hand that was attached to a micromanipulator and a thermometer probe. To cover the intestine we used a cover glass plate with a plastic ring glued on its upper surface. After a routine procedure (anaesthesia, monitoring and surgery), the intestine segment (2–3 cm long) was gently exteriorized and placed on the ‘hand’ of the organ support. A small part of the intestine formed a small ‘island’ in the bath that was filled with physiological salt solution. The cover glass was secured in place. The physiological salt solution from the superfusion tube, which was pointed to the lower surface of the cover glass, formed a ‘hanging drop’. The objective of the microscope was then immersed into distilled water that was formed by the cover glass plastic ring. The ‘hanging drop’ technique prevented any tissue quenching, ensured undisturbed microcirculation, provided for stable temperature and humidity, and permitted a clear visual field.

Vancomycin and to lesser extent tobramycin have vasomodulatory effects in experimental endotoxemia in the rat

BACKGROUND Antibiotic treatment represents a key component of therapy for severe sepsis. Apart from their antimicrobial efficacy, when choosing antibiotics in septic conditions, vasomodulatory effects should also be taken into account. OBJECTIVES Aim of this study was to evaluate the vasomodulatory effects of vancomycin (VANCO) and tobramycin (TOBRA) in experimental endotoxemia by using intravital microscopy (IVM) of the intestinal microcirculation and measurements of the arterial contractility in vitro. METHODS Endotoxemia was induced in rats by intravenous administration of lipopolysaccharide (LPS). VANCO or TOBRA were given immediately after LPS administration. Intestinal functional capillary density (FCD) and leukocyte-endothelial interactions were evaluated by IVM 2 hrs after LPS challenge. The effects of the antibiotics on the motility of aortal rings were examined in vitro. RESULTS Leukocyte adhesion was significantly potentiated in the LPS group and in both antibiotic groups as compared to control group. Roller flow in V1 and V3 venules increased in antibiotic treated groups as compared to untreated LPS animals. FCD in the longitudinal muscular and mucosal layers decreased significantly in either endotoxemia or antibiotics treated rats. However, administration of VANCO ameliorated FCD in endotoxemic rats. Both antibiotics, in higher concentration, produced moderate relaxation of the arterial smooth muscle. CONCLUSION Tobramycin and vancomycin did not affect the interaction between leukocytes and microvascular endothelium while vancomycin increased functional capillary density in the intestinal wall. Both antibiotics had direct relaxing effects on the vascular smooth muscle. Therefore, vancomycin and to lesser extent tobramycin may influence vascular tone and thereby affect microcirculation in endotoxemia and sepsis.

Combination of dehydroepiandrosterone and orthovanadate administration reduces intestinal leukocyte recruitment in models of experimental sepsis

BACKGROUND Dehydroepiandrosterone (DHEA) was shown to improve the immune function and survival in experimental sepsis. This study examined the effect of DHEA on intestinal leukocyte recruitment during experimental sepsis, considering factors of gender (male, female and ovariectomized female animals) and combined treatment using orthovanadate (OV) in two models of sepsis. METHODOLOGY/FINDINGS Male rats underwent colon ascendens stent peritonitis (CASP) or endotoxemia. DHEA was administered after induction of experimental sepsis. Changes in leukocyte adherence and capillary perfusion (measured as intestinal functional capillary density - FCD) were assessed using intravital microscopy. While DHEA increased baseline leukocyte adherence in control animals, DHEA reduced leukocyte adherence and increased FCD in male animals with CASP. These effects were also observed in DHEA-treated ovariectomized female rats with CASP. Similarly, the administration of DHEA reduced the number of adherent leukocytes to intestinal venules by 30% in the endotoxemia model. The combined treatment of DHEA and OV significantly reduced adherence of leukocytes to intestinal venules and improved FCD. CONCLUSIONS Our results indicate that DHEA is able to reduce intestinal leukocyte recruitment induced by experimental sepsis. Combination of DHEA with OV inhibits leukocyte adherence to intestinal endothelium, similar to what is achieved by the single administration of DHEA but with significantly improved FCD. These findings suggest a potential role for DHEA and OV in clinical sepsis.

Alpha-mangostin inhibits biofilm formation by Streptococcus mutans UA159

Streptococcus mutans is the primary etiological agent of human dental caries. The well-known extraordinary ability of S. mutans to adapt and survive the environment of human mouth is highly acidogenicity and strong exopolysaccharide (EPS) production, a skeleton of dental plaque (biofilm). In this research, the total biofilm biomass, as well as the contents of protein, intracellular polysaccharide (IPS), alkaline soluble polysaccharide (ASP), water soluble polysaccharide (WSP) in the biofilm treated with 150 mM α-mangostin was reduced ca. 40% compared to those of the control in ethanol as the vehicle. Two important glycosyltranferases B and C, responsible for biofilm formation by S. mutans , showed to be very sensitive to α-mangostin. Under the treatment condition, biofilm structure was clearly changed. Non-uniform and bigger microcolonies were found in the treated biofilms. Effects of α-mangostin on expression of the virulent genes and the detailed changes in biofilm structure are now under examination. The results showed that the a-mangostin is a new and potential anti-biofilm agent of S. mutans UA159.

High level expression of a gene coding for pectate lyase in Bacillus subtilis 168

Pectate lyase degrade polygalacturonate of plant cell wall productes oligogalacturonates. In recent years, pectate lyase has attracted the considerable research interest because of their potential industrial application, for instance in the textile industry, paper making. Gene pel coding for pectate lyase from Bacillus subtilis VBS11 was subcloned and fused with α-amylase promoter from B. amyloliquefaciens (BApro) . This construct was ligated into vector pMSE3 and transformated into the host strain BS168 for over expression under control of BApro promoter from B. amyloliquefaciens . rPel was producted in high level of 88,6 U/ml in Belisky minimal medium.

Screening and cloning of pectate lyase genes from Bacillus subtilis isolated in Vietnam

Pectate lyase (PL) is an important enzyme in plant pathogenesis, PL is secreted by microorganisms. PL degrades polygalacturonate of plant cell wall product oligogalacturonates. In this report, we described the screening of nine strains of B. subtilis, which isolated from Vietnam with ability to produce pectate lyase. The optimal pH for enzymatic degradation of polygalacturonate was from 8.5 to 10. The genes encoding pectate lyase from 4 different strains were cloned in E. coli and sequenced. Pectate lyase of these 4 strains contains 420 amino acid (aa). The deduced amino acid sequence of these PLs showed 98.8-99.8 % identity to PL from B. subtilis 168. There are 9 aa positions were changed in the amino acid sequencing of 4 trains in comparison between them and B. subtilis 168. The amino acid sequences of PLs of strains from plant were more conserve than those of PLs of strains from soil in comparison with PL from B. subtilis 168.