A note on an in vitro test system to compare the bactericidal properties of wound dressings

An in vitro method of comparing the antimicrobial properties of dressings used on human skin is described and has been tested with two samples of tulle gras. The method has advantages over agar diffusion methods by providing flexibility of test conditions and kinetic data for analysis.     

Novel Mesoporous Hydroxyapatite/Chitosan Composite for Bone Repair

The objective of the present investigation was to evaluate the osteogenic properties of mesoporous Hydroxyapatite/Chitosan (HA/CS) composite in vitro and in vivo.HA/CS composite was successfully prepared and synthesized using a freeze-drying method,and then characterized by Scanning Electron Microscope (SEM).Results show that the mesoporous HA/CS composite presents high surface area and porosity.The effects of mesoporous HA/CS on early adhesion,proliferation and differentiation of osteoblast cells in vitro were measured.MTT cytotoxicity test and cell adhesion test show that the composite has good biocompatibility and promotes cell viability and proliferation.In vitro tests show that osteoblast-like cells on the composite surfaces are able to adhere,proliferate,and migrate through the pores.These cells maintained similar expression levels of osteoblastic-associated markers namely Collagen type Ⅰ (COL-I),Bone Morphogenetic Protein 2(BMP-2).Histologic analysis and radiological analysis in vivo also prove that mesoporous HA/CS composite can be used to repair bone defect as a new kind of bone grafting materials.     

Adherence and colonization properties of Lactobacillus rhamnosus TB1, a broiler chicken isolate

AIMS: Selected lactic acid bacteria (LAB) isolated from intestinal tract of chicken have been studied in order to investigate their ability to adhere in vitro to Basement Membrane Matrigel (BMM). A selected strain showing a good adherence in BMM test was used for in vivo colonization assays. METHODS AND RESULTS: In vitro assessment of adhesion of broiler chicken isolates was performed using BMM assay. Among LAB strains tested, Lactobacillus rhamnosus TB1 showed a good adherence that was comparable to the one of an Escherichia coli EPEC strain used as positive control. For in vivo colonization assays this strain was fluorescently stained with the carboxyfluorescein diacetate succinimidyl ester (cFDA-SE) thus allowing its detection in different layers of intestinal tract after inoculation in broiler chicken. Further, stained L. rhamnosus were found with a highest value in rectum, jejunum and ileum both 3 and 24 h after administration. CONCLUSIONS: BMM assay is a quick method to test in vitro adhesion properties of bacterial strains and cFDA-SE-stained bacteria may be considered as an alternative method to test in vivo adhesion and colonization properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus rhamnosus TB1 was therefore showed to be able to adhere strongly in vitro to BMM and in vivo to intestinal epithelial cells of chicken and may be considered as a potential probiotic for chicken.     

检测纳米材料毒性的若干实验方法

纳米材料进入生命体和环境以后可能带来的生物安全问题需要定量的测定。现有的检测纳米材料生物安全性的方法大致可分为体内和体外实验两种,但是还没有证据表明既有的某个方法单独能作为一种检测所有纳米材料毒性的通用方法,也没有证据表明这些方法合在一起就能全面评估纳米材料的生物安全性。本文在总结既有的若干方法的同时,报道了一种基于rpsL基因的复制保真性来定量检测纳米材料毒性的方法。纳米材料对rpsL基因的体内体外复制过程保真性的影响均可方便地定量测定。     

Development of laboratory diagnostic methods for slow infections of the human central nervous system caused by nonclassical viruses

The theoretical foundations of the laboratory diagnosis of Creutzfeldt-Jakob disease and amyotrophic leukospongiosis in living patients are discussed and practical diagnostic methods are proposed on the basis of the study of the biological properties of nonclassical viruses and the pathogenesis of slow infections of the central nervous system. The use of the retrobulbar method of infecting susceptible animals has permitted, besides a considerable decrease in the incubation period in modeling slow infections, the improvement of the in vivo biological test. Thus, as the result of the multiplication of non-classical viruses in the visual nerve and the retina, experimental animals develop retinopathy which can be detected 1.5-2.5 weeks after the animals are injected with the spinal fluid or blood taken from the patient under examination. To diagnose amyotrophic leukospongiosis, the in vitro biological test, based on the capacity of the causative agent for inducing a considerable (2- to 5-fold) increase in the mitotic activity of HEp-2 cells, has been developed. The comparative studies have shown the expediency of using the method for the detection of antibodies to nerve-fiber proteins as an additional test for the differential diagnosis of slow infections of the central nervous system.     

Assessment of anti-oxidant activity of plant extracts using microbial test systems

Aims:  To evaluate the anti-oxidant properties of extracts from 20 medicinal herbs growing in western Siberia using microbial test systems and different in vitro methods.
Methods and results:  In vivo anti-oxidant activity of extracts was evaluated for their capacity to protect bacteria, Escherichia coli , against bacteriostatic and bactericidal effects of H₂O₂ and menadione, and action on anti-oxidant gene expression. In vitro anti-oxidant activity has been examined by a number of methods including: the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH^•)-scavenging assay, chelating activity and capacity to protect plasmid DNA against oxidative damage. In addition, total polyphenol content was determined. The extracts of Fragaria vesca , Rosa majalis , Pentaphylloides fruticosa , Alchemilla vulgaris and Pulmonaria mollis possessed the highest levels of anti-oxidant activity in vivo and in vitro . The protective properties were more closely related to the DPPH^• radical-scavenging activity, tannin content and action on anti-oxidant gene expression than to other parameters.
Conclusion:  The extracts of medicinal plants may have anti-oxidant effects on bacteria simultaneously through several different pathways, including direct inhibition of reactive oxygen species, iron chelation and anti-oxidant genes induction.
Significance and Impact of the Study:  Using microbial test systems, we revealed herbs that may be used as potential sources of natural anti-oxidants.     

New insights into deleterious impacts of in vivo glycation on albumin antioxidant activities

Background

Albumin constitutes the most abundant circulating antioxidant and prevents oxidative damages. However, in diabetes, this plasmatic protein is exposed to several oxidative modifications, which impact on albumin antioxidant properties.

Methods

Most studies dealing on albumin antioxidant activities were conducted on in vitro modified protein. Here we tried to decipher whether reduced antioxidant properties of albumin could be evidenced in vivo. For this, we compared the antioxidant properties of albumin purified from diabetic patients to in vitro models of glycated albumin.

Results

Both in vivo and in vitro glycated albumins displayed impaired antioxidant activities in the free radical-induced hemolysis test. Surprisingly, the ORAC method (Oxygen Radical Antioxidant Capacity) showed an enhanced antioxidant activity for glycated albumin. Faced with this paradox, we investigated antioxidant and anti-inflammatory activities of our albumin preparations on cultured cells (macrophages and adipocytes). Reduced cellular metabolism and enhanced intracellular oxidative stress were measured in cells treated with albumin from diabetics. NF-kB –mediated gene induction was higher in macrophages treated with both type of glycated albumin compared with cells treated with native albumin. Anti inflammatory activity of native albumin is significantly impaired after in vitro glycation and albumin purified from diabetics significantly enhanced IL6 secretion by adipocytes. Expression of receptor for advanced glycation products is significantly enhanced in glycated albumin-treated cells.

Conclusions and general significance

Our results bring new evidences on the deleterious impairments of albumin important functions after glycation and emphasize the importance of in vivo model of glycation in studies relied to diabetes pathology.     

Linear shear response of the upper skin layers

This study presents an in vitro experimental method to determine shear properties of the epidermis. Shear tests were performed with a parallel plate rheometer on samples of stratum corneum and the viable epidermis. The method was validated on very thin silicon sheets. Preliminary test were performed to determine the linear viscoelastic range, the effect of normal loading on the sample and the time to reach equilibrium after changes of temperature and relative humidity. The study shows that reproducible results can be obtained for the shear properties of epidermis in an in vitro set up. The dynamic shear modulus for stratum corneum ranges from about 4-12 kPa, decreasing with increasing relative humidity. The values are considerably lower than the shear modulus value based on tensile Young's moduli in the literature, indicating a considerable anisotropic material behavior. Results for the epidermis were of the same order of magnitude, but were less consistent possibly due to a less well-defined tissue composition.     

Applicability and robustness of the hen's egg test for analysis of micronucleus induction (HET-MN): results from an inter-laboratory trial

The hen's egg test for analysis of micronucleus formation (HET-MN) was developed several years ago to provide an alternative test system to the in vivo micronucleus test. In order to assess its applicability and robustness, a study was carried out at the University of Osnabrueck (lab A) and at the laboratories of Henkel AG & Co. KGaA (lab B). Following transfer of the method to lab B, a range of test substances that had been pre-tested at lab A, were tested at Henkel: the genotoxins cyclophosphamide, dimethylbenz(a)anthracene, methotrexate, acrylamide, azorubin, N-nitroso-dimethylamine and the non-genotoxins, orange G and isopropyl myristate. In a second phase, additional compounds with known in vivo properties were examined in both labs: the non-genotoxin, ampicillin, the "irrelevant" positives, isophorone and 2,4-dichlorophenol ("irrelevant" means positive in standard in vitro tests, but negative in vivo), the clastogen p-chloroaniline, and the aneugens carbendazim and vinorelbine. All substances were correctly predicted in both labs with respect to their in vivo genotoxic properties, indicating that the HET-MN may have an improved predictivity compared with current standard in vitro test systems. The results support the promising role of the HET-MN assay as a supplement to existing test batteries.     

Genotoxicity of p-aminophenol in somatic and germ line cells of Drosophila melanogaster

p-Aminophenol (PAP; as a component of, e.g., hair dyes, photographic developers, as adsorbent in gas filters, as a metabolite of various fungicides, pesticides and drugs) has been tested for genotoxicity in Drosophila by means of the sex-linked recessive lethal test (SLRLT) and the somatic mutation and recombination test (SMART) of the wing. While the SLRLT was not significant, the SMART clearly indicated that the compound has genotoxic properties in this in vivo test in agreement with a majority of mammalian short-term tests in vitro and in vivo. The reducing agent dithiothreitol enhanced the genotoxic effects of PAP in the SMART; the reasons for this interaction remain to be elucidated.     

Lack of mutagenicity of diphenylhydantoin in in vitro short-term tests

The mutagenicity of diphenylhydantoin (DPH) and its major metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), has been re-evaluated by the Ames test using Salmonella typhimurium and, for DPH only, by an in vitro cytogenetic test with human lymphocytes and a turbidimetric assay of tubulin polymerization. As negative results were obtained in all test systems used here, one has to conclude that DPH is devoid of mutagenic properties.     

用于蛋白质体外分子进化研究的DNA随机突变技术

蛋白质体外分子进化是模拟自然的进化过程,利用基因随机突变和定向筛选（选择）技术,以获得具有预期新功能的突变体分子。虽然体外进化近几年才产生,但已成为医药和工业领域中筛选具有特殊催化性质的酶的最重要的方法之一。ＤＮＡ随机突变技术是蛋白质体外分子进化研究的基础,本文将对几种最重要的突变方法：倾向错误的ＰＣＲ、ＤＮＡ重排、模板交错延伸反应和随机延伸突变的原理和应用等加以介绍。     

Multifunctional testing of PTCA balloon catheters]

New in vitro measuring methods for balloon catheters used for percutaneous transluminal coronary angioplasty (PTCA) and their verification in a complex test device are presented. This system can mimic all relevant application situations. The central element of the test device is a coronary vessel model matching the physiological situation in terms of geometrical structure and frictional properties. Reactive force sensors are used to measure the application-relevant forces exerted by the catheter on the model vessel walls and accessories, such as guide wire and guiding catheter. To generate a kink-free advancement of the catheter and permit measurement of the active forces, an alternating drive unit has been specially developed. The testing and application of the newly developed methods revealed statistically significant differences between various types of catheter. The test device closes a gap between complex but subjective clinical tests, and individual objective, but application-removed in vitro test setups for PTCA catheters. While the initial prototype had shortcomings with regard to the reproducibility of measurements, successor systems developed for industrial use are now in production. The properties of these measuring systems developed for the benefit of manufacturer and reprocessor of PTCA catheters are discussed.     

Naringenin-type flavonoids show different estrogenic effects in mammalian and teleost test systems

The estrogenic activity of several intermediary plant compounds has raised concern about possible risks of unwanted interference with endocrine regulation, but on the other hand there are potential medical benefits, in particular in treatment of menopausal symptoms or cancer. In the present study, we compare the estrogenic effects of phytoestrogens naringenin, 8-prenylnaringenin, 6-(1,1-dimethylallyl)naringenin, and the synthetic 4'-acetyl-7-prenyloxynaringenin. Two mammalian in vitro systems and a fish in vivo system were used to study the estrogenic properties with reference to genistein, 17-beta-estradiol or ethynylestradiol. Strong differences were observed between the mammalian in vitro and the fish in vivo test system. In the medaka sex reversal/vtg gene expression assay no estrogenic effects of the naringenin-type flavonoids were observed, while mammalian in vitro systems showed a similar and graded response to the test compounds.     

藻蓝蛋白色素肽光动力学抗肿瘤作用的实验研究

目的：探讨藻蓝蛋白色素肽的理化特性及光动力疗法（PDT）抗肿瘤效果。方法：用柱层析法从螺旋藻藻蛋白酶介产物中分离色素肽,并用MTT法及吖啶橙染色法检测色素肽对体外培养的小鼠肉瘤细胞S180及荷瘤小鼠PD。结果：分离出三种色素肽的分子量分别为17.4KD,7.72KD及6.6KD,它们的吸收光谱峰值分别位于618nm,580nm,605nm,荧光发射峰均在685nm处。MTT法检测证明,在浓度100μg/ml,用波长为580nm,600nm,照射剂量为28.8J/cm^2染料激光（氩离子为激光光源）照射,对肿瘤细胞的杀伤率达69．2％－80．2％;在色素肽CCP1,CCP3介导的PDT对小鼠移植瘤生长影响的实验发现,色素肽易被瘤细胞吸收,当激光照射剂量为120J/cm^2,肿瘤直径在0.5-0.7cm范围内,瘤体旁注射剂量为50μg的CPP1及CCP3光敏剂时,藻胆蛋白酶介产的的抑瘤率可达46％－81％,且被PDT作用后的细胞表现了典型的凋亡细胞特征。     

Ocular safety: a silent (in vitro) success story

Ocular irritation testing has been one of the animal test methods most criticised by animal welfare advocates. Additional criticism has arisen from within the scientific community, based on the variability of the animal test results and the questionable relevance of the extremely high dose levels employed. As a result, the Draize eye irritation test has been one of the main targets for in vitro replacement. Despite extensive efforts, however, there is still no in vitro method that is fully validated as a regulatory replacement. In spite of this, many individual companies are using diverse in vitro ocular irritation tests to gain important safety and efficacy information about their products and raw materials, eliminating the need for animal testing in the process. This is done in a safe fashion by applying intelligent testing paradigms. ECVAM has played a major role in this success, through its many programmes that have emphasised the importance of understanding the true toxicological need, and then using in vitro tests to provide that information. Thus, even in the absence of a successfully validated regulatory assay, the desired result of reducing animal testing is being met.     

Isolation of Bacillus strains from the rhizosphere of cereals and in vitro screening for antagonism against phytopathogenic, food-borne pathogenic and spoilage micro-organisms

Bacillus strains were isolated from the rhizosphere of cereals in order to be used as natural biocontrol agents (BCAs). They were screened for antagonism in vitro against various test micro-organisms. The isolates showing antagonism were identified to species level. A combination of techniques was employed for the isolation of Bacillus species. Using the direct method, only one of the 25 isolates screened showed antagonistic properties. This strain (IFS-01) was identified by means of API test strips and the ATB Plus computer programme. It proved to be Bacillus subtilis and consequently has been designated as Bacillus subtilis IFS-01. This strain produced either a broad spectrum antimicrobial compound or several compounds with different activities. The fungi and Gram-positive bacteria were more sensitive to the antagonistic isolate than the Gram-negative bacteria. A Bacillus strain producing BCAs which can be used as biopesticides or organic preservatives has been isolated and identified.     

ECVAM's activities in validating alternative tests for skin corrosion and irritation

ECVAM has funded and managed validation studies on in vitro tests for skin corrosion, resulting in the validities of four in vitro tests being endorsed by the ECVAM Scientific Advisory Committee: the rat skin transcutaneous electrical resistance (TER) assay, two tests based on the use of commercial reconstituted human skin equivalents, EPISKIN and EpiDerm, and another commercially-produced test, CORROSITEX. In the European Union (EU), a new test method on skin corrosion (B.40), incorporating the rat skin TER and human skin model assays, was included in Annex V of Directive 67/548/EEC in mid-2000, thereby making the use of in vitro alternatives for skin corrosion testing of chemicals mandatory in the EU. At the recommendation of its Skin Irritation Task Force, ECVAM has funded prevalidation studies on five in vitro tests for acute skin irritation: EpiDerm, EPISKIN, PREDISKIN, the pig-ear test, and the mouse-skin integrity function test (SIFT). However, none of the tests met the criteria (set by the Management Team for the studies) for inclusion in a large-scale formal validation study. Thus, to date, there are no validated in vitro tests for predicting the dermal irritancy of chemicals. Following further work on the EPISKIN, EpiDerm and SIFT test protocols and/or prediction models after the completion of the prevalidation studies, it appears that the modified tests could meet the performance criteria defined for progression to a validation study. This will now be assessed independently by the ECVAM Skin Irritation Task Force, with the objective of taking a decision before the end of 2002 on whether to conduct a formal validation study.     

Investigations of the antioxidant properties of plant extracts using a DNA-electrochemical biosensor

In this work, the results of a method based on an electrochemical biosensor to detect DNA damage in vitro for the evaluation of the antioxidant properties of plant extracts are reported. The biosensor consisted of a dsDNA immobilized on a screen-printed electrode surface (SPE). DNA damage was promoted by the generation of the *OH radicals via Fenton-type reaction. The interaction of the radical species with immobilised DNA in the absence and presence of antioxidants was evaluated by means of changes in the guanine oxidation peak obtained by square wave voltammetry. The results demonstrated that the DNA-based biosensor is suitable as a rapid screening test for the evaluation of antioxidant properties of samples.     

A new recipient cell line for transfection of biologically active oncogenes]

The properties of the new immortalized rat cell line (REF-1) were analyzed. These cells can be used as recipient ones in transfection assays. REF-1 cells never convert spontaneously to transformed phenotype during long-term passages in vitro unlike NIH3T3 cells. This peculiarity allowed to use REF-1 cells for identification of oncogenes, which induce slow-growing tumors. The following oncogenes were used in gene transfection experiments in order to test their effects: activated human EJ-ras; N-ras; v-myc; v-mos; activated tpr-met and c-hu-met. REF-1 cells, transfected with the members of ras family; v-mos and tpr-met were found to be transformed in vitro and induce tumors in nude mice, on the contrary of c-hu-met- and v-myc-transfected cells, which are non-tumorogenic. A number of clonal cell lines carrying different oncogenes were obtained. The detailed analyses of integration and expression of exogenous sequences of different oncogenes has been presented in 18 clonal cell lines.