Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay.Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 μg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 μg/ml for 3 and 20 h in the absence of S9, and for 3 h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 μg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 μg/kg, the last dose being administered 2 h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays.TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

Relationship between in vivo synchronicity of Plasmodium falciparum and allelic diversity

Plasmodium falciparum cells tend to grow in synchronicity during their cyclic intraerythrocytic development in vivo. Both host and parasite factors appear to be involved in this synchronization. We examined the link between mixed-allelic-family P. falciparum infection and synchronicity in parasitized red blood cells (PRBC) from symptomatic children.The distribution of rings and trophozoites in each PRBC sample was determined by standard microscopy. P. falciparum was genotyped by using a polymerase chain reaction (PCR) targeting three loci (merozoite surface proteins (MSP) 1 and 2, and 175-kD erythrocyte binding antigen (EBA), allowing us to distinguish parasite clones belonging to a single-allelic family (SAF) and those belonging to a mixed-allelic family (MAF). Parasite development was considered synchronous when peripheral blood contained at least 95% of rings or 95% of trophozoites.Parasite development was synchronous in 22 (21.2%) of the 104 children studied. Twenty (90.9%) of these infections were SAF and two (9.1%) were MAF. Rings and trophozoites predominated in respectively 12 (60%) and 8 (40%) SAF infections. Respectively 17.1% and 82.9% of the 82 asynchronous cases corresponded to SAF and MAF infection. Parasite synchronicity was therefore significantly related to single-allelic-family infection (p < 2 × 10^− 10).Twenty different MSP-1 alleles and thirteen different MSP-2 alleles were identified. Only three isolates from patients with SAF infection comprised a single allele or genotype, the other isolates harboring at least two alleles. The mean number of alleles or clones was respectively 3.0 and 10.0 in SAF and MAF infection. These results reflect the allelic diversity of the MSP loci and show that SAF infection can correspond to multiple parasite clones (or genotypes) but, in general, fewer than in MAF infection (p ≤ 0.0007).These results confirm the extensive polymorphism of P. falciparum vaccine candidates MSP-1 and -2 in southeastern Gabon and demonstrate that parasite synchronicity in vivo is strongly associated with single-allelic-family infection.     

单增李斯特菌胎盘感染的体内外模型研究进展

单核细胞增生李斯特氏菌（Listeria monocytogenes）是重要的食源性致病菌,能引发人类的李斯特菌病,是全球公共卫生问题之一。该菌易感染孕妇,引起胎儿和新生儿的侵袭性李斯特菌病,严重威胁母婴健康。因此,建立有效的单增李斯特菌感染胎盘体内外模型,解析和探究单增李斯特菌经胎盘感染机制,是预防和控制单增李斯特菌感染母婴的关键所在。本文综述了可用于研究单增李斯特菌母婴感染的体内外胎盘模型,总结和讨论了各类模型的优势和局限性;并着重分析了体外三维胎盘屏障模型在单增李斯特菌感染方面的研究进展和未来研究方向。以期为深入解析该菌经胎盘感染的途径、发病机制提供支持,并为预防和控制母婴李斯特菌病提供科学参考。     

Novel in vitro and in vivo inhibitors of prolyl endopeptidase

Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K_i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K_i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.     

Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

Background  

During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium.     

In vivo myograph measurement of muscle contraction at optimal length

Background  

Current devices for measuring muscle contraction in vivo have limited accuracy in establishing and re-establishing the optimum muscle length. They are variable in the reproducibility to determine the muscle contraction at this length, and often do not maintain precise conditions during the examination. Consequently, for clinical testing only semi-quantitative methods have been used.     

Glycation of wood frog (Rana sylvatica) hemoglobin and blood proteins: In vivo and in vitro studies

The effects of in vivo freezing and glucose cryoprotectant on protein glycation were investigated in the wood frog, Rana sylvatica. Our studies revealed no difference in the fructoselysine content of blood plasma sampled from control, 27 h frozen and 18 h thawed wood frogs. Glycated hemoglobin (GHb) decreased slightly with 48 h freezing exposure and was below control levels after 7 d recovery, while glycated serum albumin was unchanged by 48 h freezing but did increase after 7 d of recovery. In vitro exposure of blood lysates to glucose revealed that the GHb production in wood frogs was similar to that of the rat but was lower than in leopard frogs. We conclude that wood frog hemoglobin was glycated in vitro; however, GHb production was not apparent during freezing and recovery when in vivo glucose is highly elevated. It is possible that wood frog blood proteins have different in vivo susceptibilities to glycation.     

Aloe-emodin-induced DNA fragmentation in the mouse in vivo comet assay

Aloe-emodin (AE) and derivatives may be present as undesired components co-extracted during extraction of plants containing anthraquinonic derivatives for preparation of diacetylrhein. AE is a well-known in vitro mutagen, but up to now it failed to induce any clear in vivo genotoxic activity in the chromosome aberration assay in rat bone marrow or the in vivo/in vitro UDS test in liver. However, the two target organs noted during rodent carcinogenicity studies with danthron and emodin, two other well-known anthraquinone derivatives, are the colon and the kidney. Therefore, the choice of the organs for testing the genotoxicity of AE, i.e. bone marrow and liver, may be considered inadequate to demonstrate a possible in vivo genotoxic activity. In this context, the in vivo mouse comet assay was performed on both isolated kidney and colon cells in order to demonstrate a possible organospecific genotoxicity after oral administration of AE. Concurrently, the Ames test and the in vitro micronucleus assay with TK6 human lymphoblastoid cells were performed in their microscale version both with S9 from Aroclor 1254-induced liver or kidney, and without S9.AE induced primary DNA damage in the liver and in the kidney as observed between 3 and 6 h after two oral administrations at 500, 1000 and 2000 mg/kg bw, underlining an in vivo genotoxic mechanism of action. Furthermore, AE induced a clear genotoxic activity both in the Salmonella typhimurium strains TA1537 and TA98 and in the in vitro micronucleus assay in the absence as well as in the presence of metabolic activation. As no significant variation in the genotoxic activity of AE was noted when using either liver or kidney S9-mix, it seems that no quantitatively and/or qualitatively specific renal metabolism occurs. The kidney may be a target organ of AE as it is the major route of excretion. Under such conditions the separation of AE components should take place and the residual content of undesired AE derivatives should be made as low as reasonably achievable. AE present in plant extracts should be considered as an in vivo genotoxin and this property should be taken into account in the risk assessment for human exposure.     

常规培养与缺氧条件下BCG菌体内外sRNA的表达检测

近期发现细菌的sRNA在菌体内和菌体外均具有一定的生物学功能.为研究结核分枝杆菌菌体内外sRNA的表达情况,通过分析卡介苗(Bacillus Calmette-Guerin Vaccine,BCG)菌体和外泌体RNA测序结果,采用RT-qPCR法检测常规培养与缺氧条件下BCG菌体内外sRNA相对表达量,分析菌体内外sR...     

Using exomarkers to assess mitochondrial reactive species in vivo

Background

The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging.

Scope of review

One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules.

Major conclusions and general significance

Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

The molecular portrait of in vitro growth by meta-analysis of gene-expression profiles

Background  

Cell lines as model systems of tumors and tissues are essential in molecular biology, although they only approximate the properties of in vivo cells in tissues. Cell lines have been selected under in vitro conditions for a long period of time, affecting many specific cellular pathways and processes.     

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

Identifying and characterizing protein interactions are fundamental steps toward understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the colocalization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecular tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin α (Impα), and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified colocalization assay is sensitive enough to detect protein interactions with K_d values that span over four orders of magnitude (1 nM to 15 μM). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris.     

Phagocytosis by carp granulocytes; in vivo and in vitro observations

The phagocytic ability of carp (Cyprinus carpio L.) granulocytes was evaluated in vivo and in vitro. In suspensions of head kidney cells, neutrophil granulocytes incorporated both latex beads and coccidian merozoites. In intestinal tissues from carp with a Goussia carpelli infection, all granulocyte cell types (neutrophils and cells of the basophilic-eosinophilic complex) phagocytosed cell detritus and coccidian developmental stages, mainly merozoites.     

Cloning, expression, purification, distribution and kinetics characterization of the bacterial β-galactosidase fused to the cytoplasmic transduction peptide in vitro and in vivo

Cytoplasmic transduction peptide (CTP) offers exciting therapeutic opportunities for the treatment of many diseases caused by cytoplasmic functional molecules. It can transduce large, biologically active proteins into the cytoplasmic compartment of several mammalian cells. However, other intriguing features of CTP, including its activity in vitro, and distribution and tissue infiltration abilities in vivo, remain to be explored. The present study was initiated to (1) further confirm the cytoplasmic localization preference and the enzymatic activity of the transduced CTP-β-gal in vitro and (2) examine the kinetics and tissue distribution of the CTP-β-gal fusion protein in mice. A CTP-β-gal fusion protein was expressed in Escherichia coli and either transduced into BaF3-BCR/ABL cells or administered intravenously into female Balb/C mice at a dose of 100 μg per mouse. Its localization in BaF3-BCR/ABL cells was evaluated by immunocytochemistry and in situ X-gal staining, and its distribution in various tissues was analyzed both by in situ X-gal staining and quantitative enzymatic activity assay. β-Galactosidase enzyme activity was observed in BaF3-BCR/ABL cells and in all tissues tested, with peak activity occurring at 15 min in most tissues and at 24 h in brain. These data will not only allow rational selection of delivery schedules for therapeutic CTP, but will also aid the use of CTP fusion protein transduction in the development of protein therapeutics targeting the cytoplasmic compartment both in vitro and in vivo.     

Suppressive effects of swainsonine on C6 glioma cell in vitro and in vivo

Swainsonine, an extract from Astragalus membranaceus, is known for its anti-cancer effects and could prevent metastases. In order to investigate the effects and mechanisms of swainsonine in C6 glioma cells, we carry out correlated experiments in vitro and in vivo. After treatment with swainsonine, the effective dose and IC₅₀ value of swainsonine in the C6 glioma cell were examined using the MTT assay. Cell cycle distribution and apoptotic rates were analyzed using FCM and [Ca²⁺]_i was measured by LSCM. Expressions of p16 and p53 protein were evaluated by immunocytochemical methods. Simultaneously, glioma-bearing rats were administered swainsonine at doses of 2, 4 and 8 mg/kg body wt. The inhibition rate was calculated and pathological sections were observed. The results indicated that the growth of C6 glioma cells is inhibited by swainsonine in vitro, with an IC₅₀ value within 24 h of 0.05 μg/ml. Increases in swainsonine correlate with S phase percentages of 11.3%, 11.6% and 12.4%, respectively. Moreover, the expression of apoptosis inhibiting p53 and p16 protein decreases gradually. Tumor weight in vivo decreased clearly and HE dyeing of tumor tissue showed gray, its texture was soft, with necrosis and hemorrhagic concentrated inward. Swainsonine could inhibit the proliferation of C6 glioma cells in vitro and the growth of C6 glioma in vivo. The mechanisms of swainsonine-induced apoptosis may relate with the expression of apoptosis-related genes and overloading-[Ca²⁺]_i-induced endoplasmic reticulum stress.     

Experimental cryosurgery investigations in vivo

Cryosurgery is the use of freezing temperatures to elicit an ablative response in a targeted tissue. This review provides a global overview of experimentation in vivo which has been the basis of advancement of this widely applied therapeutic option. The cellular and tissue-related events that underlie the mechanisms of destruction, including direct cell injury (cryolysis), vascular stasis, apoptosis and necrosis, are described and are related to the optimal methods of technique of freezing to achieve efficacious therapy. In vivo experiments with major organs, including wound healing, the putative immunological response following thawing, and the use of cryoadjunctive strategies to enhance cancer cell sensitivity to freezing, are described.     

Adenovirus-mediated gene transfer of human butyrylcholinesterase results in persistent high-level transgene expression in vivo

Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents, pesticide intoxication, and cocaine overdose. However, its widespread application is hampered by difficulties in large-scale production of the native protein from human plasma and/or availability as a recombinant protein suitable for use in vivo. This limitation may be resolved by in vivo delivery and expression of the Hu BChE gene. In this study, recombinant (r) adenoviruses (Ads) encoding full-length and truncated rHu BChEs were tested for in vivo expression in mice. Mice injected with these rAds intraperitoneally failed to express rHu BChE. However, a single tail vein injection of both rAds resulted in persistent high serum levels of rHu BChE in BChE knockout mice, which peaked on days 4/5 at 377 ± 162 U/ml for full-length rHu BChE and 574 ± 143 U/ml for truncated rHu BChE. These activity levels are orders of magnitude higher than 1.9 U/ml of mouse BChE present in wild-type mouse serum. Thereafter, rHu BChE levels dropped rapidly and very little or no activity was detected in the serum 10 days post-virus administration. In conclusion, the present study demonstrates the potential of rAd-mediated Hu BChE gene therapy to counteract multiple lethal doses of chemical warfare nerve agent toxicity.