Translating tissue culture results into animal models: the case of Salmonella typhimurium

Investigators use both in vitro and in vivo models to better understand infectious disease processes. Both models are extremely useful in research, but there exists a significant gap in complexity between the highly controlled reductionist in vitro systems and the largely undefined, but relevant variability encompassing in vivo animal models. In an effort to understand how Salmonella initiates disease at the intestinal epithelium, in vitro models have served a useful purpose in allowing investigators to identify molecular mechanisms responsible for Salmonella invasion of host cells and stimulation of host inflammatory responses. Identification of these molecular mechanisms has generated hypotheses that are now being tested using in vivo models. Translating the in vitro findings into the context of an animal model and subsequently to human disease remains a difficult challenge for any disease process.     

DSIP affects adrenergic stimulation of rat pineal N-acetyltransferase in vivo and in vitro

The natural occurrence, sleep, and extra-sleep effects of delta sleep-inducing peptide (DSIP) have been shown by different laboratories. However, neither an in vitro assay system nor a probable mechanism of action of the peptide have been conclusively demonstrated so far. The recent finding that DSIP influences the nocturnal rise of N-acetyltransferase (NAT) activity in rat pineal led us to investigate a possible effect on pharmacologically induced NAT activity in vivo and in vitro. Stimulation of the enzyme with adrenergic drugs such as isoproterenol and phenylephrine was reduced by DSIP at doses of 150 and 300 μg/kg injected subcutaneously. In vitro, 6, 150 and 300 nM DSIP attenuated isoproterenol stimulation of the enzyme in cultured pineals, whereas 150 nM DSIP effectively reduced stimulation induced by a combination of the two drugs. The peptide alone did not influence NAT activity in vitro, but produced a slight stimulation in vivo. To our knowledge, these results represent the first report of a direct interaction of DSIP with adrenergic transmission. The in vitro system could prove useful for establishing possible mechanism(s) of action of the ‘sleep peptide.’     

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

A review on disease transmission studies in relationship to production of embryos by in vitro fertilization and to related new reproductive technologies

This paper addresses the circumstances of germplasm contamination and updates on transmission of pathogenic agents by embryos produced in vitro and by associated techniques. It has been shown that some pathogenic agents might have been associated with the follicular oocytes and oviductal cells, collected for in vitro fertilization (IVF), resulting in infected embryos. Experimental introduction of pathogenic agent with oocytes or infected semen into the IVF system allows, in most cases, for the fertilization of eggs and for the production of some transferable quality embryos. Rendering of oocytes and embryos free of infectious pathogens, using the standard sequential washing or enzymatic treatment, is inconsistent and more difficult in the presently used in vitro fertilization system as compared to in vivo produced embryos.     

Effects of Calpain on Antioxidant Enzyme Activities

The activities of superoxide dismutase, catalase and glutathione reductase were not affected by in vitro incubation with the intracellular proteinase calpain, suggesting that these enzymes are not in vivo substrates of calpain. In contrast, the activity of another important antioxidant enzyme, glutathione peroxidase, is stimulated in vitro by calpain. This may explain the correlation between elevations in glutathione peroxidase activity and calpain activity which occur in aging, exercised and dystrophic muscle. Calpain treatment in vitro caused a large decrease in the activity of carnosine synthetase which is involved in the synthesis of the putative antioxidant carnosine. This may be the reason for the in vivo correlation between elevated calpain and diminished carnosine levels in aging, hypertensive, denervated and dystrophic muscles.     

Specificity of the action of parathyroid hormone upon mitochondrial metabolism: Cyanogen bromide-derived parathyroid-hormone peptides

The mitochondrial response to cyanogen bromide-treated parathyroid hormone was studied as a means of testing further the relationship between the structure and the effects in vitro of this hormone. The treated hormone and appropriate control hormone were tested in a standard bioassay and in a mitochondrial assay system in vitro.

Reaction of more than 90 % of the methionine residues in the hormone resulted in total inactivation of the hormone both in vivo and in vitro. This result disagrees with previously published data.     

Murine T cell clones specific for Hymenolepis nana: generation and functional analysis in vivo and in vitro.

To examine the role of the T cell in protective immunity to Hymenolepis nana, H. nana-specific clonal lymphocytes were generated from mesenteric lymph nodes of BALB/c mice infected with H. nana, and some of their functions were analyzed in vitro and in vivo. Following limiting dilution techniques, five clones were generated from mesenteric lymph node cell populations. All of these clones expressed the L3T4⁺, Lyt-2.2⁻ phenotype and proliferated in vitro in response to soluble egg antigen of H. nana. Of five clones, three secreted interleukin 2 (IL-2) and interferon-γ (IFN-γ) after stimulation with egg antigen. Furthermore, these three clones conferred local delayed-type hypersensitivity to egg antigen. The remaining two clones produced interleukin 4 (IL-4) in response to egg antigen, and could not mediate local delayed-type hypersensitivity. Adoptive transfer experiments using clonal lymphocytes were also undertaken in an attempt to define cell types involved in protective immunity. Clonal lymphocytes secreting both IL-2 and IFN-γ transferred protective immunity, equivalent to that obtained by non-cultured-sensitized mesenteric lymph node cells. They were effective in very small numbers. However, clonal lymphocytes that secreted IL-4 did not transfer protective immunity. These results suggest that helper T lymphocytes, especially the Th1 subtype, are involved in protective immunity against H. nana.     

Application and prospects of high-throughput screening for in vitro neurogenesis

Over the past few decades, high-throughput screening (HTS) has made great contributions to new drug discovery. HTS technology is equipped with higher throughput, minimized platforms, more automated and computerized operating systems, more efficient and sensitive detection devices, and rapid data processing systems. At the same time, in vitro neurogenesis is gradually becoming important in establishing models to investigate the mechanisms of neural disease or developmental processes. However, challenges remain in generating more mature and functional neurons with specific subtypes and in establishing robust and standardized three-dimensional (3D) in vitro models with neural cells cultured in 3D matrices or organoids representing specific brain regions. Here, we review the applications of HTS technologies on in vitro neurogenesis, especially aiming at identifying the essential genes, chemical small molecules and adaptive microenvironments that hold great prospects for generating functional neurons or more reproductive and homogeneous 3D organoids. We also discuss the developmental tendency of HTS technology, e.g., so-called next-generation screening, which utilizes 3D organoid-based screening combined with microfluidic devices to narrow the gap between in vitro models and in vivo situations both physiologically and pathologically.     

Interaction of glucocorticoid analogues with the human glucocorticoid receptor

Transient co-transfection of receptor cDNA and suitable reporter genes was used to study human glucocorticoid receptor (hGR) function in a neutral mammalian cell background. A variety of natural and synthetic steroids were analyzed for their ability to activate gene expression through the hGR and to bind to extracts of cells expressing the hGR cDNA. There was very good correlation between these two in vitro parameters for these compounds. Furthermore, correlation of these data with reported in vivo anti-inflammatory potencies was surprisingly close, with two exceptions. The in vitro data suggest an explanation for the discrepant compounds, consistent with published data on their metabolic fate in vivo. The co-transfection assay has utility as a quantitative predictor of in vivo glucocorticoid pharmacology.     

In vitro simulation and quantification of wear within the patellofemoral joint replacement

Quantification of the wear rate in vitro is now considered an essential step in the development of a new joint replacement prior to clinical trials. However, little research exists around in vitro simulation of wear in the patellofemoral joint (PFJ) despite over 200,000 being implanted annually within the European Union. A method to simulate wear in the laboratory using four input degrees of freedom within the PFJ of total knee replacement (TKR) has been developed. Wear simulation was validated through comparison of functional kinematics and patellar surface damage modes produced in vitro to clinical outcomes. The technique has been shown to replicate the prescribed in vivo kinematics in a reproducible and repeatable manner. The wear scar areas were similar to those found in vivo. However, geometrical measurements of wear were not reliable due to creep and geometry changes. As has been found previously with tibial inserts, geometrical determination of wear volume was not found to be an effective method of comparing wear from simulators and retrievals. Change in volume calculated gravimetrically was seen to be the most repeatable measure of patellar wear in vitro.     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

Bovine babesiosis: Partial purification and characterization of blood culture-derived Babesia bovis

Morphologic, biologic and immunologic properties of corpuscular and soluble fractions of Babesia bovis purified from in vitro blood cultures were studied. Supernatant fluids obtained during routine medium exchange were studied. Supernatant fluids obtained during routine medium exchange were submitted to differential centrifugation to separate soluble and corpuscular babesial antigens from erythrocyte stroma. Extracellular babesiae were sedimented with infected and uninfected erythrocyte ghosts. The majority of babesiae were found in erythrocyte ghosts. Clumps of extracellular parasites were sometimes formed in vitro and generally could not be separated from uninfected erythrocytes. Centrifugation over a discontinuous Ficoll density gradient did not improve separations. Parasites remained viable throughout the purification procedure but were killed by freezing and rapid thawing. Both corpuscular and soluble antigen fractions elicited the production of specific anti-babesial antibodies when injected into calves. Electron microscopy of corpuscular antigen revealed the presence of intra- and extraerythrocytic babesial merozoites. A surface coat was visible loosely adhering to the plasma membrane of the parasites. Parasite suspensions and cell-free supernatant fluids obtained from in vitro cultures of B. bovis should provide a variety of unique antigens for further in vitro and in vivo studies.     

Specific effect of manganese on the allantoinase of Vigna Radiata

Vigna radiata seedlings germinated in the presence of Mn²⁺ show an unusual increase in allantoinase activity which is proportional to Mn²⁺ concentration up to 5 mM. Though Mn²⁺ is not an activator for V. radiata allantoinase, it specifically protects allantoinase against thermal as well as papain-catalysed inactivation. Evidence is presented to show that the primary effect of Mn²⁺ is a protective one, both in vitro and in vivo, and that this is reflected in the observed enhancement of allantoinase activity in Mn²⁺ grown seedlings. That this unusual effect of Mn²⁺ is a specific one is indicated by the lack of a similar effect with Mg²⁺. Cu²⁺ is shown to destabilize V. radiata allantoinase in vitro as well as in vivo.     

Synthesis and biological properties of substituted 1,4-dihydro-5-methyl-4-oxo-3-quinolinecarboxylic acids

A series of substituted 1-cyclopropyl-6-fluoro-1,4-dihydro-5-methyl-4-oxo-3-quinoline carboxylic acids was synthesized and tested for their in vitro and in vivo antibacterial activity. The introduction of a methyl group at the 5-position of quinoline nucleus enhanced characteristically the antibacterial activity against Gram-positive bacteria, including Streptococcus pneumonia, which is a major pathogen in the respiratory tract infection, while retaining Gram-negative activity. Among them, 1-cyclopropyl-6-fluoro-1,4-dihydro-5-methyl-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid hydrochloride (grepafloxacin) exhibited potent in vitro antibacterial activity against Gram-positive bacteria such as Streptococcus pneumoniae and high in vivo efficacy on the experimental systemic infections caused by the Gram-positive and -negative bacteria tested. It also showed a high distribution to the lung and bronchoalveolar lavage fluid in comparison to reference drugs and is now undergoing clinical evaluation.     

Insight into generation of induced mesenchymal stem cells from induced pluripotent cells

Mesenchymal stem cells(MSCs)have the potential for use in cell-based regenerative therapies.Currently,hundreds of clinical trials are using MSCs for the treatment of various diseases.However,MSCs are low in number in adult tissues;they show heterogeneity depending upon the cell source and exhibit limited proliferative potential and early senescence in in vitro cultures.These factors negatively impact the regenerative potential of MSCs and therefore restrict their use for clinical applications.As a result,novel methods to generate induced MSCs(iMSCs)from induced pluripotent stem cells have been explored.The development and optimization of protocols for generation of iMSCs from induced pluripotent stem cells is necessary to evaluate their regenerative potential in vivo and in vitro.In addition,it is important to compare iMSCs with primary MSCs(isolated from adult tissues)in terms of their safety and efficacy.Careful investigation of the properties of iMSCs in vitro and their long term behavior in animals is important for their translation from bench to bedside.     

A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro

Exposure of endothelium to a nominally uniform flow field in vivo and in vitrofrequently results in a heterogeneous distribution of individual cell responses. Extremes in response levels are often noted in neighboring cells. Such variations are important for the spatial interpretation of vascular responses to flow and for an understanding of mechanotransduction mechanisms at the level of single cells. We propose that variations of local forces defined by the cell surface geometry contribute to these differences. Atomic force microscopy measurements of cell surface topography in living endothelium both in vitro and in situ combined with computational fluid dynamics demonstrated large cell-to-cell variations in the distribution of flow-generated shear stresses at the endothelial luminal surface. The distribution of forces throughout the surface of individual cells of the monolayer was also found to vary considerably and to be defined by the surface geometry. We conclude that the endothelial three-dimensional surface geometry defines the detailed distribution of shear stresses and gradients at the single cell level, and that there are large variations in force magnitude and distribution between neighboring cells. The measurements support a topographic basis for differential endothelial responses to flow observed in vivo and in vitro. Included in these studies are the first preliminary measurements of the living endothelial cell surface in an intact artery.     

Ecdysterone induced sexual reproduction in Opalina sudafricana parasitic in Bufo regularis

Ecdysterone induced sexual reproduction in Opalina sudafricana parasitic in Bufo regularis. International Journal for Parasitology 3: 863–868. Ecdysterone injections (0·5 mg/ml) induced sexual reproduction in Opalina sudafricana parasitic in male or female Bufo regularis after 12 days. Hypophysectomy or gonadectomy did not prevent the hormone from producing its effect on the parasites. Ecdysterone did not induce sexual reproduction in the parasites in vitro. Urine of toads injected with ecdysterone induced the opalinids to encyst in vitro.     

Rapid increase in serum lipid peroxide 4-hydroxynonenal (HNE) through monocyte NADPH oxidase in early endo-toxemia

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for a lipid peroxide 4-hydroxynonenal (HNE), which is 100-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA) and is an easier technique to use for a large number of samples without pre-treatment. By this assay, we found that a low dose of bacterial lipo-polysaccharide (LPS), injected intra-peritoneally (0.5 mg/kg), increased serum HNE level by 28-folds, with a peak at 20 min. LPS also increased HNE in vitro to a much higher level in the monocyte-enriched plasma than in the leukocyte-enriched plasma, with a peak at 10 min. The HNE production after LPS treatment was inhibited by apocynin, a specific NADPH oxidase inhibitor in vivo and in vitro, and to a lesser extent by dimethylsulfoxide a solvent for apocynin and a hydroxyl radical scavenger in vitro. These data suggest that monocyte NADPH oxidase is involved in the lipid peroxidation (HNE formation) in the LPS-challenged rat. This is the first clear demonstration of the link between an inflammatory stimulus and lipid peroxidation in the blood.     

植物中验证蛋白相互作用的Pull-down和Co-IP技术

蛋白互作在细胞生命活动中发挥关键作用,在不同时空层面上参与多种细胞学过程,因此研究蛋白互作对理解分子调控网络至关重要。通常情况下,利用酵母双杂交系统筛选植物蛋白互作必须通过体外和体内系统进行验证。Pull-down和Co-IP是验证植物蛋白互作的常用技术。Pull-down被广泛用于体外验证蛋白间的直接互作;而在植物活体内,利用本氏烟草(Nicotiana benthamiana)叶片瞬时表达蛋白,继而通过Co-IP进行鉴定是目前验证蛋白互作最简单且最有效的方法之一。该文对GST Pull-down和烟草瞬时表达系统中Co-IP技术原理及实验方案进行详细描述,以期为验证植物蛋白互作提供参考。