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PubMed2006

Increased expression of sFlt-1 in in vivo and in vitro models of human placental hypoxia is mediated by HIF-1.

Elevated expression of soluble vascular endothelial growth factor receptor-1 (sFlt-1) in preeclampsia plays a major role in the pathogenesis of this serious disorder of human pregnancy. Although reduced placental oxygenation is thought to be involved in the pathogenesis of preeclampsia, it is unclear how oxygen regulates placental sFlt-1 expression. The aims herein were to investigate sFlt-1 expression in in vivo and in vitro physiological and pathological models of human placental hypoxia and to understand the role of hypoxia inducible factor-1 (HIF-1) in regulating the expression of this molecule. sFlt-1 expression in placental villi was significantly increased under physiological low oxygen conditions in early first-trimester and in high-altitude placentae, as well as in pathological low oxygen conditions, such as preeclampsia. In high-altitude and in preeclamptic tissue, sFlt-1 localized within villi to perivascular regions, the syncytiotrophoblast layer, and syncytial knots. In first-trimester villous explants, low oxygen, but not hypoxia-reoxygenation (HR), increased sFlt-1 expression. Moreover, exposure of villous explants to dimethyloxalyl-glycin, a pharmacological inhibitor of prolyl-hydroxylases, which mimics hypoxia by increasing HIF-1alpha stability, increased sFlt-1 expression. Conversely, HIF-1alpha knockdown using antisense oligonucleotides, decreased sFlt-1 expression. In conclusion, placental sFlt-1 expression is increased by both physiologically and pathologically low levels of oxygen. This oxygen-induced effect is mediated via the transcription factor HIF-1. Low oxygen levels, as opposed to intermittent oxygen tension (HR) changes, play an important role in regulating sFlt-1 expression in the developing human placenta and hence may contribute to the development of preeclampsia.

Adult↗
PubMed2005

In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility.

Endothelial cells (ECs) are thought to be derived mainly from the vascular endothelial growth factor receptor 2 (VEGFR-2)+ lateral mesoderm during early embryogenesis. In this study, we specified several pathways for EC differentiation using a murine embryonic stem (ES) cell differentiation culture system that is a model for cellular processes during early embryogenesis. Based on the results of in vitro fate analysis, we show that, in the main pathway, committed ECs are differentiated through the VEGFR-2+ platelet-derived growth factor receptor alpha (PDGFR-alpha)- single-positive (VSP) population that is derived from the VEGFR-2+ PDGFR-alpha+ double-positive (DP) population. This major differentiation course was also confirmed using DNA microarray analysis. In addition to this main pathway, however, ECs also can be generated from the VEGFR-2- PDGFR-alpha+ single-positive (PSP) population, which represents the paraxial mesodermal lineage and is also derived from the DP population. Our results strongly suggest that, even after differentiation from the common progenitor DP population into the VSP and PSP populations, these two populations continue spontaneous switching of their surface phenotype, which results in switching of their eventual fates. The rate of this interlineage conversion between VSP and PSP is unexpectedly high. Because of this potential to undergo fate switch, we conclude that ECs can be generated via multiple pathways in in vitro ES cell differentiation.

Animals↗
PubMed2006

A novel mouse skeletal muscle-nerve preparation and in vitro model of ischemia.

The majority of patients in pain clinics are treated for muscle pain yet methods to study it in animals are relatively weak compared to methods to study skin pain. Here we describe an in vitro muscle-nerve preparation and model of muscle ischemia and contractile fatigue in mice. Timed muscle contraction is electrically evoked, while single unit activity of muscle sensory neurons and muscle contractile force are simultaneously recorded. The muscle is placed in a small (<1 mL) chamber where oxygen levels can be manipulated, drugs can be applied, and the extracellular milieu can be highly controlled. We demonstrate that we can record from sensory afferents that have the properties expected of ischemic nociceptors. This method serves for studying the neuronal and molecular mechanisms underlying ischemic pains such as angina, intermittent claudication, and sickle cell crisis.

Action Potentials↗
PubMed1983

Primary explant culture: an in vitro model of the human prostate.

Human prostate tissue gave rise to essentially pure cultures of basal cells in vitro. Careful analysis by phase-contrast microscopy and photography suggested that these cells attempt to differentiate into secretory acinar cells. This was confirmed by immunocytochemical analysis for prekeratin which is present only in basal cells in situ and for prostatic acid phosphatase, prostate-specific antigen, and prostate fluid antigen, which are present only in secretory acinar cells in situ.

Acid Phosphatase↗
PubMed1992

Subchondral damage after acute transarticular loading: an in vitro model of joint injury.

Intact canine metacarpophalangeal and metatarsophalangeal joints were subjected to a variety of loads in vitro. Intraarticular fracture occurred in 19 joints loaded to an average force of 2.4 +/- 0.4 kN with a corresponding loading rate of 88 +/- 23 kN/s. The remaining 29 joints were without gross evidence of fracture with an average load and loading rate of 1.7 +/- 0.9 kN and 64 +/- 32 kN/s, respectively. In the fractured specimens, damage to the zone of calcified cartilage and subchondral bone was much more extensive than was initially evident by gross inspection when assessed by scanning electron microscopy. Cracks with associated step-off displacement at the zone of calcified cartilage were found distant to the gross fractures. These findings were confirmed histologically. In addition, cracks localized to the zone of calcified cartilage were commonly identified histologically in specimens loaded in the range of 1.9-2.8 kN, but were not grossly fractured. The contact area determined with pressure-sensitive film increased with increasing load up to the point of fracture. The average pressure generated at the articular cartilage surface at the time of fracture in this model is > or = 40 MPa, and the fracture occurred at the contact site. Our findings suggest that failure in acute transarticular loading begins in the zone of calcified cartilage and subsequently involves the subchondral bone and overlying cartilage. This type of injury may contribute to the development of osteoarthritis after intraarticular fracture, or at high loads that do not result in gross fracture.

Animals↗
PubMed1992

Study of thromboxane and prostacyclin metabolism in an in vitro model of first-trimester human trophoblast.

OBJECTIVES: The purpose of our study was to establish an in vitro tissue culture system to study eicosanoid metabolism in first-trimester trophoblastic tissue. Thromboxane A2, a potent vasoconstrictor, and prostacyclin, a potent vasodilator, were analyzed to evaluate their production in early pregnancy. STUDY DESIGN: Trophoblastic tissue was obtained via transabdominal chorionic villous sampling from 33 pregnancies at 9 to 12 weeks' gestation for cytogenetic diagnosis. Initially, tissue obtained from the cytogenetics lab was morphologically consistent with villous core cells. Through altering cell density and passage, the cells became morphologically consistent with cytotrophoblasts. The cell lines were exposed to arachidonic acid (50 mumol/L) and aspirin (1 to 100 mumol/L) for 24 hours. Thromboxane B2 and 6-keto prostaglandin F2 alpha were measured by radioimmunoassay. RESULTS: Villous core cells and cytotrophoblasts increased production of thromboxane A2 and prostacyclin in the presence of arachidonic acid (p < 0.002). The villous core cells produced more thromboxane A2 and prostacyclin than cytotrophoblasts (p < 0.02). A significant inhibition of both thromboxane A2 and prostacyclin production was seen in the presence of 100 mumol/L aspirin in both cell types (p < 0.05). CONCLUSIONS: This model may be useful for studying placental function in the first trimester because individual placental compartments can be evaluated in tissue culture. At the cellular level we were not able to detect a preferential decrease in thromboxane A2 production in the presence of aspirin (1 to 100 mumol/L).

6-Ketoprostaglandin F1 alpha↗
PubMed1999

Pluripotent stem cells of the mouse as a potential in vitro model for mammalian germ cells. Sister chromatid exchanges induced by MMC and ENU in undifferentiated cell lines compared to differentiated cell lines.

We tried to develop an in-vitro test system which could serve as a model for mammalian germ cells in vivo. Two pluripotent cell types were used, because they express some germ cell specific immunological and biochemical markers: (1) Embryonal carcinoma cells (ECC) of the line P19 had been isolated from a teratocarcinoma of murine primordial germ cells (PGC). (2) Embryonal stem cells (ESC) are obtained from the inner cell mass of mouse blastocysts. Sister chromatid exchanges (SCE) induced by mitomycin C and ethylnitrosourea (ENU) were analysed in the two undifferentiated cell lines, ECC and ESC, to detect differences in their sensitivity compared with differentiated cell lines of the mouse. Neither of the model cell lines have shown a greater sensibility after exposure to MMC and ENU. In contrary, the carcinoma cell line was able to tolerate higher concentrations of these genotoxicants. Therefore, SCE analysis in the ECC and ESC lines used does not provide a suitable model for genotoxicity testing on mammalian germ cells.

Animals↗
PubMedDate not supplied

An in vitro model for the in vivo mobilization of cadmium by chelating agents using 113Cd-NMR spectroscopy.

An in vitro method, based on 113Cd-NMR spectroscopy, that provides an alternative to the use of animals for an initial screening of cadmium antagonists is presented. The relative values of the effective stability constants of potential chelating antagonists for cadmium are estimated by using 113Cd-NMR spectroscopy to determine the concentrations of the cadmium species involved in appropriate competitive equilibria. This is accomplished via an examination of the competition between the proposed antagonist and EDTA (ethylenediaminetetraacetic acid) for cadmium-113; previously, EDTA has been shown to be capable of removing cadmium from such in vivo binding sites as metallothionein. The reactions proceed via the stepwise addition of three dithiocarbamate groups to the cadmium accompanied by the concurrent stepwise release of donor groups from the EDTA. The resulting 113Cd-NMR data allow for the determination of the overall stability constant for the complex formed between cadmium and N-methyl-D-glucamine dithiocarbamate, iminodiacetic acid dithiocarbamate, proline dithiocarbamate, sarcosine dithiocarbamate. The use of 113Cd-NMR spectroscopy has the potential for providing direct evidence on the effectiveness of chelate antagonists to compete with endogenous ligands for other toxic metal ions. This technique could prove very useful for other compounds that are not stable enough toward acid and/or base to be examined by standard titrimetric methods.

Cadmium↗
PubMed1983

Establishment and characterization of an in vitro model system for human adenocarcinoma of the stomach.

Ten permanent clones derived from a single biopsy specimen of an untreated human adenocarcinoma of the stomach were established and characterized in vitro. Tissue culture growth properties, doubling times, plating efficiencies, growth fractions, cell cycle phase distributions, DNA indices, modal chromosome numbers, and ploidies were determined. Growth fractions were nearly 100%, and doubling times ranged from 23 to 37 hr. The plating efficiencies were generally high for tumor cells in culture, ranging up to 70%. Modal chromosome numbers varied from 45 to 48, with a wider range of variability in about 25% of the cells studied in each clone. In addition, the parent cell line (from which the clones were isolated) was shown to grow in athymic mice and to have the same histochemical and cytological characteristics as the specimen taken from the patient. It is important to characterize human tumor cells in vitro in this detailed manner, since they serve as excellent model systems for other studies involving the heterogeneous responses to drugs and radiation. The identification of mechanisms of drug sensitivity and resistance and the testing of drug and radiation combination treatment schedules in such in vitro systems can provide valuable insight into the design of clinical protocols for treatment of stomach cancer in humans.

Adenocarcinoma↗
PubMedDate not supplied

Efficacy of sulbactam in an in vitro model of mixed aerobic/anaerobic infections.

Bacterial interactions in mixed infections may interfere with antimicrobial therapy. The in-vitro efficacy of ampicillin alone, combination sulbactam/ampicillin, and metronidazole was studied. Strains of Bacteroides fragilis, Escherichia coli, and Enterococcus faecalis, alone and in association, were tested by means of a broth dilution method. Minimal bactericidal concentrations (MBC) of ampicillin for B. fragilis 74 in association with E. coli 68 were up to 16-fold higher than for B. fragilis 74 alone (256 compared to 16 mg/l), but only 2-fold higher for sulbactam (5 mg/l)/ampicillin (0.5 and 0.25 g/l). Association of B. fragilis 45 and E. faecalis 186 increased ampicillin MBC of E. faecalis 186 from 2 to 16 mg/l, but the combination sulbactam/ampicillin restored activity of ampicillin. In association with E. faecalis, metronidazole MBCs of B. fragilis increased up to 64-fold. Strains of E. faecalis and E. coli were able to destroy 10 mg/l metronidazole within 8 to 20 h. The present experiments demonstrated effectiveness of sulbactam/ampicillin to inhibit beta-lactamases of associated pathogens. Destruction of metronidazole by E. faecalis lends additional support to the use of the combination in aerobic/anaerobic infections including E. faecalis.

Ampicillin↗
PubMedDate not supplied

How to make a good oocyte: an update on in-vitro models to study follicle regulation.

The ability to develop the technology to mature oocytes from immature oocytes in vitro is the ambition of many IVF clinics. If this can be successfully achieved then these techniques would be available to women with fertility problems. This would aid women at risk of premature ovarian failure, and possibly result in women no longer requiring an expensive drug regime and monitoring programme, which they currently have to undergo. The idea of harvesting immature oocytes for growth in vitro is not a new one, but progress has been slow in developing and optimizing techniques for use on humans and domestic species. At present, there are many technical reasons for the lack of progress in these species, such as length of culture and difficulty of follicle isolation. However, the major problem is our lack of knowledge of how the oocyte acquires developmental competence during its growth within the follicle. To date, culture systems have been developed that can support the growth and development of immature oocytes. These systems are beneficial in improving our knowledge of how autocrine/paracrine factors are involved in regulating and controlling oocyte development. However, only when we have a more in-depth understanding of what is required during development to make a viable oocyte, will we perhaps be able to develop in-vitro culture systems for clinical application. This review will focus on how analysis of early follicular growth and development, using in-vitro culture systems, has advanced our knowledge of the factors and process involved in the regulation of oocyte and somatic cell development.

Animals↗
PubMed1997

An in vitro model for infection with Leishmania major that mimics the immune response in mice.

By using a primary in vitro response specific for Leishmania major, normal T cells from resistant CBA/CaH-T6J and susceptible BALB/c mice commit to a Th1 and a Th2 response, respectively. Since commitment occurred, we measured the production of gamma interferon (IFN-gamma), interleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-10, and IL-12, prostaglandin E2 (PGE2), transforming growth factor beta (TGF-beta), and nitric oxide in the first 7 days of the response to identify factors that are critical for Th1 and Th2 development. While cells from resistant CBA mice produced more IFN-gamma, IL-10, and nitric oxide, cells from susceptible BALB/c mice produced more IL-1alpha, IL-5, PGE2, and TGF-beta. Although substantial amounts of IL-12 were detected, IL-12 did not associate with either Th1 or Th2 development. We did not anticipate that cells from resistant CBA mice would make more IL-10 in vitro. However, this also occurred in vivo since CBA mice produced substantial amounts of IL-10 following infection with L. major. Moreover, adding anti-IL-10 to primary in vitro responses enhanced production of IFN-gamma and nitric oxide by cells from CBA and BALB/c mice. Therefore, IL-10 cannot be regarded as a cytokine that associates with susceptibility to infection with L. major. Finally, the data presented here suggest that a collection of factors that can be produced by accessory cells influence Th commitment (e.g., IL-1, PGE2, and TGF-beta favor Th2 development).

Animals↗
PubMed2001

Neuroblastoma cell lines--a versatile in vitro model in neurobiology.

Neuroblastoma (NB) cell lines are transformed, neural crest derived cells, capable of unlimited proliferation in vitro. These cell lines retain the ability of differentiation into neuronal cell types on treatment with various agents. This ability of NB cells to proliferate as well as to differentiate makes it an excellent in vitro system for various studies. This review article focuses on the applications and potential uses of murine and human NB cell lines. NB cells are extensively used for testing neurotoxicity of putative drugs such as antimalarial or anticancer agents. NB cell lines have wide applications in virus research to understand various aspects of virus-host cell interactions at the molecular and cellular levels. They have been used to dissect the relationships between proliferation, differentiation and apoptosis. This feature has been useful in understanding the pediatric cancer--neuroblastoma and for development of newer therapies.

Animal Testing Alternatives↗
PubMed1982

Effects of xanthine: xanthine oxidase on membrane function: an in vitro model of endothelial damage.

Endothelial damage caused by the oxidative species from activated granulocytes has been studied in vitro using the cell-free oxygen radical generating system, xanthine: xanthine oxidase and cultured porcine aortic endothelium. Increasing concentrations of xanthine oxidase caused dose dependent effects on the following functions, 86Rb efflux, prostaglandin production, release of 3H from adenine nucleotides, adenine uptake, 51chromium release and cell morphology. The threshold concentration of xanthine oxidase required for these effects varied by approximately 30 fold, 86Rb+ efflux being affected first at 0.003 units/ml of enzyme. Xanthine oxidase alone had a dose-dependent effect on 86Rb+ efflux which was heat labile and potentiated by xanthine. Neither superoxide dismutase (SOD) nor catalase (CAT) affected the response to xanthine: xanthine oxidase but a mixture of SOD and CAT reduced it by approximately 50% to the response to enzyme alone. Therefore the effect of xanthine: xanthine oxidase has at least two components, one due to enzyme alone and one due to the oxidation of xanthine, probably resulting from an interaction of oxygen radicals and hydrogen peroxide.

Adenine↗
PubMed1996

Comparative in vivo and in vitro models to approach the cellular basis of endotoxic shock. The role of sinusoidal liver cells.

During endotoxic shock, the liver exerts a lipopolysaccharide (LPS) clearance function with the participation of both parenchymal and sinusoidal cells. Liver damage could be caused by LPS direct action, hypoxia and/or inflammatory mediators released by Kupffer cells. The aim of this study is to establish an experimental model that could allow us to understand the direct E. coli 011:B4 LPS action on sinusoidal cells. A comparative study was carried out, in vivo and in vitro, using either a rat reversible endotoxic shock model or sinusoidal cell cultures. The LPS was found to induce important and similar morphological alterations both in vivo and in vitro, specially in Kupffer cells. These cells present mitochondrial damage, nuclear membrane swelling, and increased number of phagosomes, including lamellar bodies. An immunocolloidal gold technique shows, in vitro, the LPS mainly located on Kupffer cell membrane and in phagosomes. The LPS binding to membrane, as a primary step of Kupffer cell activation, increases the phagocytosis. This effect could be related to a decrease of fluidity on the external membrane portion.

Animals↗
PubMed1991

[Free microsurgical latissimus dorsi muscular flaps in swine. In-vitro model for the experimental continuous development of microsurgical reconstruction in the head-and-neck area].

An in-vitro system for research into microsurgical transfer technology in head and neck reconstruction is presented for the first time: the free, muscular latissimus-dorsi-flap of the pig. This in-vitro-system is useful for training in microsurgical techniques, for biomechanical tests of vascular connections, and for the simulation of extracorporal circulatory systems in free flaps. This in-vitro system corresponds to human surgical conditions and anatomical proportions. It helps reduce the number of animals needed in microsurgical research and therefore complies with present legislation. The article outlines the surgical technique of raising and replanting the free flap and its cold storage.

Animals↗
PubMed1981

The effect of sodium salicylate on the rat embryo in culture: an in vitro model for the morphological assessment of teratogenicity.

The optimum teratogenic dose of subcutaneously administered sodium salicylate was determined in vivo at 9.5 days of gestation. Fetuses from rats injected with this dose at 9.5 days were examined at 11.5 days and its propensities for producing resorption as well as deformities noted. Next, maternal serum levels 3-3.5 hours and 18.5 hours after salicylate injection were determined. Having established the feasibility of determining an effect due to salicylate at 11.5 days, after injection at 9.5 days, the following experiments were performed in vitro in animals cultured between 9.5 and 11.5 days by the method of New et al. (1976a): (1) Sodium salicylate was added to the culture serum, at levels equivalent to those obtained 3-3.5 hours after maternal injection of the optimum teratogenic dose, for 24 of the 48 hours culture period. (2) Rats were cultured for the first 24 hours of the 48 hours culture period in serum taken from rats injected 3.5 hours previously with the optimum teratogenic dose. (3) Rats were cultured for 24 out of 48 hours in serum from animals which had been injected with the optimum teratogenic dose 18.5 hours before bleeding. (4) Rats were cultured for 24 out of 48 hours in serum containing salicylate added to make up levels normally associated with (3). (5) A control culture was performed. The experiment indicates great similarity between the results obtained from animals cultured in serum with salicylate added and results from culture in serum of salicylate treated rats. It appears therefore that sodium salicylate - acting directly on the feto-placental unit - is the active teratogen rather than any of its metabolites.

Abnormalities, Drug-Induced↗