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PubMed2001

Effects of sevoflurane on dopamine, glutamate and aspartate release in an in vitro model of cerebral ischaemia.

Release of excitatory amino acids and dopamine plays a central role in neuronal damage after cerebral ischaemia. In the present study, we used an in vitro model of ischaemia to investigate the effects of sevoflurane on dopamine, glutamate and aspartate efflux from rat corticostriatal slices. Slices were superfused with artificial cerebrospinal fluid at 34 degrees C and episodes of 'ischaemia' were mimicked by removal of oxygen and reduction in glucose concentration from 4 to 2 mmol litre(-1) for < or = 30 min. Dopamine efflux was monitored in situ by voltammetry while glutamate and aspartate concentrations in samples of the superfusate were measured by HPLC with fluorescence detection. Neurotransmitter outflow from slices was measured in the absence or presence of sevoflurane (4%). After induction of ischaemia in control slices, there was a mean (SEM) delay of 166 (7) s (n = 5) before sudden efflux of dopamine which reached a maximum extracellular concentration of 77.0 (15.2) micromol litre(-1). Sevoflurane (4%) reduced the rate of dopamine efflux during ischaemia (6.90 (1.5) and 4.73 (1.76) micromol litre(-1) s(-1) in controls and sevoflurane-treated slices, respectively; P<0.05), without affecting its onset or magnitude. Excitatory amino acid efflux was much slower. lschaemia-induced glutamate efflux had not reached maximum after 30 min of ischaemia. Basal (pre-ischaemic) glutamate and aspartate efflux per slice was 94.8 (24.8) and 69.3 (31.5) nmol litre(-1) superfusate (n = 4) and was not significantly reduced by 4% sevoflurane. lschaemia greatly increased glutamate and aspartate efflux (to a maximum of 919 (244)% and 974 (489)% of control, respectively). However, ischaemia-induced efflux of both glutamate and aspartate was significantly reduced by 4% sevoflurane (P < 0.001 for glutamate, P < 0.01 for aspartate). In summary, sevoflurane may owe part of its reported neuroprotective effect to a reduction of ischaemia-induced efflux of excitatory amino acids and, to a lesser extent, dopamine.

Anesthetics, Inhalation↗
PubMed1994

Tumor cell-specific loss of p53 protein in a unique in vitro model of human breast tumor progression.

Mutations of the p53 gene are the most frequent genetic lesion in breast cancer. Here, we examined p53 expression in a unique in vitro model of tumor progression derived from a single breast cancer patient (21T series). While the normal mammary epithelial, fibroblast and mesothelial cells derived from this patient expressed easily detectable functional p53 protein, the primary as well as metastatic tumor cell lines demonstrated a lack of p53 protein synthesis. 21T tumor cells failed to exhibit G1 cell cycle arrest upon exposure to gamma-irradiation, and their growth was suppressed by transfection of a normal p53 cDNA, demonstrating a lack of p53-mediated function in these cells. No p53 gene deletion or rearrangements were detectable. PCR and sequence analysis of the entire coding region of p53 gene revealed a novel mutation, an insertion of a single T within codon 33, which resulted in a frame-shift and early termination. The same mutation was observed in all 21T tumor cell lines. These results demonstrate a tumor cell-specific loss of p53 protein due to a frame-shift mutation, and suggest that p53 loss may occur at a relatively early step in breast tumorigenesis before metastatic seeding or emergence of tumor heterogeneity. In addition, the availability of normal and tumor-derived epithelial cells with known p53 sequences from a single breast cancer patient should facilitate understanding of the p53 regulation in mammary cells.

Breast↗
PubMed1992

A quantitative in vitro model for silicone oil emulsification. Role of blood constituents.

To understand why some patients seem to be protected from emulsification and others are not, the authors developed an in vitro model for quantitative analysis of silicone oil emulsification. The pro-emulsifying potential of substances and blood components that may have access to the vitreous cavity in a patient's eye was analyzed. In this model, red blood cell ghosts had the highest emulsifying effect; plasma and lymphocytes also had a significant emulsifying effect. Phospholipids in membranes and other soluble blood components may play important roles in this process. These results suggest the importance of avoiding and removing hemorrhage and avoiding inflammation when silicone oil is used in vitreoretinal surgery.

Blood Cells↗
PubMed1993

Pharmacodynamic effects of extended dosing intervals of imipenem alone and in combination with amikacin against Pseudomonas aeruginosa in an in vitro model.

The pharmacodynamic effects of extended imipenem dosing intervals were studied against two strains of Pseudomonas aeruginosa (ATCC 27853 and an imipenem-resistant mutant, 27853R) in an in vitro model of infection. Imipenem was administered as monotherapy (simulated 1-g bolus every 8 or every 12 h) and in combination with amikacin (7.5-mg/kg bolus every 12 h or a 15-mg/kg bolus once). Monotherapy with imipenem administered every 8 h was equally bactericidal at 24 h compared with regimens combined with amikacin for ATCC 27853. Imipenem administered every 12 h against the sensitive strain and both imipenem monotherapy regimens against the resistant strain demonstrated regrowth at 24 h. Although both amikacin regimens administered as monotherapy resulted in rapid bacterial killing activity with respect to time to a 99.9% reduction in log10 CFU/milliliter, regrowth at 24 h was observed at levels reaching or exceeding the initial inoculum. All combination regimens resulted in no detectable growth by 24 h regardless of dosing interval for either drug or initial susceptibility to imipenem. Results from this study indicate the potential for several novel dosing regimens against P. aeruginosa. Monotherapy with imipenem, 1 g every 8 h, was effective against a sensitive strain of P. aeruginosa. Combination therapy with imipenem and once-daily or twice-daily amikacin resulted in increased killing activity against imipenem-resistant P. aeruginosa. Once-daily or twice-daily amikacin in combination therapy, regardless of P. aeruginosa susceptibility, allowed for extension of imipenem dosing intervals.

Amikacin↗
PubMed1997

Axenically cultured amastigote forms as an in vitro model for investigation of antileishmanial agents.

Using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide microassay, previously described as a means of quantifying Leishmania amazonensis in vitro at the amastigote stage (D. Sereno and J. L. Lemesre, Parisitol. Res., in press), we have compared the activities of seven drugs, including those currently used to treat leishmaniasis, against axenically grown amastigote and promastigote forms of three Leishmania species (L. amazonensis, L. mexicana, and L. infantum, responsible for diffuse cutaneous, cutaneous, and visceral leishmaniasis, respectively). The ability of axenically cultured amastigote organisms to be used in an investigation of antileishmanial agents was first evaluated. We have confirmed the toxicities of sodium stibogluconate (Pentostam), pentamidine, and amphotericin B to active and dividing populations of axenically cultured amastigotes. The toxicity of potassium antimonyl tartrate trihydrate, which is generally higher than that of Pentostam, seemed to indicate that pentavalent antimony can be metabolized in vivo to compounds, possibly trivalent in nature, which are more active against the amastigote organisms. When the drug susceptibilities of parasites at both stages were compared, great variations were found for all the drugs studied. These major differences, which show the specific chemosusceptibility of the parasite at the mammalian stage, demonstrate the potential of using cultured amastigotes instead of promastigotes in a drug-screening procedure for early detection. This in vitro model may help in the isolation of active compounds, particularly those with low-grade activities, against the mammalian stage of the parasite.

Animals↗
PubMed1999

Pharmacodynamic comparisons of levofloxacin, ciprofloxacin, and ampicillin against Streptococcus pneumoniae in an in vitro model of infection.

The increasing frequency of penicillin-resistant pneumococcus continues to be of concern throughout the world. Newer fluoroquinolone antibiotics, such as levofloxacin, have shown enhanced in vitro activity against Streptococcus pneumoniae. In this study, the bactericidal characteristics and pharmacodynamic profiles of levofloxacin, ciprofloxacin, and ampicillin against four isolates of S. pneumoniae were compared by using an in vitro model of infection. Standard antibiotic dosing regimens which simulated the pharmacokinetic profile observed in humans were used. Control and treatment models were sampled for bacterial CFU per milliliter over the duration of each 24- or 48-h experiment. In addition, treatment models were sampled for MIC determinations and drug concentration. Regrowth of all isolates as well as an increase in MICs throughout the study period was observed in the ciprofloxacin experiments. A limited amount of regrowth was noted during levofloxacin therapy for one isolate; however, no change in MIC was detected for any isolate. Ampicillin showed rapid and sustained bactericidal activity against all isolates. In this study, ratios of effective fluoroquinolone area under the concentration-time curve (AUC):MIC values ranged from 30 to 55. Levofloxacin, owing to its larger AUC0-24 values, has excellent and sustained activity against different pneumococcal strains superior to that of ciprofloxacin.

Ampicillin↗
PubMed2006

hTERT-immortalized prostate epithelial and stromal-derived cells: an authentic in vitro model for differentiation and carcinogenesis.

Prostate cancer is the most commonly diagnosed type of cancer in men, and there is no available cure for patients with advanced disease. In vitro model systems are urgently required to permit the study of human prostate cell differentiation and malignant transformation. Unfortunately, human prostate cells are particularly difficult to convert into continuously growing cultures. We report here the successful immortalization without viral oncogenes of prostate epithelial cells and, for the first time, prostate stromal cells. These cells exhibit a significant pattern of authentic prostate-specific features. In particular, the epithelial cell culture is able to differentiate into glandular buds that closely resemble the structures formed by primary prostate epithelial cells. The stromal cells have typical characteristics of prostate smooth muscle cells. These immortalized cultures may serve as a unique experimental platform to permit several research directions, including the study of cell-cell interactions in an authentic prostate microenvironment, prostate cell differentiation, and most significantly, the complex multistep process leading to prostate cell transformation.

Aged↗
PubMed1995

Conditional transformation of mouse liver epithelial cells. An in vitro model for analysis of genetic events in hepatocarcinogenesis.

Primary rodent and human hepatocytes have a very limited lifespan in culture and are not readily applicable to transformation studies in vitro. To facilitate the investigation of early genetic events involved in hepatocarcinogenesis, we examined a transformation assay system utilizing conditionally immortalized mouse liver epithelial cells as an alternative to primary hepatocytes. By infecting primary mouse hepatocytes with a recombinant retrovirus carrying a temperature-sensitive simian virus 40 large T antigen gene, two mouse liver epithelial cell lines, CHST8 and CHST10-2.1, were established. Because of the heat-labile nature of the large T antigen, the cell lines proliferated rapidly at 33 degrees C, but were growth-arrested at 39 degrees C. Because activated c-H-ras and c-myc oncogenes are frequently found to be involved in mouse hepatocarcinogenesis in vivo, we assessed whether those oncogenes can complement the immortalizing function of the large T antigen at the nonpermissive temperature. When CHST8 cells were doubly transfected with activated c-H-ras and c-myc at 33 degrees C, they exhibited clonal growth ability even after shifting the temperature to 39 degrees C. However, neither c-H-ras nor c-myc alone allowed growth at 39 degrees C. On the other hand, c-H-ras alone was sufficient for overcoming the growth defect of CHST10-2.1 cells at 39 degrees C, whereas c-myc alone was again ineffective. Northern blot studies revealed that endogenous c-myc expression was significantly downregulated in the parental CHST8 cells after a temperature shift from 33 to 39 degrees C. In contrast, in the parental CHST10-2.1 cells, appreciable c-myc expression was observed at both temperatures. These results indicate that c-H-ras and c-myc can cooperate in complementing the ability of the temperature-sensitive large T antigen to immortalize mouse liver cells at the nonpermissive temperature. In addition, the mutant c-H-ras, but not c-myc, cooperated with the functional T antigen at 33 degrees C to allow growth in soft agarose of the CHST8 and CHST10-2.1 cell lines. However, cell lines carrying mutant c-H-ras and overexpressing c-myc were unable to grow in soft agarose at 39 degrees C. Thus, the two cellular oncogenes were insufficient for full transformation of the liver epithelial cells. The present in vitro model should be useful for investigating molecular events involved in both early and late stages of hepatocarcinogenesis.

Animals↗
PubMed1991

Blood-brain barrier alterations in bacterial meningitis: development of an in vitro model and observations on the effects of lipopolysaccharide.

To further examine the effects of purified Haemophilus influenzae type b lipopolysaccharide (LPS) on blood-brain barrier permeability, we have developed an in vitro model of the BBB. Microvascular endothelial cells were isolated from rat cerebral cortices by enzymatic digestion, dextran centrifugation, and separation on percoll gradients. The cells were determined to be endothelial in origin by positive fluorescent staining for Factor VIII-related antigen and the ability to take up acetylated low density lipoproteins, and their cerebral origin by the formation of junctional complexes in vitro. Cells were seeded onto semipermeable polycarbonate filters and permeability assessed by measuring traversal of radioactive albumin across the monolayer. Treatment of the cells with LPS at concentrations of 1.0 microgram/ml and 0.1 microgram/ml for 4 h led to statistically significant increases in albumin permeability of 4.6% (P = 0.001) and 5.6% (P less than 0.001), respectively, without evidence of cell death as assessed by release of lactate dehydrogenase into the media. These results indicate that LPS significantly increases albumin permeability across a monolayer of cerebral microvascular endothelial cells in the absence of host inflammatory cells. Future studies on the effects of LPS on intracellular regulation will determine the mechanisms responsible for these alterations.

Albumins↗
PubMed1991

A mammalian in vitro model to study gangliogenesis from neural crest cells.

In spite of considerable advances towards understanding lineages derived from neural crest cells using amphibian and avian embryos, the molecular mechanisms involved in the formation of mammalian peripheral ganglia remain largely unknown, mainly because of the lack of experimental systems that will allow their in vitro manipulation. Here, we present a novel mammalian in vitro model permitting to study gangliogenesis from neural crest cells. This model allowed us to manipulate molecules involved in cell-cell interactions. Our data are in favour of the existence of a hierarchy among adhesion molecules.

Animals↗
PubMed2005

A probable role for trail-induced apoptosis in the pathogenesis of marrow failure. Implications from an in vitro model and from marrow of aplastic anemia patients.

Aplastic anemia (AA) is a syndrome of hematopoietic failure involving increased apoptosis of stem cells. In order to investigate the molecular mechanisms participated in the process of marrow failure, we created an in vitro model of hematopoietic cell suppression, by continuous addition of TNF-alpha and IFN-gamma in an vitro long-term bone marrow culture system. An up-regulation of Fas expression was observed in CD34+ cells in cytokine treated cultures, compared to controls. This was accompanied by significant TRAIL and decreased caspase 3 mRNA expression, whereas the expression of Bcl-2 family members was low (Bcl-xl) or absent (Bcl-2, Bax). The expression of these apoptotic genes was also investigated in aplastic anemia patients. Apart from Fas mRNA expression in total marrow and/or CD34+ cells, TRAIL mRNA expression was found only in CD34+ cells in active disease while in total marrow cell compartment this remains a constant finding even in patients in remission. The above data are in agreement with previous studies proposing a major role for the extrinsic apoptosis pathway in the pathogenesis of aplastic anemia and additionally introduce TRAIL as a probable important molecule in the process.

Adult↗
PubMed1994

A comparison of in-vitro models to study hepatic lipid and lipoprotein metabolism.

Removal of the liver from its physiological environment provides the opportunity to examine its response when challenged directly with substances which, in vivo, are associated with changes in lipid and lipoprotein metabolism. Many in-vitro models are currently available, ranging from intact, perfused livers to transformed cell lines. Selection of the most suitable model depends upon the precise experimental objective(s) and, in most cases, involves trading off one advantage in favour of another. This is particularly true in selecting a human model, and in this case, complementary studies involving more than one model system may provide the best results.

Animals↗
PubMed1995

Functional and antiischaemic effects of Monoacetyl-vitexinrhamnoside in different in vitro models.

1. Functional and antiischaemic effects of monoacetyl-vitexinrhamnoside (AVR), a flavonoid with phosphodiesterase (PDE)-inhibitory properties contained in Crataegus species (Hawthorn, Rosaceae) were studied in several in-vitro models. 2. In rabbit isolated femoral artery rings, AVR concentration-dependently reduced developed tension. Vasodilation by AVR was reduced after inhibiting EDRF formation by L-NG-nitro arginine. 3. In spontaneously-beating Langendorff-guinea pig hearts, AVR concentration-dependently enhanced heart-rate, contractility, lusitropy and coronary flow. 4. In isolated electrically-driven Langendorff-rabbit hearts, acute regional ischemia (MI) was induced by coronary artery occlusion and quantified from epicardial NADH-fluorescence photography. AVR (5 x 10(-5) mol/l) induced a slight numerical increase of left ventricular pressure and coronary flow (p > 0.05). MI was reduced (p < 0.05). 5. Monoacetyl-vitexinrhamnoside is an inodilator whose vasodilatory action may be mediated in part by EDRF in addition to PDE-inhibition. Monoacetyl-vitexinrhamnoside does possess marked antiischemic properties even in isolated hearts, suggesting an improvement of myocardial perfusion.

Abortifacient Agents↗
PubMed1991

Pharmacological properties of cloned muscarinic receptors expressed in A9 L cells; comparison with in vitro models.

The effects of a series of muscarinic agonists and antagonists at cloned m1 and m3 muscarinic receptors expressed in mouse fibroblast A9 L cells have been compared with their effects in in vitro models of M1 (rat superior cervical ganglion) and M3 (guinea-pig ileum) muscarinic receptors. A good correlation existed between the potencies of muscarinic agonists at cloned m1 muscarinic receptors and the M1 sites in rat ganglion (r = 0.80) as well as at cloned m3 receptors and guinea-pig ileum M3 receptors (r = 0.87). However, cross correlations of potencies in rat ganglion and cloned m3 receptors as well as in guinea-pig ileum and in cloned m1 receptors also yielded relatively high correlation coefficients (0.71 and 0.91, respectively). Low correlation coefficients were found for the maximal responses of muscarinic agonists in rat ganglion and cloned m1 receptors (0.53) and in guinea-pig ileum and cloned m3 receptors (0.36). A high correlation between pA2 values of muscarinic antagonists at cloned m1 receptors and in rat ganglion (r = 0.97) and between cloned m3 receptors and guinea-pig ileum (r = 0.98) was found. Cross correlation of pA2 values in rat ganglion and cloned m3 receptors and in guinea-pig ileum and cloned m1 receptors yielded correlation coefficients of 0.82 and 0.72, respectively. The data indicate that the cloned muscarinic receptor sites seem similar to the corresponding endogenous sites. The good correlations in corresponding but also non-corresponding receptor models reflect the relatively low selectivity of the majority of the compounds investigated.

Animals↗
PubMed2002

Influence of antiseptic agents on interleukin-8 release and transmigration of polymorphonuclear neutrophils in a human in vitro model of peritonitis.

BACKGROUND: The aim of this study was to evaluate the influence of taurolidine (TAU) and polyhexanid (POLY) on basic inflammatory reactions during peritonitis by using an in vitro model of human peritoneum. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVEC) and human peritoneal mesothelial cells (HPMC; concentration: 2x10(5)/cm2) were brought on a collagen-coated filter insert with 3-microm pore size (HUVEC on the bottom, HPMC on the top), thus resulting in a two-chamber peritoneal model. After 5 days, confluence of the cells was reached, and HPMC were stimulated with 0.5 mL of TNF-alpha (10 microg/mL) for 4 h. Afterwards, 0.5 mL of TAU (1% and 2%) or 0.5 mL of POLY (0.1% and 0.2%) solution were added to the upper (HPMC) compartment. Polymorphonuclear neutrophils (PMN, 10(6)/mL) were placed in the lower compartment 1 h later. After 2 and 6 h, aliquots were taken from the upper compartment and transmigrated PMN were counted. Interleukin-8 (IL-8) concentrations were measured in both compartments by chemiluminescent enzyme immunometric assay. Expression of the adhesion molecules P-selectin and intercellular adhesion molecule-1 (ICAM-1) was assessed by immunohistochemistry. Controls were either TNF-alpha-stimulated HPMC without any antiseptic agents, or stimulated HPMC where TNF-alpha had been substituted by culture medium. Each experiment was performed in triplicate. RESULTS: Stimulation with TNF-alpha led to a time-dependent increase of IL-8 secretion to the apical compartment resulting in a gradient between both chambers, as well as to a time-dependent increase of PMN transmigration and expression of adhesion molecules. IL-8 gradients and PMN migration were significantly higher as compared to the other groups (p<0.05). After substitution of the stimulus by culture medium, significantly less IL-8 was measured in both compartments. PMN transmigration was almost absent (p<0.05). Addition of POLY and TAU led to comparable low IL-8 gradients with concomitant low PMN transmigration. The initially detected expression of adhesion molecules significantly decreased during the observation time. The IL-8 gradient in all groups correlated significantly with PMN transmigration (r=0.74226; p<0.0001). CONCLUSION: The diminished IL-8 response together with low PMN transmigration rates after addition of TAU and POLY may reflect either antiinflammatory effects or cellular damage.

Anti-Infective Agents, Local↗
PubMed1977

[Zinc uptake of normal and leukaemic lymphocytes in an in vitro model (author's transl)].

Zinc uptake and/or possible exchange by isolated lymphocytes from normal persons and from patients with chronic lymphatic leukemia and chronic myeloid leukemia were studied in an in vitro model. The uptake of zinc per 10(9) lymphocytes of normal persons was 60 +/- 10 nanomoles and 120 +/- 15 nanomoles during incubation for 30 and 60 minutes, respectively. Two out of the 3 patients with chronic myeloid leukemia showed normal zinc uptake in this test system, whereas zinc uptake by lymphocytes from patients with chronic lymphatic leukemia was significantly decreased (22+/-8 nanomoles in 30 minutes and 35+/-10 nanomoles in 60 minutes).

Adult↗
PubMed2002

In vitro models for the identification and characterization of tumor-promoting and protective factors for colon carcinogenesis.

The present review aims to give a short overview of the existing cell culture models and their characteristics. While both the induction and the prevention of tumors can only be unequivocally demonstrated in vivo, in vitro models of colorectal cell growth are essential tools for the identification of candidate compounds and for the analysis of underlying mechanisms. Unfortunately normal and premalignant colorectal epithelial cells that represent the target cells of tumor promoters like desoxycholic acid or 1,2-diglyceride and could be used to investigate their cell biological effects are difficult to obtain. Cell line studies have mostly used carcinoma cell lines or rat small intestinal epithelial cells. Some normal immortalized cell lines have been established but are not widely used, and premalignant adenoma cell lines are extremely rare. However, those that do exist are useful both in mechanistic studies of cell growth and for the analysis of tumor promoters and chemoprotectors of colorectal carcinogenesis.

Adenoma↗
PubMed2001

Characterization of the maturation of human pro-apolipoprotein A-I in an in vitro model.

The reaction conditions and the protein structural features involved in the maturation of pro-apolipoprotein A-I (cleavage of pro-peptide) were investigated in an in vitro model. ProapoA-I, mutants and wild type, were expressed in the PGEX/E. coli expression system as fusion proteins with glutathione S-transferase (GST). Use of GST-proapoA-I and truncated forms of proapoA-I enabled quantitation of the amount of GST and apoA-I formed as a result of cleavage following incubation with human serum. Deletion of the pro-peptide (GST-apoA-I) resulted in complete inhibition of the reaction. Truncation of proapoA-I to residues 222, 150, 135, and 25 as well as substitution of residues -6, -5, and -4 with alanine did not affect the reaction. Substitution of residues -1, -2, 1, 3, and 4 with alanine either completely blocked or substantially inhibited cleavage of the pro-peptide. The reaction was inhibited by addition of EDTA, o-phenanthroline, dithiothreitol, and beta-mercaptoethanol and to a lesser extent by p-chloromercuriphenylsulfonic acid, but not by leupeptin, N-ethylmaleimide, PMSF, pepstatin A, or trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane. Calcium was essential for the activation of the cleavage enzyme, but it had a biphasic effect on the cleavage, activating it at concentrations below 1.5 mM and inhibiting at concentrations above 1.75 mM. Manganese alone was not essential for activation of the enzyme nor did it modify the effect of low concentration of calcium. However, a high concentration of manganese partially reverted the inhibitory effect of a high calcium concentration. Thus, residues within -2 to +4 are involved in forming the cleavage site for the maturation enzyme. The reaction of maturation is inhibited by metalloprotease inhibitors and is dependent upon calcium.

Apolipoprotein A-I↗