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PubMed2001

Alveolar macrophage cell line MH-S is valuable as an in vitro model for Legionella pneumophila infection.

Alveolar macrophages are the preferential site for growth of Legionella pneumophila (Lp) during infection. However, the study of Lp infection in alveolar macrophages is difficult due to the limitation of available primary alveolar macrophages. In the present study, we established an in vitro Lp infection model in alveolar macrophages using a continuous cell line of murine alveolar macrophages designated MH-S. Infection of both MH-S cells and primary mouse alveolar macrophages obtained by alveolar lavage with virulent L. pneumophila (Lp-V) showed vigorous growth of the bacteria, but infection with avirulent L. pneumophila (Lp-Av) resulted in only minimum growth. Cytokine message expression determination in the MH-S cells after infection showed strong induction of interleukin (IL)-6, IL-10, and tumor necrosis factor-alpha messages induced by Lp-V but minimal induction of these cytokines by Lp-Av infection. IL-1 alpha protein secretion and the message levels for IL-1 alpha were also analyzed, and remarkable induction of IL-1 alpha was evident in both macrophage types when infected with Lp-V. Analysis of IL-12 p40 responses of both macrophage types to Lp-V infection assessed by reverse transcriptase/polymerase chain reaction revealed induction of increased message levels, but significant levels were induced only slowly. Determination of IL-12 protein secretion by enzyme-linked immunosorbent assay of culture supernatants from both macrophage types infected with either Lp-V or Lp-Av showed only minimum production. Thus, MH-S alveolar macrophages showed a similar response to Lp infection compared with primary alveolar macrophages and can be a useful in vitro model system to study Lp infection. The study also revealed the restricted IL-12 protein secretion of alveolar macrophages by Lp infection.

Animals↗
PubMed1992

Percutaneous absorption of nicotinic acid, phenol, benzoic acid and triclopyr butoxyethyl ester through rat and human skin in vitro: further validation of an in vitro model by comparison with in vivo data.

The in vitro percutaneous absorption of three model compounds, nicotinic acid, phenol and benzoic acid, and the herbicide triclopyr butoxyethyl ester (triclopyr BEE) has been investigated in flow-through diffusion cells using skin from male Fischer 344 rats and humans. After the application of the four chemicals to the epidermal surface of unoccluded full-thickness rat skin, the absorption of each compound across the skin and into the receptor fluid at 72 hr reached 3.7 +/- 0.3, 5.7 +/- 0.6, 26.7 +/- 3.7 and 48.3 +/- 1.2% (mean +/- SD, n = 2-7) of the applied dose for triclopyr BEE, nicotinic acid, phenol and benzoic acid, respectively. After the application of the four chemicals to the epidermal surface of unoccluded full-thickness human skin, the absorption of each compound across the skin and into the receptor fluid at 72 hr was significantly (P < 0.05) less than through rat skin, reaching 0.7 +/- 0.1, 0.7 +/- 0.2, 18.8 +/- 1.3 and 37.8 +/- 6.9% (mean +/- SD, n = 2-7) of the applied dose for triclopyr BEE, nicotinic acid, phenol and benzoic acid, respectively. Occlusion of the skin surface with teflon caps often significantly (P < 0.05) enhanced the percutaneous absorption of the model compounds, although this effect was not uniform, varying with the compound under study and the skin (rat or human) used. When rat skin was occluded with teflon caps, the extent of absorption at 72 hr reached 8.6 +/- 0.8, 36.2 +/- 1.7 and 51.8 +/- 3.3% (mean +/- SD, n = 3-4) for nicotinic acid, phenol and benzoic acid, respectively. Corresponding values for human skin occluded with teflon caps were 3.3 +/- 1.6, 47.1 +/- 0.5 and 65.5 +/- 7.1% (mean +/- SD, n = 3-4). The experiments on the absorption of each model compound through rat and human skin were repeated and there was generally good agreement between the results from the two sets of experiments. The in vitro data reported compare favourably with data obtained by other workers using both in vitro and in vivo methodologies. The in vitro: in vivo correlation supports the use of the flow-through diffusion cell system as a model for the prediction of percutaneous absorption in vivo in the rat and in humans.

Absorption↗
PubMed2004

Differential invasion of Candida albicans isolates in an in vitro model of oral candidosis.

The study assessed the ability of Candida albicans isolates to invade an in vitro oral tissue model. The extent and pattern of isolate invasion was then correlated with the infection origin of the isolate to identify characteristics that may be restricted to specific forms of oral infection, particularly chronic hyperplastic candidosis (CHC). Reconstituted human oral epithelium was infected with C. albicans isolated from normal oral mucosa (n = 4), CHC (n = 7), non-CHC oral candidoses (n = 4) and squamous cell carcinoma (SCC; n = 4). After infection for 24 h, histological analysis revealed yeast adhesion, hyphal extension, and invasion of the epithelium. Differential patterns of invasion were evident and, whilst consistent for a given isolate, did not relate to the infection origin of the isolate. Two principal patterns of invasion were evident and described as either a 'localised' or a 'uniform' distribution of invading hyphae. Several isolates also exhibited superficial infection with limited hyphal invasion. In conclusion, the use of the in vitro tissue model allowed the assessment of the invasive capabilities of isolates of C. albicans. However, the apparent differences in invasive characteristics did not appear to be related to the clinical origin of isolates.

Candida albicans↗
PubMed2007

A variant of recombinant factor VIIa with enhanced procoagulant and antifibrinolytic activities in an in vitro model of hemophilia.

OBJECTIVE: Recombinant factor VIIa (rFVIIa, NovoSeven) has proven efficacy in treating bleeding in hemophilia patients with inhibitors. A rFVIIa analog with mutations V158D/E296V/M298Q (NN1731) exhibits increased procoagulant activity in in vitro and in vivo models. The aim of this work was to define the effects of NN1731 toward factor X activation, platelet activation, thrombin generation, and fibrin clot formation and stability. METHODS AND RESULTS: In a cell-based in vitro model of hemophilia, rFVIIa and NN1731 similarly increased factor X activation on tissue factor-bearing cells; however, NN1731 exhibited 30-fold higher factor Xa generation on platelets than similar rFVIIa concentrations. NN1731-mediated thrombin generation depended on platelet activation, but NN1731 did not directly activate platelets. NN1731 produced 4- to 10-fold higher maximal thrombin generation rates than equal rFVIIa concentrations. Both rFVIIa and NN1731 shortened clotting times in the absence of factors IX and VIII; however, NN1731 did so at 50-fold lower concentrations than were required of rFVIIa. In fibrinolytic conditions, both rFVIIa and NN1731 increased fibrin formation and stability; however, NN1731 was effective at 50-fold lower concentrations than were required of rFVIIa. CONCLUSIONS: By increasing factor Xa generation, NN1731 promotes the formation of thrombin and a stable clot to a greater degree than rFVIIa.

Antifibrinolytic Agents↗
PubMed2005

Evaluation of drug penetration into the brain: a double study by in vivo imaging with positron emission tomography and using an in vitro model of the human blood-brain barrier.

The blood-brain barrier (BBB) permeabilities of 11 compounds were measured both in vitro with a newly developed coculture-based model of human BBB and in vivo with positron emission tomography (PET). The 11 compounds were fluoropyridinyl derivatives labeled with the positron-emitter fluorine-18, [(18)F]F-A-85380 [2-[(18)F]fluoro-3-[2(S)-2 azetidinylmethoxy]pyridine], and 10 selected N-substituted-azetidinyl and pyrrolidinyl closely related [(18)F]fluoropyridinyl derivatives (including [N'-aromatic/aliphatic]-thioureas, -ureas, and -amides). The in vitro BBB model, a new coculture system of primary human brain endothelial cells and astrocytes, was used to measure the permeability coefficient for each compound. Dynamic PET studies were performed in rats with the same compounds, and a two-compartment model analysis was used to calculate their in vivo permeability coefficients. The 11 derivatives differed in their degree of BBB passage and transport mechanism. The analysis of PET data showed a significant cerebral uptake for six derivatives, for which the in vitro evaluation indicated active influx or free diffusion. Five derivatives displayed low in vivo cerebral uptake, in agreement with the observation of an in vitro active efflux. Overall, there was a remarkable correlation between the in vitro and in vivo permeability coefficients (r = 0.99). This double study proves a close correlationship between the assessment of the BBB passage in vitro and in vivo. The in vitro model of human BBB offers the possibility of subtle discrimination of various BBB permeability degrees and transport mechanisms. Conversely, small animal PET imaging appears suitable to screen directly in vivo brain targeting of drugs or radiopharmaceutical candidates.

Algorithms↗
PubMed1998

Comparative pharmacodynamics of meropenem using an in-vitro model to simulate once, twice and three times daily dosing in humans.

An in-vitro pharmacokinetic model was used to study the antibacterial activity of meropenem. Strains of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were exposed to meropenem concentrations likely to be produced in an adult by rapid iv infusion of 3 g once a day (q24h regimen), 1.5 g twice a day (ql2h regimen) or 1 g three times a day (q8h regimen). Each of these three dosing regimens produced a rapid reduction in viable bacterial count over the first 16 h after dosing. There were no differences in the pattern of reduction in viable count with the q24h, q12h or q8h regimens with any of the three bacterial strains tested over the first 16 h. However, reduction in viable counts was poorer at 24 h with the q24h than q12h or q8h regimens. A simulation lasting 48 h of the q24h dosing regimen indicated a reduction in bacterial count below the limit of detection from the model with E. coli but not with P. aeruginosa or S. aureus. The time for which the meropenem concentration was higher than the MIC for the bacteria correlated best with the reduction in viable bacterial count at 24 h. The AUC for the bacterial time-kill curve, which may be a better measure of antibacterial efficacy, was not related to the length of time for which the concentration was above the MIC or the peak concentration/MIC ratio. The antibacterial effect of the conventional q8h dosing simulation was indistinguishable from that given by the q12h simulation, and both the q8h and q12h regimens offered minor advantages over a q24h regimen. Dosing of meropenem 12 hourly or 24 hourly in humans should be investigated.

Adult↗
PubMed2000

Assessment of different antibacterial effect measures used in in vitro models of infection and subsequent use in pharmacodynamic correlations for moxifloxacin.

A dilutional culture in vitro pharmacodynamic model of infection was used to assess the best measure of antibacterial effect for moxifloxacin at simulated human doses of 400 mg 24 hourly for 48 h. This was then related to two pharmacodynamic parameters, the drug area under curve: MIC ratio (AUC/MIC) and the length of time that the drug concentration remained above the MIC of the bacterium (T > MIC). Twenty-one bacterial strains (Streptococcus pneumoniae n = 6; Haemophilus influenzae n = 6; Moraxella catarrhalis n = 3; beta-haemolytic streptococci n = 3; Staphylococcus aureus n = 3; MIC range 0.06-3.6 mg/L) were tested in 69 individual simulations. The measures or parameters of antibacterial effect considered were log change in viable count in the initial inoculum at 12 h (triangle up12), 24 h (triangle up24), 36 h (triangle up36), 48 h (triangle up48), maximum reduction in count (triangle up(max)); time for bacterial counts to reduce by 100-fold from the initial density (T99) or 1,000-fold (T99.9); and area under the bacterial kill curve from 0 to 24 h (AUBKC(24)) or from 0 to 48 h (AUBKC(48)). triangle up12, triangle up24, triangle up36, triangle up48, triangle up(max), T99, T99.9 did not vary over the complete range of MICs; at high MICs, especially with Gram-positive bacteria the T99 and T99.9 values were >48 h while at low MICs, especially with Gram-negative bacteria, bacterial counts were reduced below the limit of detection with triangle up12, triangle up24, triangle up36, triangle up48 and triangle up(max) exceeding >6.5 log reduction. AUBKC(24) and AUBKC(48) varied more completely over the range of MICs and more importantly had the best within-strain reproducibility (median percentage coefficient of variation <15%). The relationship between the transformed AUBKC(24) and AUC/MIC could be described by a sigmoid Emax model but the relationship with T > MIC could not. Use of weighted least squares regression to examine the combined effect of AUC/MIC and T > MIC on AUBKC(24) indicated that AUC/MIC provided a good fit to the data (r(2) = 0.94) and adding T > MIC did not improve the model fit. Cox proportional hazards regression indicated that AUC/MIC was predictive of T99 and in a multivariate model although AUC/MIC predicted outcome after fitting AUC/MIC, T > MIC was not significant. AUBKC was thus shown to be the optimum measure of antibacterial effect to use in pharmacodynamic studies of moxifloxacin and AUC/MIC the best predictor of antibacterial effect as measured by AUBKC(24) or T99. These results are in good agreement with animal data on moxifloxacin pharmacodynamics and human data for some other fluoroquinolones.

Animals↗
PubMed2004

An in vitro model system that can differentiate the stages of DNA replication affected by anticancer agents.

We have previously reported on the potential use of a novel in vitro human cell-derived model system to investigate the mechanism of action of anticancer agents that directly affect the process of DNA replication. Our cell-free system uses a multiprotein DNA replication complex (designated the DNA synthesome) that has been isolated, characterized, and extensively purified from a wide variety of mammalian cells and tissues. The DNA synthesome is competent to orchestrate simian virus 40 (SV40) origin-specific and large T antigen-dependent DNA replication in vitro. In this study, the synthesome-based cell-free system was tested to evaluate the mechanism of action of 1-beta-d-arabinofuranosylcytosine (ara-C), camptothecin (CPT), and doxorubicin (DOX). Using a novel synthesome-based in vitro kinetic assay, we demonstrated that DNA replication mediated by the synthesome is initiated within the SV40 replication origin and proceeds bidirectionally in a manner analogous to that occurring within the cell. Ara-CTP, CPT, and DOX have been found to affect different stages of the in vitro DNA replication process mediated by the complex. Ara-CTP inhibited both the initiation and elongation stages, whereas CPT produced most of its effects by inhibiting the elongation phase of DNA replication. DOX inhibited the termination stage of DNA synthesis mediated by the synthesome. The data presented here support our contention that the DNA synthesome represents a highly effective in vitro model system for investigating the mechanism by which some anticancer agents can directly affect the process of DNA replication.

Antineoplastic Agents↗
PubMed2006

A simple in vitro model to study the stability of acylglucuronides.

INTRODUCTION: Compounds containing the carboxylic functional group (e.g. non-steroidal anti-inflammatory drugs) can be metabolized to form acylglucuronides. Acylglucuronides are intrinsically reactive metabolites capable of undergoing hydrolysis, intra-molecular rearrangement, and formation of covalent adducts with proteins, which may generate potential toxicity. The purpose of this study is to develop an in vitro screening model to assess degradation kinetics of acylglucuronides. METHOD: Zomepirac, ibuprofen, gemfibrozil, and compounds A, B, C, and D were incubated in the presence of rat microsomal protein and uridine 5'-diphosphoglucuronic acid (UDPGA), followed by addition of human plasma to evaluate degradation kinetics of the acylglucuronides. As a comparison, authentic acylglucuronide standards of zomepirac, ibuprofen, gemfibrozil, and compounds A, B, C, and D were chemically synthesized and were evaluated for degradation kinetics. RESULTS: The results demonstrate that degradation half-life values of acylglucuronides of zomepirac, ibuprofen, gemfibrozil, and compounds A, B, C, and D determined by the in vitro formation/degradation model were in the same rank-order with those of the authentic acylglucuronide standards. DISCUSSION: For the seven compounds tested, the model placed the stability of the acylglucuronides formed in vitro in a rank-order consistent with authentic acylglucuronide standards. The method allows for a rapid assessment of the stability of acylglucuronides.

Animals↗
PubMed2000

Astrocytes increase the functional expression of P-glycoprotein in an in vitro model of the blood-brain barrier.

PURPOSE: To investigate the influence of astrocytes on P-glycoprotein (Pgp) expression and intracellular accumulation of Pgp substrates, separate from their net transcellular transport across the blood-brain barrier (BBB). METHODS: An in vitro BBB model was used, comprising of brain capillary endothelial cells (BCEC) monolayers or BCEC co-cultured with astrocytes. RESULTS: BCEC+astrocyte co-cultures seemed to express a higher level of Pgp compared to BCEC monolayers. Inhibition of Pgp results in an increased intracellular accumulation of Pgp substrates in both BCEC monolayers and BCEC+astrocyte co-cultures, and increased the sensitivity for vinblastine mediated disruption of the in vitro BBB (called the vinblastine exclusion assay). BCEC monolayers were more sensitive to vinblastine mediated disruption compared to BCEC+astrocyte co-cultures. In the latter, but not in BCEC monolayers, an inhibitable polar transport of Pgp substrates was only found from the brain to the blood side of the filter. CONCLUSIONS: Astrocytes increase the functional expression of Pgp in our in vitro BBB model. These results also illustrate that an important role for Pgp on the BBB is to protect the barrier against intracellular accumulation of cytotoxic BBB disrupting compounds.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Characterization of metabolites of hydroxycinnamates in the in vitro model of human small intestinal epithelium caco-2 cells.

Hydroxycinnamic acids are antioxidant phenolic compounds which are widespread in plant foods, contribute significantly to total polyphenol intakes, and are absorbed by humans. The extent of their putative health benefit in vivo depends largely on their bioavailability. However, the mechanisms of absorption and metabolism of these phenolic compounds have not been described. In this study, we used the in vitro Caco-2 model of human small intestinal epithelium to investigate the metabolism of the major dietary hydroxycinnamates (ferulate, sinapate, p-coumarate, and caffeate) and of diferulates. The appearance of metabolites in the medium versus time was monitored, and the various conjugates and derivatives produced were identified by HPLC-DAD, LC/MS, and enzyme treatment with beta-glucuronidase or sulfatase. Enterocyte-like differentiated Caco-2 cells have extra- and intracellular esterases able to de-esterify hydroxycinnamate and diferulate esters. In addition, intracellular UDP-glucuronosyltransferases and sulfotransferases existing in Caco-2 cells are able to form the sulfate and the glucuronide conjugates of methyl ferulate, methyl sinapate, methyl caffeate, and methyl p-coumarate. However, only the sulfate conjugates of the free acids, ferulic acid, sinapic acid, and p-coumaric acid, were detected after 24 h. The O-methylated derivatives, ferulic and isoferulic acid, were the only metabolites detected following incubation of Caco-2 cells with caffeic acid. These results show that the in vitro model system differentiated Caco-2 cells have the capacity to metabolize dietary hydroxycinnamates, including various phase I (de-esterification) and phase II (glucuronidation, sulfation, and O-methylation) reactions, and suggests that the human small intestinal epithelium plays a role in the metabolism and bioavailability of these phenolic compounds.

Caco-2 Cells↗
PubMed1992

Use of norfloxacin to study colonization ability of Escherichia coli in in vivo and in vitro models of the porcine gut.

The colonization resistance conveyed by the intestinal microbiota can prevent colonization of the intestinal system by new strains. In this study, this resistance was partly circumvented by use of the antimicrobial drug norfloxacin. The colonization abilities of two closely related Escherichia coli strains, which were resistant to nalidixic acid and rifampin, respectively, were investigated in minipigs and a two-stage continuous-flow in vitro gut model. Whereas both strains were unable to colonize the intact enteric system in vivo and in vitro, a 3-day norfloxacin treatment modified both systems to allow colonization by the nalidixic acid-resistant strain but not the rifampin-resistant strain. The results indicate the usefulness of norfloxacin to circumvent the normal colonization resistance while keeping a fairly normal microbiota in the gut. The results also indicate that it could be possible to construct in vitro gut models which could distinguish between strains with different gut colonization abilities. Both of these possibilities could come to be used in the study of the colonization and effects in the gut of new bacterial strains, i.e., genetically modified microorganisms.

Animals↗
PubMed2001

The organotypic culture of HPV-transformed keratinocytes: an effective in vitro model for the development of new immunotherapeutic approaches for mucosal (pre)neoplastic lesions.

The purpose of this study is to develop a reliable in vitro human model to test new immunotherapeutic approaches for squamous cell carcinoma that develop on mucosal surfaces. The organotypic (raft) culture permits cells to proliferate and differentiate at an air-liquid interface on a dermal equivalent support. Normal keratinocytes stratify and fully differentiate in a manner similar to the normal squamous epithelial tissues, while human papillomavirus-immortalized and established squamous carcinoma cell lines exhibit dysplastic morphologies similar to (pre)neoplastic lesions seen in vivo. We have demonstrated the ability of these organotypic cultures to be manipulated by altering the epithelial stratification with cytokines (interferon-gamma and tumor necrosis factor-alpha) and by integrating activated lymphocytes or dendritic cells into the in vitro formed epithelial sheet. This model may provide a useful tool to investigate the factors contributing to the presence and function of immunocompetent cells within a neoplastic epithelium that develops on a mucosal surface.

Cancer Vaccines↗
PubMed1986

In vitro models for the study of combination antibiotic therapy in neutropenic patients.

Neutropenic patients are at risk of serious infection caused by gram-negative bacilli and staphylococci. The mortality rate associated with gram-negative bacteremia in these patients is extremely high, especially in those with persistent and profound granulocytopenia. In these latter patients, the best results have been obtained by administering combinations of antibiotics in which both agents are active and/or show in vitro synergism against the infecting organism. Most combinations include an aminoglycoside such as amikacin and a broad-spectrum beta-lactam antibiotic, such as azlocillin, mezlocillin, piperacillin, or ceftazidime. The International Antimicrobial Therapy Project Group of the European Organization for Research and Treatment of Cancer has completed several studies evaluating various antibiotic combinations in the empiric treatment of febrile neutropenic patients. These trials have evaluated cephalothin plus gentamicin, carbenicillin plus gentamicin, and cephalothin plus carbenicillin; carbenicillin plus amikacin and carbenicillin plus amikacin plus cefazolin; azlocillin plus amikacin, ticarcillin plus amikacin, and cefotaxime plus amikacin; and azlocillin plus amikacin versus ceftazidime plus long- or short-course amikacin. The preclinical evaluation of antibiotic combinations usually involves the in vitro testing of antibiotics alone and in combination by the checkerboard method or with the use of time-kill curves. However, these methods expose the bacterial culture to a static or constant concentration of the drugs. During the in vivo treatment of infections, bacteria are exposed to changing concentrations of antibiotics, which are contingent on the individual pharmacokinetics of these drugs. We have designed a two-compartment in vitro pharmacokinetic model that allows the simultaneous study of the activity of two antibiotics with similar or different half-lives against a number of bacteria. Amikacin and azlocillin have been studied alone and in combination in this model against Pseudomonas aeruginosa, a frequent cause of bacteremia in neutropenic patients. In pharmacologically relevant doses, amikacin alone produced rapid bacterial killing, followed by regrowth of resistant subpopulations. Azlocillin alone produced a more gradual reduction of the bacterial inoculum, with ultimate bacteriostasis. Amikacin plus azlocillin produced rapid and complete eradication of the organism. In vitro pharmacokinetic models may prove to be more predictive of clinical outcome than are traditional static in vitro methods used to study antibiotic combinations.

Agranulocytosis↗
PubMedDate not supplied

Kinetics of bromocriptine release from microspheres: comparative analysis between different in vitro models.

This paper describes how the use of different in vitro experimental systems can influence the determination of (a) the drug release profile from microparticles and (b) the interpretation of the release mechanism(s). We employed, as model dosage form, the Parlodel LA, a recently marketed microsphere system especially designed for bromocriptine-controlled delivery. The release kinetics of bromocriptine from microspheres were determined by using two different experimental approaches: a dialysis method and a flow-through cell method. From the comparison of the obtained data it clearly appears that different in vitro experimental models lead to distinct results in terms of drug availability. On the contrary both series of data can be convincingly fitted with the same mathematical equation, giving almost identical results in terms of postulated release mechanism. Taken together these results indicate that different experimental approaches should always be employed to determine drug release kinetics from microparticles in order to obtain more reliable information on the therapeutic dose (bioavailable drug, for in vivo experiments) and on the uniformity of different batches of microspheres.

Bromocriptine↗
PubMedDate not supplied

Interleukin-7 production by B lymphocytes affects the T cell-dependent osteoclast formation in an in vitro model derived from human periodontitis patients.

Periodontitis is characterized by irreversible destruction of alveolar bone and connective tissue attachment in the periodontium. We recently reported that T cells support the osteoclastogenesis by the overproduction of nuclear factor-kappa-B-ligand (RANKL) and Tumor Necrosis Factor-alpha (TNF-alpha) in an in vitro osteoclastogenesis model from periodontitis patients (Pp). It is known that IL-7 stimulates the production of osteoclastogenic factors by T cells and IL-6 potentiates IL-7 expression. Thus, we studied the involvement of IL-6 and IL-7 in the T cell regulation of osteoclast (OC) formation, in an in vitro osteoclastogenesis model from Pp. We demonstrated high levels of IL-7 in both the media collected from PBMC cultures of Pp and the sera of the same patients. We also demonstrated that freshly isolated B cells from PBMCs of Pp were the source of IL-7 in our model. B cells, in fact, overexpressed IL-7 at mRNA and protein levels, and this production was up-regulated by IL-6. Moreover, the OC formation decreased in the presence of anti-IL-6 and IL-7 functional antibodies in PBMC cultures from Pp. These data suggest that B cells could be responsible for the T cell-dependent osteoclastogenesis in periodontitis through the involvement of IL-6 and IL-7.

Adult↗
PubMed2003

Synaptic background noise controls the input/output characteristics of single cells in an in vitro model of in vivo activity.

In vivo, in vitro and computational studies were used to investigate the impact of the synaptic background activity observed in neocortical neurons in vivo. We simulated background activity in vitro using two stochastic Ornstein-Uhlenbeck processes describing glutamatergic and GABAergic synaptic conductances, which were injected into a cell in real time using the dynamic clamp technique. With parameters chosen to mimic in vivo conditions, layer 5 rat prefrontal cortex cells recorded in vitro were depolarized by about 15 mV, their membrane fluctuated with a S.D. of about 4 mV, their input resistances decreased five-fold, their spontaneous firing had a high coefficient of variation and an average firing rate of about 5-10 Hz. Brief changes in the variance of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) synaptic conductance fluctuations induced time-locked spiking without significantly changing the average membrane potential of the cell. These transients mimicked increases in the correlation of excitatory inputs. Background activity was highly effective in modulating the firing-rate/current curve of the cell: the variance of the simulated gamma-aminobutyric acid (GABA) and AMPA conductances individually set the input/output gain, the mean excitatory and inhibitory conductances set the working point, and the mean inhibitory conductance controlled the input resistance. An average ratio of inhibitory to excitatory mean conductances close to 4 was optimal in generating membrane potential fluctuations with high coefficients of variation. We conclude that background synaptic activity can dynamically modulate the input/output properties of individual neocortical neurons in vivo.

Animals↗
PubMedDate not supplied

Expression of matrix proteins in an in vitro model of airway remodeling in asthma.

Acute asthma is characterized by a decrease in the pH of the exhaled breath condensate and bronchoconstriction. These perturbations may injure the epithelium in a chronic, intermittent pattern, leading to subepithelial fibrosis. We used an in vitro three-dimensional model of the bronchial mucosa to elucidate the response to a repeated chemical or physical insult to the epithelium in the postcontraction phase. We used enzyme-linked immunosorbent assay and reverse transcriptase--polymerase chain reaction to assess the production of the following proteins: matrix metalloproteinase (MMP) 3, MMP-9, tissue inhibitor of MMP-1, transforming growth factor beta 1, thrombospondin 1, tenascin, and fibronectin. The presence of the epithelium enhanced the degree of tissue contraction (50.1 +/- 4.4% of original area versus 75.4 +/- 2.3%). In the absence of injury, tenascin, fibronectin, MMP-3, and tissue inhibitor of MMP-1 are actively expressed. However, the chronic chemical wound markedly inhibited the expression of all proteins. We conclude that the epithelium, wound type, and age of the tissue (contracting versus postcontraction) impact the expression of key proteins in an in vitro model of subepithelial fibrosis in asthma.

Apoptosis↗