Human Lung Slices Research Articles

Gene transfer to adult human lung tissue ex vivo.

The potential of gene therapy for treatment of lung disease remains unrealised. Early model systems often resulted in promising efficiency of gene transfer, only to prove irreproducible in the clinic. While problems such as induction of host immune responses and duration of expression also need to be addressed, it is now widely believed that alternative, relevant models which more accurately reflect gene transfer efficiencies in human lungs are urgently required. We report here on a human lung slice culture system to assess gene transfer to adult lung epithelium. A lacZ-expressing adenovirus (AdCA35lacZ) was used as a reporter vector. A solution of AdCA35lacZ was instilled via bronchioles into resected lung tissue, a route analogous to clinical administration. Following a 1 h incubation, the tissue was inflated with a 0.4% agarose solution, instilled via the same bronchioles. Once solidified, 500 microm slices of the tissue were prepared and cultured for 4 days. beta-Galactosidase staining revealed lacZ transgene expression in bronchiolar and alveolar cells of the lung slices throughout the 4 days in culture. This system, which can also be used to study other viral and liposome vectors, could prove to be a useful alternative model for assessing gene delivery to adult human lung epithelium.

In vitro modulation of the P450 activities of hamster and human lung slices.

The respiratory tract is a portal of entry for many environmental chemicals. The respiratory tract plays an important role in the detoxification or metabolic activation of these chemicals, e.g., via cytochrome P450 enzymes. Alterations in the capabilities of these enzymes to metabolize inhaled compounds can, therefore, affect the toxicity of the chemicals. The pulmonary cytochrome P450 activity has been studied in many species, but relatively little is known about this activity in the human lung tissue. In this limited study, we have investigated the possibility of modulating in vitro the P450 activity in lung slices from hamsters and humans. The alkoxyresorufin-O-dealkylase activity was measured in the S9 fraction of lung slices incubated for 24 h with 10(6) mol/L 20-methylcholanthrene (3MC) or beta-naphthoflavone (beta N). The ethoxyresorufin-O-deethylase (EROD) activity was increased by 3MC and beta N in lung slices of both species. The benzyloxyresorufin-O-deethylase (BROD) activity was decreased after incubation with 3MC but increased with beta N. These data show that in vitro modulation in lung slices is feasible, although technical improvement is still needed, particularly in relation to the viability of the slices.

In vitro expression of pro-inflammatory cytokines in human lung slices

In vitro expression of pro-inflammatory cytokines in human lung slices

In vitro expression of pro-inflammatory cytokines in human lung slices

In vitro expression of pro-inflammatory cytokines in human lung slices

Metabolism of [ring-U- 14C] agaritine by precision-cut rat, mouse and human liver and lung slices

Agaritine {(β- N-[γ- l(+)glutamyl]-4-hydroxymethylphenylhydrazine)} is present in the common cultivated mushroom Agaricus bisporus and several agaritine derivatives have been shown to produce tumours in experimental animals. In this investigation the metabolism of [ring-U- 14C]agaritine has been studied in precision-cut rat, mouse and human liver slices and in precision-cut rat and mouse lung slices. To confirm the functional viability of the tissue slice preparations, the metabolism of 7-ethoxycoumarin was also studied. Liver and lung slices from all species metabolized 50 μM 7-ethoxycoumarin to 7-hydroxycoumarin, which was conjugated with D-glucuronic acid and sulfate. Incubation of rat, mouse and human liver slices, and rat and mouse lung slices with 25 μM [ 14C]agaritine resulted in a time-dependent formation of metabolite(s), which bound covalently to tissue slice proteins. Agaritine metabolite covalent binding was greater in mouse liver than in rat and human liver slices and was greater in mouse lung than in rat lung slices. No correlation was observed between agaritine metabolite covalent binding and tissue slice γ-glutamyltransferase activity. Additional studies with mouse liver slices showed that [ 14C]agaritine was also metabolized to a number of unknown polar metabolites. These results demonstrate that agaritine can be metabolized by enzymes present in mammalian liver and lung.

Cyclosporin a metabolism in human and rat lung slices, and human bronchial epithelial cells

Cyclosporin a metabolism in human and rat lung slices, and human bronchial epithelial cells

Putrescine and paraquat uptake in human lung slices and isolated type II pneumocytes

Paraquat is accumulated into the lungs of various species by an active uptake system which also appears to mediate the uptake of endogenous polyamines, such as putrescine. The accumulation of putrescine in the human lung has been previously shown to be mainly located in the type II cells. In the present study, we have studied the mutually competitive inhibition of putrescine and paraquat in human lung slices and the inhibition of putrescine by paraquat or cystamine in isolated human type II pneumocytes. Peripheral lung tissue taken from patients undergoing pneumectomy or lobectomy was used. The initial steps of the cell isolation procedure differed from the literature in that the tissue was first sliced in 0.7 mm thick slices, which were washed in phosphate buffered saline without calcium and magnesium (PBS −), followed by incubations with trypsin. The type II cells were purified and isolated by differential adherence on plastic followed by Percoll gradient centrifugation. Uptake was determined 48 hr after cell isolation. The accumulation of radiolabelled putrescine showed saturation kinetics, with the following apparent kinetic parameters: k n 6.7 and 6.2–7.6μM and V max2.7 and 3.0–3.4μmol/g prot/hr for slices and isolated cells, respectively. In the presence of paraquat, putrescine uptake was reduced, in both systems, in a manner compatible with competitive inhibition, with calculated inhibition constants ( k i) of 549–614 and 659–895 μM paraquat for slices and isolated cells, respectively. The accumulation of putrescine in isolated human pneumocytes was strongly reduced in the presence of cystamine, with calculated k i of 3.7 μM cystamine. These data indicate that putrescine, paraquat and cystamine accumulate in the human lung by the same uptake system, but that the affinities for the three substrates differ. The presence of an uptake system for putrescine in cultured human pulmonary type II is probably useful as a functional viability test.

The Use of Human Lung Slices in Toxicology

1. Successful use of agar-filled precision-cut rat lung slices in dynamic organ culture prompted the use of this technology with human lung. 2. The larger tissue mass of a human lung required that the trachea be cannulated with a balloon catheter and subsequently inflated with 4 liters of warm agar/medium mixture and then cooled before being precision-cut into 500 microns thick slices. 3. To characterize the human lung slices, viability and the effects of acrolein and nitrofurantoin were assessed over a period of 24 h using protein synthesis and nonprotein sulfhydryl content. 4. Control human lung slices synthesized protein at a linear rate and maintained a stable nonprotein sulfhydryl content for 24 h. 5. Slices incubated with acrolein exhibited no significant decrease in protein synthesis or nonprotein sulfhydryl levels until 24 h. 6. Incubation with nitrofurantoin exhibited a definite time- and dose-dependent inhibition of protein synthesis, and depletion of the cellular thiol pool. 7. These results indicate that this human lung tissue slice system may be used as an in vitro model to identify and screen pneumotoxicants.

Effect of platelet-activating factor on beta-adrenoceptors in human lung

We examined the effect of platelet-activating factor (PAF) on beta-adrenoceptors in the membranes of human lung and the functional responses to methacholine, histamine and isoproterenol in human lung parenchyma and tracheal strips. Preincubation of the human lung slices with 0.1 uM PAF decreased the density of beta-adrenoceptors without a change in the affinity. In the functional studies, a subthreshold dose of PAF (0.1 uM) significantly reduced the potency of isoproterenol to reverse methacholine or histamine-induced contraction. BN 52021, a PAF antagonist, protected against these PAF-induced changes. These findings suggest that PAF-induced down-regulation of beta-adrenoceptors could be a mechanism of the non-specific airway hyperreactivity in asthma.

Interferon enhances tryptophan metabolism by inducing pulmonary indoleamine 2,3-dioxygenase: its possible occurrence in cancer patients.

Human lungs bearing cancer (n = 27) exhibited up to an approximately 20-fold [on average approximately 5-fold (P less than 0.005)] increase in the enzyme activity that degrades tryptophan to form formylkynurenine, in comparison with lungs with benign lesions (blebs) (n = 7) taken as controls. On the basis of molecular and kinetic properties, this activity was ascribed to indoleamine 2,3-dioxygenase (IDO) [indoleamine:oxygen 2,3-oxidoreductase (decyclizing)]. In vitro studies with human lung slices revealed that human interferon gamma (IFN-gamma) induced the de novo synthesis of IDO dose dependently (10-10(4) units/ml), and at maximum the activity reached nearly 100 times that in the control lungs described above. Human IFN-alpha also served as an inducer, but it was two to three orders of magnitude less potent than IFN-gamma relative to the antiviral titers, suggesting that IFN-gamma is the main mediator of the IDO induction. IDO thus induced in slices avidly metabolized tryptophan in situ: Upon a 24-hr incubation of lung slices pretreated with varied doses of IFN-gamma (10-10(3) units/ml), up to 96% of the tryptophan in the slices was depleted and up to 70% of the tryptophan in the medium was converted, mainly to formylkynurenine, kynurenine, or both. The foregoing results suggest that an IFN-mediated induction of IDO also takes place in vivo in human lungs as a response to cancer, leading to metabolic consequences such as depletion of tryptophan and accumulation of (formyl)kynurenine, which may provide a unique host defense mechanism.

Potentiation of histamine release by sodium cromoglycate

Human lung slices passively sensitized with allergic serum released histamine when incubated with specific antigen and anti-IgE but anti-IgG had no effect. Sodium cromoglycate (SCG) inhibited antigen induced histamine release but the dose-response curve was bell-shaped. Inhibition of anti-IgE induced release was linearly related to dose, whereas that induced by anti-IgG was potentiated by increasing doses of SCG. After sensitization with allergic serum in which IgE had been inactivated by heating, specific antigen released little or no histamine but this was potentiated by SCG. It is concluded that SCG inhibits IgE mediated but potentiates IgG mediated allregic reactions thus explaining its characteristic dose-response curve in vitro .

Metabolism of dehydroisoandrosterone and androstenedione by the human lung in vitro

3β-Hydroxysteroid oxidoreductase-Δ 5→4-isomerase and 7α-hydroxylase activities are present in human lung and this is reflected in the rates of formation at initial velocities of androstenedione (∼110 pmol/100mg protein/h) and of 7α-hydroxydehydroisoandrosterone (∼100 pmol/100mg protein/h) as the principal metabolites obtained from incubations of human lung slices with [7- 3H]-dehydroisoandrosterone. The 5α-reduced metabolites 5α-androstanedione and androsterone were formed with total conversion rates of ∼75 pmol/100 mg protein/h, and in addition, the 17β-hydroxysteroids 5-androstene-3β, 17β-diol and testosterone were also formed (∼25 pmol/100 mg protein/h and ∼5 pmol/100 mg protein/h, respectively). The incubation of human lung slices with [1,2,6,7- 3H]-androstenedione resulted in the formation of 5α-androstanedione, androsterone and isoandrosterone with total conversion rates of ∼ 44 pmol/100 mg protein/h, and of testosterone (∼ 22 pmol/100 mg protein/h). 5β-Reduced-C 19-steroids were not identified among the metabolites. The human lung is a potential site for the conversion of circulating dehydroisoandrosterone to androstenedione, to testosterone and to 5α-reduced steroids. The resulting steroids may exert their activities in situ or elsewhere, conceivably after further metabolism to more potent androgens.