In vitro effect of air pollutants on human bronchi

A useful approach for constructing dose–response relationships and for studying the underlying mechanisms by which a xenobiotic agent enhances airway reactivity is to measure the response of an isolated airway following ex vivo exposure to a pollutant. We have in this way determined the dose–response relationship between ex vivo exposure to pollutants such as nitrogen dioxide (NO₂), the aldehyde acrolein, and ozone (O₃) and the reactivity to agonists in human isolated bronchial smooth muscle. We have also investigated the underlying alteration in the cellular mechanisms of airway smooth-muscle contraction induced by such exposure and found that it is related to alteration in calcium signaling at the site of the airway smooth-muscle cell. Finally, although there is epidemiological evidence that an increase in allergic diseases such as asthma may be linked to air pollution, there are few experimental data to address this issue. The final aim of this study was therefore to investigate the interaction between passive sensitization and exposure to pollutants in human isolated airways. We have examined (i) the effect of a pre-exposure to pollutants on the contraction of sensitized bronchi in response to a specific antigen and (ii) the effect of passive sensitization on the contraction in response to nonspecific agonists in bronchi pre-exposed to pollutants. The results indicate a combined effect of immunological sensitization and exposure to pollutants; that is, passive sensitization and exposure to pollutants act in a synergistic manner on human bronchial smooth-muscle reactivity in response to both specific antigens and nonspecific agonists. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro effect of actinomycin D on human neutrophil function

The effect of actinomycin D (ACT-D) on human neutrophil chemotaxis, chemiluminescence (CL), superoxide (O2-) production, phagocytic uptake, and intracellular bacterial killing has been examined. The viability of the ACT-D-treated neutrophils was 98% even at a concentration of 10 micrograms/ml for 4 hr. Using fMLP as the chemotactic factor, depressed chemotaxis was demonstrated following ACT-D (1-10 micrograms/ml) pretreatment of neutrophils as compared with the non-treated controls. Similar ACT-D pretreatment produced the depressed responses in phorbol myristate acetate-induced CL and superoxide production by neutrophils. Moreover, using heat-inactivated human serum as an opsonin for Salmonella enteritidis (NCTC 6676), there was a significant difference in intracellular killing (P less than 0.01) but no difference in phagocytic uptake between ACT-D-treated and non-treated neutrophils. These studies indicate that ACT-D profoundly impairs both intracellular bacterial killing by human neutrophil through an effect on respiratory burst activity and directed cell migration of human neutrophils.     

In vitro effect of lead acetate on human erythropoietic progenitors

Lead is known to induce hematological disturbances resulting from abnormalities in cell differentiation and hemoglobin synthesis during hematopoiesis. The aim of the present work was to study human erythropoiesisin vitro in the presence of lead. Human erythroblastic progenitors, burst-forming units–erythroid (BFU-E), were exposed to lead acetate at increasing concentrations during 14 days of culture. Hematotoxicity was evaluatedin vitro according to proliferation and differentiation of cell colonies arising from BFU-E development. The ability of cells to synthesize proteins, porphyrins, and hemoglobin was measured by spectrophotometric tests and by high-pressure liquid chromatography (HPLC). Results showed that in the presence of 10^–3 mol/L lead acetate, no hemoglobinized cells were observed in culture and no fluorescent porphyrins were detected in cells. Up to 10^–3 mol/L, lead acetate is not cytotoxic, i.e., it does not induce cell destruction. The present work demonstrates that lead acetate interferes with the porphyrin synthesis of human erythroblastic progenitors in vitro. The decrease of porphyrin content with 10^–5 mol/L lead acetate suggest that δ-aminolevulinic acid dehydratase can be inhibited by lead acetate duringin vitro erythropoiesis. In vivo erythropoiesis occurs in the bone marrow. As about 95% of the body burden of lead in adults is located in the bones with a biological half-life of some years, the concentration of lead acetate found to block porphyrin synthesis in vitro has to be compared with in situ bone marrow lead concentrations. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro posttranslational modification of lamin B cloned from a human T-cell line.

Autoimmune diseases are characterized by spontaneously occurring autoantibodies which have proven to be useful reagents for the characterization of specific nuclear proteins. Using a monoclonal autoantibody (72B9) derived from a murine lupus strain, we have cloned a cDNA from the human T-cell line MOLT-4, which encodes nuclear lamin B. The identity of the encoded protein as lamin B was established by both biochemical and immunological criteria. Inspection of the deduced amino acid sequence of lamin B revealed the presence in coil 1B of the alpha-helical domain of a leucine heptad repeat region. Analysis of mRNA in HL60 and MOLT-4 cells, which express only lamin B, or HeLa cells, which express all three major lamins (A, B, and C), together with the comigration of in vitro-translated product with isolated HeLa cell lamin B by two-dimensional gel electrophoresis, suggests that a single lamin B is expressed in mammalian somatic cells. In vitro translation with the cDNA clone revealed an EDTA-sensitive posttranslational modification which resulted in an increase in the apparent molecular weight to that equivalent to the native in vivo-synthesized lamin B protein. This in vitro modification included incorporation of a product of mevalonolactone and required an intact carboxy terminus.     

In vitro effect of larval stages of Ascaris lumbricoides on human blood clotting

The effects of larval stages of Ascaris lumbricoides on human blood clotting was studied in vitro. Extracts and excretory/secretory products of third-stage larvae (L3) and late third-stage larvae (LL3) cultured from ova obtained from infected patients were analysed for anti-coagulant activity. Prothrombin time (PT) was prolonged by the addition of either whole extract of L3/LL3 or ES products of L3/LL3 as compared to controls. Partial thromboplastin time with kaolin (PTTK) was also prolonged on the addition of either extracts of ES products of L3/LL3. The prolongation of PTTK was significantly higher with extracts/ES products of L3 when compared to the extracts/ES products of LL3 (p less than 0.005). Thrombin time (TT) was prolonged by extracts of L3/LL3 and their ES products.     

In vitro activation of the IkappaB kinase complex by human T-cell leukemia virus type-1 Tax

Human T-cell leukemia virus type-I expresses Tax, a 40-kDa oncoprotein that activates IkappaB kinase (IKK), resulting in constitutive activation of NFkappaB. Herein, we have developed an in vitro signaling assay to analyze IKK complex activation by recombinant Tax. Using this assay in combination with reporter assays, we demonstrate that Tax-mediated activation of IKK is independent of phosphatases. We show that sustained activation of the Tax-mediated activation of the NFkappaB pathway is dependent on an intact Hsp90-IKK complex. By acetylating and thereby preventing activation of the IKK complex by the Yersinia effector YopJ, we demonstrate that Tax-mediated activation of the IKK complex requires a phosphorylation step. Our characterization of an in vitro signaling assay system for the mechanism of Tax-mediated activation of the IKK complex with a variety of mutants and inhibitors results in a working model for the biochemical mechanism of Tax-induced activation.     

In vitro polydeoxyribonucleotide effects on human pre-adipocytes

Abstract. Objectives: Adipose tissue is the most abundant and accessible source of adult stem cells. Human processed lipoaspirate contains pre‐adipocytes that possess one of the a characteristic pathways of multipotent adult stem cells and are able to differentiate in vitro into mesenchymal and also neurogenic lineages. Because stem cells have great potential for use in tissue repair and regeneration, it would be significant to be able to obtain large amounts of these cells in vitro. As demonstrated previously, purine nucleosides and nucleotides mixtures can act as mitogens for several cell types. The aim of this study was to evaluate the effects of polydeoxyribonucleotides (PDRN), at appropriate concentrations, on human pre‐adipocytes grown in a controlled medium, also using different passages, so as to investigate the relationship between the effect of this compound and cellular senescence, which is the phenomenon when normal diploid cells lose the ability to divide further. Materials and methods: Human pre‐adipocytes were obtained by liposuction. Cells from different culture passages (P6 and P16) were treated with PDRN at different experimental times. Cell number was evaluated for each sample by direct counting after trypan blue treatment. DNA assay and the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide test were also carried out in all cases. Results and Conclusions: PDRN seemed to promote proliferation of human pre‐adipocytes at both passages, but cell population growth increased in pre‐adipocyte at P16, after 9 days as compared to control. Our data suggest that PDRN could act as a pre‐adipocyte growth stimulator.     

In vitro synthesis of prostaglandins and related lipids by populations of human peripheral blood mononuclear cells

This report focuses on the identification of the human peripheral blood mononuclear cells that do or do not produce prostaglandins (PGs) and related arachidonic acid metabolites. Our results, using two different assay systems, indicate that the monocyte/macrophage (MØ) is the major and possibly sole source of thromboxane (TXB₂) and prostaglandin E₂ (PGE₂) among peripheral blood mononuclear cells. Adherent peripheral blood monocytes (> 95% esterase positive) produced substantial amounts of these compounds. Quantitation of products which had incorporated exogenous ¹⁴C-arachidonic acid and radioimmunoassay of adherent cell culture fluids demonstrated that the amount of TXB₂ produced by these cells was appreciably greater than the amount of PGE₂ produced. Additional confirmation of TXB₂ synthesis was shown by abolishing the TXB₂ peak on TLC and TXB₂ activity detected by RIA by treating cells with a specific inhibitor of thromboxane synthetase. In contrast, non-adherent T cells failed to synthesize either PGE₂ or TXB₂. Non-adherent B cells (95% Ig positive) incubated with ¹⁴C-arachidonic acid produced a small peak of radioactivity co-chromatographing with TXB₂, and no PGE₂. All three cell populations incorporated similar amounts of ¹⁴C-arachidonic acid into hydroxy-fatty acids. We were unable to detect 6-keto-F_1α, the hydrolysis product of prostacyclin (PGI₂) in any of the cell types tested. The absence of PG synthesis among normal peripheral blood T and B cells was also noted among established human lymphoid cell lines. Neither a human T (CCRF), nor a human B-cell line (GM-130), produced PGE₂ or TXB₂. Three murine macrophage cell lines, P388D₁, J774.2, and WHI-3 produced PGE₂ and the latter TXB₂ as well.     

In vitro effect of phencyclidine and other psychomotor stimulants on serotonin uptake in human platelets

Phencylidine, ketamine and fluoxetine inhibited serotonin (5-HT) uptake in a non-competitive manner in human blood platelets whereas d- and 1-amphetamine produced a competitive inhibition of 5-HT uptake. Phencyclidine (IC₅₀, 2.5 μM) was one-hundredth as potent as fluoxetine (IC₅₀, 22 νM) but ten times more potent than ketamine (IC₅₀, 25 μM) and d-amphetamine (IC₅₀, 24 μM) and three times more potent than 1-amphetamine (IC₅₀, 80 μM) in inhibition of 5-HT uptake by human blood platelets. The possibility that inhibition of 5-HT may contribute to some of the proposed serotonergic effects of psychomotor stimulants is discussed.     

In vitro prooxidant effect of caffeine

Caffeine is a methylxanthine compound that acts as a stimulant in humans. It is the most widely consumed behaviourally active substance in the western world and it affects calcium influx and release in living cells. Caffeine is present in various substances such as tea, coffee and some medications. This article is focused on the impact of caffeine on oxidation. Caffeine promoted the peroxidation of linoleic acid emulsions used by 32.5, 48.9, and 54.3%, respectively, at 15, 30 and 45 μg/mL concentrations. Standard antioxidants such as α-tocopherol and trolox, a water-soluble analogue of tocopherol, inhibited 76.2 and 93.2% peroxidation of linoleic acid emulsion at 45 μg/mL concentration. Also, PC₅₀ value (caffeine concentration that produced a 50% increase in linoleic acid peroxidation) of caffeine was found to be 185 μM.     

In vitro prooxidant effect of caffeine

Caffeine is a methylxanthine compound that acts as a stimulant in humans. It is the most widely consumed behaviourally active substance in the western world and it affects calcium influx and release in living cells. Caffeine is present in various substances such as tea, coffee and some medications. This article is focused on the impact of caffeine on oxidation. Caffeine promoted the peroxidation of linoleic acid emulsions used by 32.5, 48.9, and 54.3%, respectively, at 15, 30 and 45 microg/mL concentrations. Standard antioxidants such as alpha-tocopherol and trolox, a water-soluble analogue of tocopherol, inhibited 76.2 and 93.2% peroxidation of linoleic acid emulsion at 45 microg/mL concentration. Also, PC(50) value (caffeine concentration that produced a 50% increase in linoleic acid peroxidation) of caffeine was found to be 185 microM.