Surfactant protein D modulates allergen particle uptake and inflammatory response in a human epithelial airway model

Background Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was investigated. Methods SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and advanced computer-controlled analysis. Finally, human primary CD4+ T-Cells were added to the epithelial airway model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array. Results SPP were taken up by epithelial cells, macrophages, and dendritic cells. This uptake coincided with secretion of pro-inflammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific way (e.g. increased number of macrophages and epithelial cells, which participated in allergen particle uptake) and led to a decreased secretion of pro-inflammatory cytokines. Conclusion These results display a possible mechanism of how SP-D can modulate the inflammatory response to inhaled allergen.

High salt intake down-regulates colonic mineralocorticoid receptors, epithelial sodium channels and 11β-hydroxysteroid dehydrogenase type 2

Besides the kidneys, the gastrointestinal tract is the principal organ responsible for sodium homeostasis. For sodium transport across the cell membranes the epithelial sodium channel (ENaC) is of pivotal relevance. The ENaC is mainly regulated by mineralocorticoid receptor mediated actions. The MR activation by endogenous 11β-hydroxy-glucocorticoids is modulated by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Here we present evidence for intestinal segment specific 11β-HSD2 expression and hypothesize that a high salt intake and/or uninephrectomy (UNX) affects colonic 11β-HSD2, MR and ENaC expression. The 11β-HSD2 activity was measured by means of 3H-corticosterone conversion into 3H-11-dehydrocorticosterone in Sprague Dawley rats on a normal and high salt diet. The activity increased steadily from the ileum to the distal colon by a factor of about 3, an observation in line with the relevance of the distal colon for sodium handling. High salt intake diminished mRNA and protein of 11β-HSD2 by about 50% (p<0.001) and reduced the expression of the MR (p<0.01). The functionally relevant ENaC-β and ENaC-γ expression, a measure of mineralocorticoid action, diminished by more than 50% by high salt intake (p<0.001). The observed changes were present in rats with and without UNX. Thus, colonic epithelial cells appear to contribute to the protective armamentarium of the mammalian body against salt overload, a mechanism not modulated by UNX.

Impaired innate interferon induction in severe therapy resistant atopic asthmatic children

Deficient type I interferon-β and type III interferon-λ induction by rhinoviruses has previously been reported in mild/moderate atopic asthmatic adults. No studies have yet investigated if this occurs in severe therapy resistant asthma (STRA). Here, we show that compared with non-allergic healthy control children, bronchial epithelial cells cultured ex vivo from severe therapy resistant atopic asthmatic children have profoundly impaired interferon-β and interferon-λ mRNA and protein in response to rhinovirus (RV) and polyIC stimulation. Severe treatment resistant asthmatics also exhibited increased virus load, which negatively correlated with interferon mRNA levels. Furthermore, uninfected cells from severe therapy resistant asthmatic children showed lower levels of Toll-like receptor-3 mRNA and reduced retinoic acid inducible gene and melanoma differentiation-associated gene 5 mRNA after RV stimulation. These data expand on the original work, suggesting that the innate anti-viral response to RVs is impaired in asthmatic tissues and demonstrate that this is a feature of STRA.

Effect of remifentanil on mitochondrial oxygen consumption of cultured human hepatocytes

During sepsis, liver dysfunction is common, and failure of mitochondria to effectively couple oxygen consumption with energy production has been described. In addition to sepsis, pharmacological agents used to treat septic patients may contribute to mitochondrial dysfunction. This study addressed the hypothesis that remifentanil interacts with hepatic mitochondrial oxygen consumption. The human hepatoma cell line HepG2 and their isolated mitochondria were exposed to remifentanil, with or without further exposure to tumor necrosis factor-α (TNF-α). Mitochondrial oxygen consumption was measured by high-resolution respirometry, Caspase-3 protein levels by Western blotting, and cytokine levels by ELISA. Inhibitory κBα (IκBα) phosphorylation, measurement of the cellular ATP content and mitochondrial membrane potential in intact cells were analysed using commercial ELISA kits. Maximal cellular respiration increased after one hour of incubation with remifentanil, and phosphorylation of IκBα occurred, denoting stimulation of nuclear factor κB (NF-κB). The effect on cellular respiration was not present at 2, 4, 8 or 16 hours of incubation. Remifentanil increased the isolated mitochondrial respiratory control ratio of complex-I-dependent respiration without interfering with maximal respiration. Preincubation with the opioid receptor antagonist naloxone prevented a remifentanil-induced increase in cellular respiration. Remifentanil at 10× higher concentrations than therapeutic reduced mitochondrial membrane potential and ATP content without uncoupling oxygen consumption and basal respiration levels. TNF-α exposure reduced respiration of complex-I, -II and -IV, an effect which was prevented by prior remifentanil incubation. Furthermore, prior remifentanil incubation prevented TNF-α-induced IL-6 release of HepG2 cells, and attenuated fragmentation of pro-caspase-3 into cleaved active caspase 3 (an early marker of apoptosis). Our data suggest that remifentanil increases cellular respiration of human hepatocytes and prevents TNF-α-induced mitochondrial dysfunction. The results were not explained by uncoupling of mitochondrial respiration.

In vitro effects of thiazolides on Giardia lamblia WB clone C6 cultured axenically and in coculture with Caco2 cells

The thiazolides represent a novel class of anti-infective drugs, with the nitrothiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] (NTZ) as the parent compound. NTZ exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. In vivo, NTZ is rapidly deacetylated to tizoxanide (TIZ), which exhibits similar activities. We have here comparatively investigated the in vitro effects of NTZ, TIZ, a number of other modified thiazolides, and metronidazole (MTZ) on Giardia lamblia trophozoites grown under axenic culture conditions and in coculture with the human cancer colon cell line Caco2. The modifications of the thiazolides included, on one hand, the replacement of the nitro group on the thiazole ring with a bromide, and, on the other hand, the differential positioning of methyl groups on the benzene ring. Of seven compounds with a bromo instead of a nitro group, only one, RM4820, showed moderate inhibition of Giardia proliferation in axenic culture, but not in coculture with Caco2 cells, with a 50% inhibitory concentration (IC50) of 18.8 microM; in comparison, NTZ and tizoxanide had IC50s of 2.4 microM, and MTZ had an IC50 of 7.8 microM. Moreover, the methylation or carboxylation of the benzene ring at position 3 resulted in a significant decrease of activity, and methylation at position 5 completely abrogated the antiparasitic effect of the nitrothiazole compound. Trophozoites treated with NTZ showed distinct lesions on the ventral disk as soon as 2 to 3 h after treatment, whereas treatment with metronidazole resulted in severe damage to the dorsal surface membrane at later time points.

Vitamin E reduces antidepressant-related beta-adrenoceptor down-regulation in cultured cells. Comparable effects on St. John's wort and tricyclic antidepressant treatment

The mode of action of antidepressants is still a matter of debate. Acute inhibition of neurotransmitter reuptake in central neuronal synapses, followed by a down-regulation of central postsynaptic beta-adrenoceptor (beta-AR) numbers were consistently observed in vivo, while a reduction in surface beta-AR density was found in cell cultures. Effects of the tricyclic antidepressant desipramine (DMI) were abolished by vitamin E (alpha-TOC) in vitro as well as in vivo. Alpha-TOC interfered with antidepressant-induced changes of cellular plasma membrane properties and with recycling of beta-AR. St. John's wort (SJW) extract reduced beta-AR numbers in cultured cells to a similar extent as DMI or the selective serotonin re-uptake inhibitor fluoxetine. We chronically co-exposed cell cultures to SJW extract and to alpha-TOC. Receptor down-regulation following exposure to the plant extract was inhibited in the presence of alpha-TOC suggesting a mode of action of SJW extract comparable to that of synthetic antidepressants. Inhibition of cell proliferation by the plant extract was also significantly reduced by alpha-TOC.

GDNF family ligands display distinct action profiles on cultured GABAergic and serotonergic neurons of rat ventral mesencephalon

Glial-cell-line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN), known as the GDNF family ligands (GFLs), influence the development, survival and differentiation of cultured dopaminergic neurons from ventral mesencephalon (VM). Detailed knowledge about the effects of GFLs on other neuronal populations in the VM is essential for their potential application as therapeutic molecules for Parkinson's disease. Hence, in a comparative study, we investigated the effects of GFLs on cell densities and morphological differentiation of gamma-aminobutyric acid-immunoreactive (GABA-ir) and serotonin-ir (5-HT-ir) neurons in primary cultures of E14 rat VM. We observed that all GFLs [10 ng/ml] significantly increased GABA-ir cell densities (1.6-fold) as well as neurite length/neuron. However, only GDNF significantly increased the number of primary neurites/neuron, and none of the GFLs affected soma size of GABA-ir neurons. In contrast, only NRTN treatment significantly increased 5-HT-ir cells densities at 10 ng/ml (1.3-fold), while an augmentation was seen for GDNF and PSPN at 100 ng/ml (2.4-fold and 1.7-fold, respectively). ARTN had no effect on 5-HT-ir cell densities. Morphological analysis of 5-HT-ir neurons revealed a significant increase of soma size, number of primary neurites/neuron and neurite length/neuron after GDNF exposure, while PSPN only affected soma size, and NRTN and ARTN failed to exert any effect. In conclusion, we identified GFLs as effective neurotrophic factors for VM GABAergic and serotonergic neurons, demonstrating characteristic individual action profiles emphasizing their important and distinct roles during brain development.

Effects of daunorubicin, mitomycin C, azathioprine and cyclosporin A on human retinal pigmented epithelial, corneal endothelial and conjunctival cell lines

BACKGROUND: We wished to investigate the toxicity of four immunosuppressant and antimetabolic drugs, which are known to influence postoperative wound healing, on three different human ocular cell lines. METHODS: Acute toxicity to cyclosporin A, azathioprine, mitomicyn C and daunorubicin was assessed in Chang cells by monitoring their uptake of propidium iodide during a 3-h period. Chronic toxicity was assessed by monitoring the proliferation and viability of subconfluent cultures of Chang cells, human corneal endothelial cells (HCECs) and retinal pigmented epithelial (RPE) cells after continuous exposure to the drugs for 7 days. RESULTS: Acute toxicity testing revealed no obvious effects. However, the chronic toxicity tests disclosed a narrow concentration range over which cell proliferation decreased dramatically but calcein metabolism was sustained. Although the three lines reacted similarly to each agent, HCECs were the most vulnerable to daunorubicin and mitomycin. At a daunorubicin concentration of 0.05 microg/ml, a 75% decrease in calcein metabolism (P < 0.001) and a > or = 95% cell loss (P < 0.001) were observed. At a mitomycin concentration of 0.01 mug/ml, cell density decreased by 61% (P < 0.001) without a change in calcein metabolism, but at 0.1 microg/ml, the latter parameter decreased to 12% (P = 0.00014). At this concentration the proliferation of Chang and RPE cells decreased by more than 50%, whilst calcein metabolism was largely sustained. Cyclosporin inhibited cell proliferation moderately at lower concentrations (< 5 microg/ml; P=0.05) and substantially at higher ones, with a corresponding decline in calcein metabolism. Azathioprine induced a profound decrease in both parameters at concentrations above 5 microg/ml. CONCLUSION: Daunorubicin, cyclosporin and azathioprine could be used to inhibit excessive intraocular scarring after glaucoma and vitreoretinal surgery without overly reducing cell viability. The attributes of immunosuppressants lie in their combined antiproliferative and immunomodulatory effects.

Epithelial transformations in the establishment of the blood-gas barrier in the developing chick embryo lung

The tall epithelium of the developing chick embryo lung is converted to a squamous one, which participates in formation of the thin blood-gas barrier. We show that this conversion occurred through processes resembling exocrine secretion. Initially, cells formed intraluminal protrusions (aposomes), and then transcellular double membranes were established. Gaps between the membranes opened, thus, severing the aposome from the cell. Alternatively, aposomes were squeezed out by adjacent cells or were spontaneously constricted and extruded. As a third mechanism, formation and fusion of severed vesicles or vacuoles below the aposome and their fusion with the apicolateral plasma membrane resulted in severing of the aposome. The atria started to form by progressive epithelial attenuation and subsequent invasion of the surrounding mesenchyme at regions delineated by subepithelial alpha-smooth muscle actin-positive cells. Further epithelial attenuation was achieved by vacuolation; rupture of such vacuoles with resultant numerous microfolds and microvilli, which were abscised to accomplish a smooth squamous epithelium just before hatching.