The Human Tissue-Biomaterial Interface : A Role for PPARγ-Dependent Glucocorticoid Receptor Activation in Regulating the CD163(+) M2 Macrophage Phenotype

In vivo studies of implanted acellular biological scaffolds in experimental animals have shown constructive remodeling mediated by anti-inflammatory macrophages. Little is known about the human macrophage response to such biomaterials, or the nature of the signaling mechanisms that govern the macrophage phenotype in this environment. The cellular events at the interface of a tissue and implanted decellularized biomaterial were examined by establishing a novel ex vivo tissue culture model in which surgically excised human urinary tract tissue was combined with porcine acellular bladder matrix (PABM). Evaluation of the tissue-biomaterial interface showed a time-dependent infiltration of the biomaterial by CD68(+) CD80(-) macrophages. The migration of CD68(+) cells from the tissue to the interface was accompanied by maturation to a CD163(hi) phenotype, suggesting that factor(s) associated with the biomaterial or the wound edge was/were responsible for the active recruitment and polarization of local macrophages. Glucocorticoid receptor (GR) and peroxisome proliferator activated receptor gamma (PPARγ) signaling was investigated as candidate pathways for integrating inflammatory responses; both showed intense nuclear labeling in interface macrophages. GR and PPARγ activation polarized peripheral blood-derived macrophages from a default M1 (CD80(+)) toward an M2 (CD163(+)) phenotype, but PPARγ signaling predominated, as its antagonism blocked any GR-mediated effect. Seeding on PABM was effective at polarizing peripheral blood-derived macrophages from a default CD80(+) phenotype on glass to a CD80(-) phenotype, with intense nuclear localization of PPARγ. These results endorse in vivo observations that the infiltration of decellularized biological scaffolds, exemplified here by PABM, is pioneered by macrophages. Thus, it appears that natural factors present in PABM are involved in the active recruitment and polarization of macrophages to a CD163(+) phenotype, with activation of PPARγ identified as the candidate pathway. The harnessing of these natural matrix-associated factors may be useful in enhancing the integration of synthetic and other natural biomaterials by polarizing macrophage activation toward an M2 regulatory phenotype

Monitoring the initial pulmonary absorption of two different beclomethasone dipropionate aerosols employing a human lung reperfusion model

BACKGROUND: The pulmonary residence time of inhaled glucocorticoids as well as their rate and extend of absorption into systemic circulation are important facets of their efficacy-safety profile. We evaluated a novel approach to elucidate the pulmonary absorption of an inhaled glucocorticoid. Our objective was to monitor and compare the combined process of drug particle dissolution, pro-drug activation and time course of initial distribution from human lung tissue into plasma for two different glucocorticoid formulations. METHODS: We chose beclomethasone dipropionate (BDP) delivered by two different commercially available HFA-propelled metered dose inhalers (Sanasthmax(®)/Becloforte™ and Ventolair(®)/Qvar™). Initially we developed a simple dialysis model to assess the transfer of BDP and its active metabolite from human lung homogenate into human plasma. In a novel experimental setting we then administered the aerosols into the bronchus of an extracorporally ventilated and reperfused human lung lobe and monitored the concentrations of BDP and its metabolites in the reperfusion fluid. RESULTS: Unexpectedly, we observed differences between the two aerosol formulations Sanasthmax(®)/Becloforte™ and Ventolair(®)/Qvar™ in both the dialysis as well as in the human reperfusion model. The HFA-BDP formulated as Ventolair(®)/Qvar™ displayed a more rapid release from lung tissue compared to Sanasthmax(®)/Becloforte™. We succeeded to explain and illustrate the observed differences between the two aerosols with their unique particle topology and divergent dissolution behaviour in human bronchial fluid. CONCLUSION: We conclude that though the ultrafine particles of Ventolair(®)/Qvar™ are beneficial for high lung deposition, they also yield a less desired more rapid systemic drug delivery. While the differences between Sanasthmax(®)/Becloforte™ and Ventolair(®)/Qvar™ were obvious in both the dialysis and lung perfusion experiments, the latter allowed to record time courses of pro-drug activation and distribution that were more consistent with results of comparable clinical trials. Thus, the extracorporally reperfused and ventilated human lung is a highly valuable physiological model to explore the lung pharmacokinetics of inhaled drugs

Single and multiple dose pharmacokinetics of maritime pine bark extract (Pycnogenol) after oral administration to healthy volunteers

BACKGROUND: Since plant extracts are increasingly used as phytotherapeutics or dietary supplements information on bioavailability, bioefficacy and safety are warranted. We elucidated the plasma kinetics of genuine extract components and metabolites after single and multiple ingestion of the standardized maritime pine bark extract Pycnogenol (USP quality) by human volunteers. METHODS: Eleven volunteers received a single dose of 300 mg pine bark extract, five volunteers ingested 200 mg daily for five days to reach steady state concentrations. Plasma samples were obtained before and at defined time points after intake of the extract. Samples were analyzed by HPLC with ion-pair reagents and simultaneous UV and electrochemical detection. RESULTS: We quantified total plasma concentrations of catechin, caffeic acid, ferulic acid, taxifolin and the metabolite M1 (δ-(3,4-dihydroxy-phenyl)-γ-valerolactone). Additionally, we describe plasma time courses and steady state appearance of ten so far unknown compounds, U1 to U10. After single ingestion, compounds derived from the extract were rapidly absorbed and the majority of them were detectable over whole experimental period of 14 h. The analysis of steady state plasma samples revealed significant phase II metabolism. CONCLUSION: We present the first systematic pharmacokinetic analysis of compounds derived from maritime pine bark extract. Beyond the known constituents and metabolites we uncovered the plasma time courses of ten unknown compounds. In concert with our previous detection of anti-inflammatory bioefficacy of these plasma samples ex vivo we suggest that constituents and metabolites of Pycnogenol bear potential for disclosure of novel active principles

Smoking status and anti-inflammatory macrophages in bronchoalveolar lavage and induced sputum in COPD

<p>Abstract</p> <p>Background</p> <p>Macrophages have been implicated in the pathogenesis of COPD. M1 and M2 macrophages constitute subpopulations displaying pro- and anti-inflammatory properties. We hypothesized that smoking cessation affects macrophage heterogeneity in the lung of patients with COPD. Our aim was to study macrophage heterogeneity using the M2-marker CD163 and selected pro- and anti-inflammatory mediators in bronchoalveolar lavage (BAL) fluid and induced sputum from current smokers and ex-smokers with COPD.</p> <p>Methods</p> <p>114 COPD patients (72 current smokers; 42 ex-smokers, median smoking cessation 3.5 years) were studied cross-sectionally and underwent sputum induction (M/F 99/15, age 62 ± 8 [mean ± SD] years, 42 (31-55) [median (range)] packyears, post-bronchodilator FEV₁63 ± 9% predicted, no steroids past 6 months). BAL was collected from 71 patients. CD163⁺macrophages were quantified in BAL and sputum cytospins. Pro- and anti-inflammatory mediators were measured in BAL and sputum supernatants.</p> <p>Results</p> <p>Ex-smokers with COPD had a higher percentage, but lower number of CD163⁺macrophages in BAL than current smokers (83.5% and 68.0%, p = 0.04; 5.6 and 20.1 ×10⁴/ml, p = 0.001 respectively). The percentage CD163⁺M2 macrophages was higher in BAL compared to sputum (74.0% and 30.3%, p < 0.001). BAL M-CSF levels were higher in smokers than ex-smokers (571 pg/ml and 150 pg/ml, p = 0.001) and correlated with the number of CD163⁺BAL macrophages (Rs = 0.38, p = 0.003). No significant differences were found between smokers and ex-smokers in the levels of pro-inflammatory (IL-6 and IL-8), and anti-inflammatory (elafin, and Secretory Leukocyte Protease Inhibitor [SLPI]) mediators in BAL and sputum.</p> <p>Conclusions</p> <p>Our data suggest that smoking cessation partially changes the macrophage polarization <it>in vivo </it>in the periphery of the lung towards an anti-inflammatory phenotype, which is not accompanied by a decrease in inflammatory parameters.</p

Earthworms Use Odor Cues to Locate and Feed on Microorganisms in Soil

Earthworms are key components of temperate soil ecosystems but key aspects of their ecology remain unexamined. Here we elucidate the role of olfactory cues in earthworm attraction to food sources and document specific chemical cues that attract Eisenia fetida to the soil fungi Geotrichum candidum. Fungi and other microorganisms are major sources of volatile emissions in soil ecosystems as well as primary food sources for earthworms, suggesting the likelihood that earthworms might profitably use olfactory cues to guide foraging behavior. Moreover, previous studies have documented earthworm movement toward microbial food sources. But, the specific olfactory cues responsible for earthworm attraction have not previously been identified. Using olfactometer assays combined with chemical analyses (GC-MS), we documented the attraction of E. fetida individuals to filtrate derived from G. candidum colonies and to two individual compounds tested in isolation: ethyl pentanoate and ethyl hexanoate. Attraction at a distance was observed when barriers prevented the worms from reaching the target stimuli, confirming the role of volatile cues. These findings enhance our understanding of the mechanisms underlying key trophic interactions in soil ecosystems and have potential implications for the extraction and collection of earthworms in vermiculture and other applied activities

Cellular pharmacodynamic effects of Pycnogenol® in patients with severe osteoarthritis: a randomized controlled pilot study

Background: The standardized maritime pine bark extract (Pycnogenol$^{®}$) has previously shown symptom alleviating effects in patients suffering from moderate forms of knee osteoarthritis (OA). The cellular mechanisms for this positive impact are so far unknown. The purpose of the present randomized pilot controlled study was to span the knowledge gap between the reported clinical effects of Pycnogenol$^{®}$ and its in vivo mechanism of action in OA patients. Methods: Thirty three patients with severe OA scheduled for a knee arthroplasty either received 100 mg of Pycnogenol$^{®}$ twice daily or no treatment (control group) three weeks before surgery. Cartilage, synovial fluid and serum samples were collected during surgical intervention. Relative gene expression of cartilage homeostasis markers were analyzed in the patients' chondrocytes. Inflammatory and cartilage metabolism mediators were investigated in serum and synovial fluid samples. Results: The oral intake of Pycnogenol$^{®}$ downregulated the gene expression of various cartilage degradation markers in the patients' chondrocytes, the decrease of MMP3, MMP13 and the pro-inflammatory cytokine IL1B were statistically significant (p ≤ 0.05). Additionally, protein concentrations of ADAMTS-5 in serum were reduced significantly (p ≤ 0.05) after three weeks intake of the pine bark extract. Conclusions: This is the first report about positive cellular effects of a dietary supplement on key catabolic and inflammatory markers in patients with severe OA. The results provide a rational basis for understanding previously reported clinical effects of Pycnogenol$^{®}$ on symptom scores of patients suffering from OA

Interplacental uterine expression of genes involved in prostaglandin synthesis during canine pregnancy and at induced prepartum luteolysis/abortion

BACKGROUND: In the non-pregnant dog, ovarian cyclicity is independent of a uterine luteolysin. This is in contrast to pregnant animals where a prepartum increase of luteolytic PGF2alpha occurs, apparently originating in the pregnant uterus. Recently, the placenta as a source of prepartum prostaglandins (PGs) was investigated, indicating fetal trophoblast cells as the likely main source. However, the possible contribution of uterine interplacental tissues to the production of these hormones has not yet been thoroughly examined in the dog. METHODS: Several key factors involved in the production and/or actions of PGs were studied: cyclooxygenase 2 (COX2, PTGS2), PGF2alpha-synthase (PGFS/AKR1C3), PGE2-synthase (PGES), and the respective receptors FP (PTGFR), EP2 (PTGER2) and EP4 (PGTER4), 15-hydroxyprostaglandin dehydrogenase (HPGD), PG-transporter (PGT, SLCO2A1) and progesterone receptor. Their expression and localization patterns were assessed by Real Time PCR and immunohistology in the interplacental uterine sites from pregnant dogs during the pre-implantation period (days 8-12), post-implantation (days 18-25), mid-gestation (days 35-40) and during antigestagen-induced luteolysis/abortion. RESULTS: Whereas only low COX2 expression was observed in uterine samples at all the selected time points, expression of PGFS/AKR1C3 strongly increased post-implantation. A gradual increase in PGES-mRNA expression was noted towards mid-gestation. FP-mRNA expression decreased significantly with the progression of pregnancy until mid-gestation. This was associated with clearly detectable expression of HPGD, which did not change significantly over time. The expression of FP and EP2-mRNA decreased significantly over time while EP4-mRNA expression remained unaffected. The antigestagen-treatment led to a significant increase in expression of COX2, PGES, EP2 and PGT (SLCO2A1) mRNA. COX2 was localized predominantly in the myometrium. The expression of PGFS/AKR1C3, which was unchanged, was localized mostly to the surface luminal epithelium. The expression of EP4, PGT and HPGH did not change during treatment, they were co-localized with PGES and EP2 in all uterine compartments. CONCLUSIONS: The data clearly demonstrate the basic capability of the canine pregnant uterus to produce and respond to PGs and suggests their functions both as local regulatory factors involved in the establishment and maintenance of pregnancy, as well as potential contributors to the process of parturition, supporting the myometrial contractility associated with fetal expulsion