Cigarette smoke increases pro-inflammatory markers and inhibits osteogenic differentiation in experimental exposure model.

Tobacco smokers have slowed bone growth and regeneration and more frequent implant failures than non-smokers, but the effect of cigarette smoking on the host response to bone-dwelling biomaterials is poorly understood. Macrophages and mesenchymal stem cells (MSCs) are essential in the healing response after implant placement. This study examined the effects of an experimental model of cigarette smoke exposure using cigarette smoke extract (CSE) on bone architecture in vivo and differentiation and inflammatory cytokine production on clinically relevant microstructured surfaces in vitro. CSE was prepared by bubbling mainstream smoke from one research cigarette (3R4F) in 1 mL phosphate-buffered saline. For in vivo studies, bone morphometry was examined in femurs isolated from mice injected with diluted CSE for 25 days. For in vitro studies, osteogenic markers and interleukins were measured in human MSCs and murine macrophages cultured on rough or rough-hydrophilic titanium (Ti) surfaces in culture media ±â€¯CSE for seven days. In vivo, CSE exposure decreased in bone area, volume, and interconnectivity in a dose-dependent manner. In vitro, macrophages exposed to CSE increased production of pro-inflammatory cytokines, abolishing the increase in anti-inflammatory cytokines typically seen on rough-hydrophilic surfaces. MSCs exposed to CSE had lower mRNA expression of osteoblast differentiation markers, increased levels of pro-inflammatory mRNA, and reduced production of osteogenic proteins. Our results demonstrate that CSE decreases osteogenic differentiation and anti-inflammatory interleukin production while increasing pro-inflammatory interleukin production in macrophages and MSCs, suggesting that compounds in CSE strongly affect stem cell differentiation and may compromise bone formation following biomaterial placement. STATEMENT OF SIGNIFICANCE: The study of implantable materials' interaction with biological systems occurs nearly exclusively in healthy cell and animal models. However, 15% of the US population smokes cigarettes, which is known to modulate immune response and tissue regeneration. To explore this interaction, we created a method of capturing smoke compounds as CSE for in vivo and in vitro use. We found chronic injection into mice produced an osteoporotic, pro-inflammatory phenotype similar to direct smoke models. Furthermore, CSE attenuated osteogenic differentiation and promoted a pro-inflammatory profile in MSCs and macrophages, respectively, when cultured on titanium surfaces. These results demonstrate that this CSE model may be useful for predicting how chronic tobacco exposure may adversely affect the outcome of biomedical implants in pre-clinical models.

A double-blind, placebo-controlled trial of montelukast in adult atopic eczema.

BACKGROUND: Montelukast is an antagonist of cys-leukotriene receptors used mainly in the treatment of asthma- and seasonal-allergic rhinitis. Initial reports concerning the use of montelukast in atopic dermatitis (AD) have been encouraging, although not consistent. OBJECTIVES: We have undertaken a randomized, double-blind, parallel-group, placebo-controlled trial to investigate further the efficacy of montelukast in the treatment of atopic eczema. METHODS: Following a screening visit, subjects received placebo treatment for 2 weeks in a single-blind phase, followed after visit 2 by an 8-week, double-blind period of treatment with montelukast 10 mg daily or placebo. Subjects were patients aged 16-60 years under our care for treatment of AD of moderate severity, defined by a six-area, six-sign atopic dermatitis (SASSAD) score in the range 12-50. Response to treatment was assessed by investigators and by subjects using a seven-point scale, with response defined as marked improvement or better. In addition, the SASSAD score was used to monitor the severity of clinical signs. The proportion of skin involved was estimated and visual analogue scales were used to record the severity of pruritus and sleep disturbance. Topical corticosteroid usage was recorded using a five-point scale. Adverse events were recorded. RESULTS: Sixty subjects were recruited and 54 completed the study. The treatment groups were well matched for disease severity at baseline (SASSAD scores were 25 and 29 in the montelukast and placebo groups, respectively). There were no significant differences between the treatment groups in any of the parameters used to assess treatment response. The improvement in mean SASSAD score from baseline (visit 2) to the end of treatment was marginally superior in the placebo group, 1.41 points on montelukast vs. 1.76 on placebo, a difference of 0.35 (95% confidence interval -6.1 to 6.8). Adverse events were generally of a mild nature except for a brief septicaemic illness in one subject receiving montelukast. CONCLUSIONS: The data do not support previous reports of efficacy of montelukast in treatment of AD.

A study examining inter- and intrarater reliability of three scales for measuring severity of psoriasis: Psoriasis Area and Severity Index, Physician's Global Assessment and Lattice System Physician's Global Assessment.

BACKGROUND: There is a lack of consensus as to the best way of monitoring psoriasis severity in clinical trials. The Psoriasis Area and Severity Index (PASI) is the most frequently used system and the Physician's Global Assessment (PGA) is also often used. However, both instruments have some drawbacks and neither has been fully evaluated in terms of 'validity' and 'reliability' as a psoriasis rating scale. The Lattice System Physician's Global Assessment (LS-PGA) scale has recently been developed to address some disadvantages of the PASI and PGA. OBJECTIVES: To evaluate the inter-rater and intrarater reliability of the PASI, PGA and LS-PGA. METHODS: On the day before the study, 14 dermatologists (raters), with varied experience of assessing psoriasis, received detailed training (2.5 h) on use of the scales. On the study day, each rater evaluated 16 adults with chronic plaque psoriasis in the morning and again in the afternoon. Raters were randomly assigned to assess subjects using the scales in a specific sequence, either PGA, LS-PGA, PASI or PGA, PASI, LS-PGA. Each rater used one sequence in the morning and the other in the afternoon. The primary endpoint was the inter-rater and intrarater reliability as determined by intraclass correlation coefficients (ICCs). RESULTS: All three scales demonstrated 'substantial' (a priori defined as ICC > 80%) intrarater reliability. The inter-rater reliability for each of the PASI and LS-PGA was also 'substantial' and for the PGA was 'moderate' (ICC 75%). CONCLUSIONS: Each one of the three scales provided reproducible psoriasis severity assessments. In terms of both intrarater and inter-rater reliability values, the three scales can be ranked from highest to lowest as follows: PASI, LS-PGA and PGA.

Efficacy and tolerability of borage oil in adults and children with atopic eczema: randomised, double blind, placebo controlled, parallel group trial.

OBJECTIVE: To study the efficacy and tolerability of borage oil, which contains a high concentration of gamma linolenic acid, in children and adults with atopic eczema. DESIGN: Single centre, randomised, double blind, placebo controlled, parallel group trial. SETTING: Acute district general hospital in Nuneaton, England. PARTICIPANTS: 151 patients, of whom 11 failed to return for assessment, leaving an evaluable population of 140 (including 69 children). INTERVENTION: Adults received four capsules of borage oil twice daily (920 mg gamma linolenic acid), and children received two capsules twice daily, for 12 weeks. MAIN OUTCOME MEASURES: Change in total sign score at 12 weeks measured with the six area, six sign, atopic dermatitis (SASSAD) score (primary endpoint); symptom scores, assessed on visual analogue scales; topical corticosteroid requirement, assessed on a five point scale; global assessment of response by participants; adverse events and tolerability. RESULTS: The mean SASSAD score fell from 30 to 27 in the borage oil group and from 28 to 23 in the placebo group. The difference between the mean improvements in the two groups was 1.4 (95% confidence interval -2.2 to 5.0) points in favour of placebo (P = 0.45). No significant differences occurred between treatment groups in the other assessments. Subset analysis of adults and children did not indicate any difference in response. The treatments were well tolerated. CONCLUSION: Gamma linolenic acid is not beneficial in atopic dermatitis.

Azathioprine in severe adult atopic dermatitis: a double-blind, placebo-controlled, crossover trial.

BACKGROUND: There is a limited range of treatments for severe atopic dermatitis (AD). Azathioprine has often been used but there has been no randomized controlled trial of this drug to confirm its efficacy in AD. OBJECTIVES: To establish or refute the efficacy of azathioprine in severe AD. To investigate the safety and tolerability of azathioprine in this patient population. METHODS: We performed a double-blind, randomized, placebo-controlled, crossover trial of azathioprine in adult patients with severe AD. Each treatment period was of 3 months' duration. Treatments were azathioprine 2.5 mg kg(-1) day(-1) and matched placebo. Disease activity was monitored using the SASSAD sign score. In addition, severity of pruritus, sleep disturbance and disruption of work/daytime activity were monitored using visual analogue scales. Adverse events were recorded and haematological and biochemical monitoring was performed. RESULTS: Thirty-seven subjects were enrolled, mean age 38 years (range 17-73). Sixteen were withdrawn, 12 during azathioprine treatment and four during placebo treatment. The SASSAD score fell by 26% during treatment with azathioprine vs. 3% on placebo (P < 0.01). Pruritus, sleep disturbance and disruption of work/daytime activity all improved significantly on active treatment but not on placebo. The difference in mean improvement between azathioprine and placebo was significant for disruption of work/daytime activity (P < 0.02) but not for pruritus or sleep disturbance. Gastrointestinal disturbances were reported by 14 patients during azathioprine treatment and four were withdrawn as a result of severe nausea and vomiting. Leukopenia was observed in two patients and deranged liver enzymes in eight during treatment with azathioprine. CONCLUSIONS: Azathioprine is an effective and useful drug in severe AD although it is not always well-tolerated. Monitoring of the full blood count and liver enzymes is advisable during treatment.

Novel 6-substituted uracil analogs as inhibitors of the angiogenic actions of thymidine phosphorylase.

Thymidine phosphorylase (TP) catalyzes the reversible phosphorolysis of thymidine and other pyrimidine 2'-deoxyribonucleosides. In addition, TP has been shown to possess angiogenic activity in a number of in vitro and in vivo assays, and its angiogenic activity has been linked to its catalytic activity. A series of 5- and 6-substituted uracil derivatives were synthesized and evaluated for their abilities to inhibit TP activity. Among the most active compounds was a 6-amino-substituted uracil analog, 6-(2-aminoethyl)amino-5-chlorouracil (AEAC), which was a competitive inhibitor with a K(i) of 165 nM. The inhibitory activity of AEAC was selective for TP, as it did not inhibit purine nucleoside phosphorylase or uridine phosphorylase at concentrations up to 1 mM. Human recombinant TP induced human umbilical vein endothelial cell (HUVEC) migration in a modified Boyden chamber assay in vitro, and this action could be abrogated by the TP inhibitors. The actions of the inhibitors were specific for TP, as they had no effect on the chemotactic actions of vascular endothelial growth factor (VEGF). HUVEC migration was also induced when TP-transfected human colon and breast carcinoma cells were co-cultured in the Boyden chamber assay in place of the purified angiogenic factors, and a TP inhibitor blocked the tumor cell-mediated migration almost completely. These studies suggest that inhibitors of TP may be useful in pathological conditions that are dependent upon TP-driven angiogenesis.

Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane.

Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1balpha presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by approximately 50% and PDI and GP1balpha were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1balpha. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1balpha on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1balpha that resulted in exposure of a free thiol(s).

Exposure of the cryptic Arg-Gly-Asp sequence in thrombospondin-1 by protein disulfide isomerase.

Thrombospondin-1 is a matrix protein that inhibits proliferation, motility and sprouting of endothelial cells in vitro and angiogenesis in vivo. One mechanism by which thrombospondin-1 may influence endothelial cell biology is through interaction with the endothelial cell alphav beta3 integrin receptor. This interaction is mediated via a cryptic Arg-Gly-Asp sequence in the C-terminal Ca2+-binding region of thrombospondin-1. Exposure of the Arg-Gly-Asp sequence is controlled by disulfide interchange events in the Ca2+-binding loops and C-globular domain. Limited reduction of thrombospondin-1 by dithiothreitol exposes the Arg-Gly-Asp sequence which can bind to the alphav beta3 integrin receptor and support endothelial cell spreading (X. Sun, K. Skorstengaard, D.F. Mosher, J. Cell Biol. 118 (1992) 693-701). Our aim was to identify possible physiological reductants that can mediate Arg-Gly-Asp exposure. We now report that protein disulfide isomerase, which is known to catalyze disulfide interchange in thrombospondin-1 and change its enzyme inhibitory properties and its binding to monoclonal antibodies, was secreted by bovine aortic endothelial cells and deposited on the cell surface. There was an average of approximately 2.2 fg of protein disulfide isomerase on the surface of a bovine aortic endothelial cell. Treatment of thrombospondin-1 with purified protein disulfide isomerase enhanced adhesion of endothelial cells to thrombospondin-1 in an Arg-Gly-Asp-dependent manner through the alphav beta3 integrin receptor and supported cell spreading. Both Ca2+-depleted and Ca2+-replete thrombospondin-1 were substrates for protein disulfide isomerase. These results suggest that endothelial cell derived protein disulfide isomerase may regulate Arg-Gly-Asp-dependent binding of thrombospondin-1.

Interaction of platelet-derived growth factor with thrombospondin 1.

Key factors that mediate vascular smooth muscle cell proliferation and migration are platelet-derived growth factor (PDGF) and thrombospondin 1 (TSP1). We now report that PDGFBB bound tightly and specifically to TSP1, that this interaction was markedly dependent on the disulphide bond arrangement in TSP1, and that binding of PDGFBB to TSP1 did not preclude PDGFBB from binding to its receptor on rat aortic vascular smooth-muscle cells. At physiologic ionic strength and pH, PDGFBB bound to Ca2+-depleted TSP1 with a dissociation constant of 11 +/- 2 nM and to Ca2+-replete TSP1 with a dissociation constant of 32 +/- 5 nM. Binding was specific, as both soluble TSP1 and unlabelled PDGFBB competed for binding of iodinated PDGFBB to immobilized TSP1, whereas other platelet alpha-granule proteins did not compete. The tertiary structure of TSP1 is regulated by intramolecular disulphide interchange; we found that catalysis of disulphide interchange in TSP1 by protein disulphide isomerase ablated the binding of PDGFBB. The interaction of PDGFBB with TSP1 was weakened by increasing salt concentration and essentially ablated at 0.65 ionic strength; it was inhibited by heparin with a half-maximal effect at 20 i.u./ml, implying that the binding was mediated largely by ionic interactions. An anti TSP1 monoclonal antibody decreased the binding of iodinated PDGFBB to PDGF receptor on rat aortic vascular smooth-muscle cells by 37 +/- 2%, whereas platelet TSP1 non-competitively inhibited binding of iodinated PDGFBB. Uncomplexed PDGFBB bound to PDGF receptor with an affinity 5 +/- 2 times that of PDGFBB-TSP1 complexes. These results suggest that TSP1 might assist in the targeting of PDGF to its receptor on vascular smooth-muscle cells.

Newborn cerebrovascular responses after first trimester moderate maternal ethanol exposure in sheep.

Fetal alcohol syndrome is one of the leading causes of mental retardation in the United States, but the pathogenesis of the associated brain damage is unknown. We tested the hypothesis that neonatal cerebrovascular responses to CO2 and/or hypoxia may be altered by moderate chronic maternal ethanol exposure early in gestation. We studied 26 newborn lambs (1-4 d old). Their mothers had received daily i.v. infusions of either ethanol (1 g/kg; ethanol concentration = 167 +/- 3 mg/dL; mean +/- SEM) or a similar volume of saline for 3 wk during the first trimester. In nine lambs, we studied cerebral responses to CO2 (saline, n = 4; ethanol, n = 5) and in 17 lambs, cerebral responses to hypoxia (saline, n = 7; ethanol, n = 10). Cerebrovascular responses to CO2 were not different between the groups. However, the cerebral vasodilatory response to hypoxemia was significantly attenuated in the ethanol lambs, such that cerebral O2 delivery was not maintained. During severe hypoxia (arterial PO2 = 30 mm Hg), cerebral blood flow increased 106 +/- 23% (mean +/- SEM) above baseline in the saline-treated group, but increased only 32 +/- 15% above baseline in the ethanol-treated group (p < 0.02). Similarly, cerebrovascular resistance in the saline group decreased 52 +/- 6% from baseline, but decreased only 16 +/- 11% in the ethanol group (p < 0.02). We conclude that moderate maternal ethanol infusion early in pregnancy attenuates neonatal hypoxic, but not CO2, cerebrovascular responsivity.

Catalysis of disulfide isomerization in thrombospondin 1 by protein disulfide isomerase.

Thrombospondin 1 is a multidomain glycoprotein from platelets and most cells that participates in diverse biological processes. The structure and some functional properties of thrombospondin 1 are regulated by disulfide interchange in the Ca(2+)-binding repeats and C-globular domain. The recent identification of the enzyme, protein disulfide isomerase, on the platelet surface suggested that protein disulfide isomerase may catalyze disulfide isomerization in platelet thrombospondin 1. Protein disulfide isomerase was found to form disulfide-linked complexes with thrombospondin 1, which is consistent with protein disulfide isomerase-mediated rearrangement of disulfide bonds in thrombospondin 1. To quantitate disulfide interchange in thrombospondin 1, perturbation of the enzyme inhibitory properties of platelet thrombospondin 1 were measured, specifically changes in the apparent dissociation constant for inhibition of neutrophil cathepsin G by thrombospondin 1. The inhibition constant increased > or = 10-14-fold following incubation of either Ca(2+)-replete or Ca(2+)-depleted thrombospondin 1 with protein disulfide isomerase and reduced glutathione. The rate of protein disulfide isomerase-catalyzed disulfide interchange in thrombospondin 1 increased linearly with protein disulfide isomerase concentration and the K(m) for reduced glutathione was 0.4 +/- 0.2 mM. Disulfide isomerization in both platelet and fibroblast thrombospondin 1 was probed by measuring perturbation in epitopes for two anti-thrombospondin 1 monoclonal antibodies. Antibody D4.6 binds to the C-terminal Ca(2+)-binding domains which are involved in disulfide interchange, whereas antibody HB8432 binds toward the N-terminus of the thrombospondin 1 subunit. In accordance with the location of these epitopes, incubation of platelet thrombospondin 1 or fibroblast thrombospondin 1 with protein disulfide isomerase and reduced glutathione resulted in 2-fold enhancement of binding of D4.6, whereas binding of HB8432 did not significantly change. In summary, protein disulfide isomerase catalyzes disulfide interchange in thrombospondin 1 which alters binding of neutrophil cathepsin G and antibody D4.6 to thrombospondin 1.

Inhibition of heparin activity in plasma by soluble fibrin: evidence for ternary thrombin-fibrin-heparin complex formation.

The ability of heparin to dramatically enhance the inactivation of thrombin (IIa) by antithrombin III (ATIII) in buffer is negated through formation of a IIa-fibrin-heparin ternary complex (Hogg and Jackson, Proc Natl Acad Sci USA 86:3619, 1989; Hogg and Jackson, J Biol Chem 265:241, 1990). IIa, in this ternary complex, is protected from inactivation by ATIII. Our aim was to determine whether fibrin also compromises heparin efficacy in plasma. We found that soluble fibrin ablated the heparin-mediated prolongation of the thrombin time with half-maximal effect at 60 nmol/L fibrin. The heparin-mediated prolongation of the activated partial thromboplastin time (APTT) was also reduced by fibrin with half-maximal effects at 140 nmol/L fibrin using 0.12 U/mL heparin and 500 nmol/L fibrin using 0.25 U/mL heparin. The mechanism of inhibition of heparin activity by fibrin in plasma was determined by measuring IIa-ATIII complexes by enzyme-linked immunosorbent assay (ELISA). Fibrin was found to inhibit the heparin-catalyzed inactivation of IIa by ATIII with half-maximal effect at 97 +/- 19 nmol/L fibrin. Fibrin had no effect on the heparin-catalyzed inactivation of factor Xa by ATIII in plasma, using either standard heparin, a heparinoid preparation (Orgaran; Organon, Lane Cove, Sydney, Australia), or low-molecular weight heparin. These findings imply that fibrin is a potent modulator of heparin activity in vivo by inhibiting heparin-catalyzed IIa-ATIII complex formation through formation of ternary IIa-fibrin-heparin complexes.

Cerebral responses to acute maternal alcohol intoxication in immature fetal sheep.

Previous studies in mature fetal sheep have shown that alcohol depresses cerebral blood flow (CBF), cerebral O2 consumption (CMRO2), and cerebral glucose consumption (CMRglu). This effect earlier in gestation might contribute to the pathogenesis of fetal alcohol syndrome. Physiologic studies of immature fetal sheep have demonstrated lower CBF, CMRO2, and CMRglu as well as a blunted vasodilatory response to hypoxia compared with mature fetal sheep. The purpose of this study was to determine whether immature fetal responses to alcohol are blunted compared with near-term fetal responses. We studied seven immature fetal sheep in utero at 92 +/- 1 d gestation (term = 147 d) 2 d after placement of vascular catheters. Pure ethanol (1 g/kg) was infused i.v. to the mother over 1 h. We measured CBF and myocardial blood flow by radioactive microspheres and calculated CMRO2 and CMRglu using arterial and sagittal sinus O2 and glucose concentrations. At a fetal ethanol concentration of 33 + 8 mmol/L (150 +/- 37 mg/dL), there were no significant changes in CBF, CMRO2, or CMRglu. There was mild hypoglycemia (glucose concentration = 1.05 +/- 0.2 versus 1.33 +/- 0.2 mM baseline) and lactic acidemia (lactate concentration = 1.29 +/- 0.3 versus 1.07 +/- 0.2 mM baseline). Cardiovascular variables were unchanged as was myocardial blood flow. The immature fetal sheep brain shows no significant cerebrovascular and metabolic response to acute alcohol intoxication compared with mature fetal sheep. Mild hypoglycemia and lactic acidemia did develop. The reason for the developmental differences in response to alcohol and their relationship to fetal alcohol syndrome remain to be elucidated.