Plasma vitamin D levels and inflammation in the aortic wall of patients with coronary artery disease with and without inflammatory rheumatic disease.

OBJECTIVES: Vitamin D modulates inflammation, and this may explain the observed associations between vitamin D status and disorders driven by systemic inflammation, such as coronary artery disease (CAD) and inflammatory rheumatic diseases (IRDs). The aims of this study were to assess vitamin D status in patients with CAD alone and in patients with CAD and IRD, and to explore potential associations between vitamin D status and the presence of mononuclear cell infiltrates (MCIs) in the aortic adventitia of these patients. METHOD: Plasma levels of 25-hydroxyvitamin D3 [(25(OH)D3] were determined by radioimmunoassay and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by enzyme immunoassay in the 121 patients from the Feiring Heart Biopsy Study (FHBS) who had available histology data on adventitial MCIs; 53 of these had CAD alone and 68 had CAD and IRD. RESULTS: In the crude analysis, vitamin D levels were similar in CAD patients with and without IRD. After adjustment for potential confounders, IRD was associated with an increase of 8.8 nmol/L [95% confidence interval (CI) 1.0-16.6; p = 0.027] in 25(OH)D3 and an increase of 18.8 pmol/L (95% CI 4.3-33.3; p = 0.012) in 1,25(OH)2D3, while MCIs in the aortic adventitia were associated with lower levels of 1,25(OH)2D3 (ß = -18.8, 95% CI -33.6 to -4.0; p = 0.014). CONCLUSIONS: IRD was associated with higher levels of both 25(OH)D3 and 1,25(OH)2D3. These findings argue against the hypothesis that patients with high systemic inflammatory burden (CAD+IRD) should have lower vitamin D levels than those with less inflammation (CAD only). Of note, when controlled for potential confounders, low 1,25(OH)2D3 levels were associated with adventitial aortic inflammation.

Cytokine Levels After Consumption of a Medicinal Agaricus blazei Murill-Based Mushroom Extract, AndoSan™ , in Patients with Crohn's Disease and Ulcerative Colitis in a Randomized Single-Blinded Placebo-Controlled Study.

Ingestion of the Agaricus blazei Murill-based mushroom extract AndoSan™ has been shown in randomized placebo-controlled studies to improve symptoms in Crohn's disease (CD) and ulcerative colitis (UC) and also fatigue and quality of life in the latter patients. The aim was to examine whether this clinical impact of AndoSan™ intake could be explained by influence on foremost pro-inflammatory cytokines in the patients. Fifty patients with symptomatic UC and CD were randomized and blinded for oral daily intake of AndoSan™ or placebo. Blood samples taken before (visit 1) and after 21 days' (visit 3) consumption were analysed for cytokines IL-1ß, IL-2, IL-4-8, IL-10, IL-12-13, IL-17, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1ß and TNF-α. Baseline cytokine levels were similar in CD and UC. In CD, cytokine levels at visit 1 versus visit 3 were unaltered within the AndoSan™ and the placebo groups. Only IL-2 was significantly reduced at visit 3 in the Andosan™ compared with the placebo group. However, when combining IL-1ß, IL-6 and G-CSF in the patients with CD, the cytokine levels were significantly lower in the AndoSanTM - versus the placebo group, visit 3. In UC, levels of IL-2, IL-5 and MIP-1ß were reduced within the AndoSan™ group. IL-5 was also reduced at visit 3 compared with placebo. Generally, the effect on reduction in systemic cytokine levels by consumption of AndoSan™ was limited and supported only marginally anti-inflammatory effects in these patients. Therefore, other explanations behind the clinical anti-inflammatory effects than the contribution of cytokines seem more pertinent, including anti-allergic and antioxidant activities.

Effects of serum-free storage on morphology, phenotype, and viability of ex vivo cultured human conjunctival epithelium.

The use of amniotic membrane (AM) represents one of the major developments in ocular surface reconstruction. However, in a study on patients with primary pterygium, transplantation of AM with ex vivo expanded human conjunctival epithelial cells (HCjE) promoted earlier epithelialization than AM alone. We previously showed that cultured human limbal epithelial cells maintain their morphology, phenotype, and viability for one week when stored at 23°C. The current study investigates the feasibility of storing HCjE in HEPES-MEM and Optisol-GS at 23°C for 4 and 7 days, respectively. The five experimental groups were analyzed by light microscopy, immunohistochemistry, transmission electron microscopy, and a viability assay. The ultrastructural integrity of cultured HCjE was well preserved following 4 days of storage, however, 7 days of storage resulted in some loss of cell-cell contacts and epithelial detachment from the amniotic membrane. The number of microvilli in cultured HCjE not subjected to storage was 2.03±0.38 microvilli/µm. In comparison, after 4 and 7 days of HEPES-MEM storage this number was 1.69±0.54 microvilli/µm; P=0.98 and 0.89±1.0 microvilli/µm; P=0.28, respectively. After Optisol-GS storage for 4 and 7 days, the mean number of microvilli was 1.07±1.0 microvilli/µm; P=0.47 and 0.07±0.07 microvilli/µm; P=0.03, respectively. The number of cell layers in cultured HCjE not subjected to storage was 4.4±0.3 cell layers, as opposed to 4.0±0.9 cell layers; P=0.89 after 4 days of HEPES-MEM storage and 2.8±0.6 cell layers; P=0.01 after 7 days of storage in HEPES-MEM. The number of cell layers after 4 and 7 days of storage in Optisol-GS was 3.7±0.2 cell layers; P=0.46 and 3.4±0.4 cell layers; P=0.18, respectively. The expression of markers for undifferentiated cells (ΔNp63α, ABCG2 and p63), proliferating cells (Ki67 and PCNA), goblet cells (Ck7 and MUC5AC), stratified squamous epithelial cells (Ck4), and apoptotic cells (caspase-3) in cultured HCjE appeared to be unchanged after 4 and 7 days of HEPES-MEM and Optisol-GS storage. The percentage of viable cells in cultured HCjE not subjected to storage (91.4%±3.2%) was sustained after 4 and 7 days of storage in HEPES-MEM (94.1%±4.5%; P=0.99 and 85.1%±13.7%; P=0.87, respectively) as well as after 4 and 7 days of storage in Optisol-GS (87.7%±15.2%; P=0.97 and 79.8%±15.7%; P=0.48, respectively). We conclude that cultured HCjE may be stored for at least 4 days in serum-free conditions at 23°C while maintaining the phenotype and viability. HEPES-MEM appears to be comparable to Optisol-GS for serum-free storage with preservation of the ultrastructure for at least 4 days.

Effect of an extract based on the medicinal mushroom Agaricus blazei Murill on expression of cytokines and calprotectin in patients with ulcerative colitis and Crohn's disease.

An immunomodulatory extract (AndoSan™) based on the medicinal mushroom Agaricus blazei Murill (AbM) has shown to reduce blood cytokine levels in healthy volunteers after 12 days' ingestion, pointing to an anti-inflammatory effect. The aim was to study whether AndoSan™ had similar effects on cytokines in patients with ulcerative colitis (UC) and Crohn's disease (CD). Calprotectin, a marker for inflammatory bowel disease (IBD), was also measured. Patients with CD (n = 11) and with UC (n = 10) consumed 60 ml/day of AndoSan™. Patient blood plasma was harvested before and after 6 h LPS (1 ng/ml) stimulation ex vivo. Plasma and faecal calprotectin levels were analysed using ELISA and 17 cytokines [IL-2, IFN-γ, IL-12 (Th1), IL-4, IL-5, IL-13 (Th2), IL-7, IL-17, IL-1ß, IL-6, TNF-α, IL-8, MIP-1ß, MCP-1, G-CSF, GM-CSF and IL-10] by multiplex assay. After 12 days' ingestion of AndoSan™, baseline plasma cytokine levels in UC was reduced for MCP-1 (40%) and in LPS-stimulated blood for MIP-1ß (78%), IL-6 (44%), IL-1ß (41%), IL-8 (30%), G-CSF (29%), MCP-1 (18%) and GM-CSF (17%). There were corresponding reductions in CD: IL-2 (100%), IL-17 (55%) and IL-8 (29%) and for IL-1ß (35%), MIP-1ß (30%), MCP-1 (22%), IL-8 (18%), IL-17 (17%) and G-CSF (14%), respectively. Baseline concentrations for the 17 cytokines in the UC and CD patient groups were largely similar. Faecal calprotectin was reduced in the UC group. Ingestion of an AbM-based medicinal mushroom by patients with IBD resulted in interesting anti-inflammatory effects as demonstrated by declined levels of pathogenic cytokines in blood and calprotectin in faeces.

An extract based on the medicinal mushroom Agaricus blazei Murill stimulates monocyte-derived dendritic cells to cytokine and chemokine production in vitro.

The edible mushroom Agaricus blazei Murill (AbM), which has been used in traditional medicine against a range of diseases and possess immunomodulating properties, probably due to its high content of beta-glucans. Others and we have demonstrated stimulatory effects of extracts of this mushroom on different immune cells. Dendritic cells are major directors of immune function. We wanted to examine the effect of AbM stimulation on signal substance release from monocyte-derived dendritic cells (MDDC). After 6d incubation with IL-4 and GM-CSF, the cells were true MDDC. Then the cells were further incubated with up to 10% of the AbM-based extract, AndoSan, LPS (0.5 microg/ml) or PBS control. We found that the AbM extract promoted dose-dependent increased levels of IL-8, G-CSF, TNFalpha, IL-1beta, IL-6 and MIP-1beta, in that order. The synthesis of IL-2, IL-8 and IFNgamma were similar for the AbM extract and LPS. However, AndoSan induced a 10- to 2-fold higher production than did LPS of G-CSF, TNFalpha and IL-1beta, respectively. AbM did not induce increased synthesis of Th2 or anti-inflammatory cytokines or the Th1 cytokine IL-12. We conclude that stimulation of MDDC with an AbM-based extract resulted in increased production of proinflammatory, chemotactic and some Th1-type cytokines in vitro.

Kinetics of local tissue and regional lymph node IL-2 and IFN-gamma responses in experimental oral mucosa and skin contact sensitivity in mice.

Using ELISA, we have quantified the levels of IL-2 and IFN-gamma in the oral mucosa, ear skin and regional and distant lymph nodes in an experimental murine model of contact sensitivity (CS), induced by the hapten oxazolone (OXA). Compared to normal conditions, the levels of IL-2 peaked early (4-6 h) after hapten exposure in the hapten-exposed tissues analysed both during the first hapten exposure (sensitization) and the second (elicitation) phase, thereafter quickly to subside. The oral mucosa displayed maximal 24-fold increase in IL-2 levels after sensitization and 39-fold increase after elicitation. Respective figures for ear skin were x27 and x35 and for regional lymph nodes x8 and x9, respectively. The distant lymph nodes displayed only minor cytokine increases at any time. IFN-gamma-levels did not increase after sensitization with OXA. An increase in IFN-gamma was seen after the second exposure, peaking at 8-24 h, thereafter quickly subsiding. The oral mucosa IFN-gamma increased x14 after elicitation, the ear skin x8 and regional lymph nodes x37. The weight of the four regional lymph nodes increased from 10 to 38 mg, and the total number of cells in these lymph nodes was increased x11, peaking 48 h after the elicitation. We conclude that in CS reactions, tissue levels of IL-2 increased in buccal mucosa, ear skin and in regional lymph nodes after hapten exposure and re-exposure, IFN-gamma appeared only after re-exposure to the hapten. The increased weight of the regional lymph nodes was mainly attributed to cell proliferation. The common ectodermal origin and the similarity of the CS reactions on skin and in buccal mucosa indicate that these tissues share common immunological patterns of Th1 cell reactivity, at least in dealing with haptens like OXA.

Effect of an extract based on the medicinal mushroom Agaricus blazei murill on release of cytokines, chemokines and leukocyte growth factors in human blood ex vivo and in vivo.

An immunostimulatory extract based on the medicinal mushroom Agaricus blazei Murill (AbM) has been shown to stimulate mononuclear phagocytes in vitro to produce pro-inflammatory cytokines, and to protect against lethal peritonitis in mice. The present aim was to study the effect of AbM on release of several cytokines in human whole blood both after stimulation ex vivo and in vivo after oral intake over several days in healthy volunteers. The 17 signal substances examined were; T helper 1 (Th1) cytokines [interleukin (IL)-2, interferon (IFN)-gamma and IL-12], T helper 2 cytokines (IL-4, IL-5 and IL-13), pleiotropic (IL-7, IL-17), pro-inflammatory [IL-1beta, IL-6, tumour necrosis factor (TNF)-alpha (mainly produced by Th1 cells)]--and anti-inflammatory (IL-10) cytokines, chemokines [IL-8, macrophage inhibitory protein (MIP)-1beta and monocyte chemoattractant protein (MCP)-1] and leukocyte growth factors [granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor]. After stimulation of whole blood ex vivo with 0.5-5.0% of a mushroom extract, AndoSan mainly containing AbM, there was a dose-dependent increase in all the cytokines studied, ranging from two to 399-fold (TNF-alpha). However, in vivo in the eight volunteers who completed the daily intake (60 ml) of this AbM extract for 12 days, a significant reduction was observed in levels of IL-1beta (97%), TNF-alpha (84%), IL-17 (50%) and IL-2 (46%). Although not significant, there was a trend towards reduced levels for IL-8, IFN-gamma and G-CSF, whilst those of the remaining nine cytokines tested, were unaltered. The discrepant results on cytokine release ex vivo and in vivo may partly be explained by the antioxidant activity of AbM in vivo and limited absorption of its large, complex and bioactive beta-glucans across the intestinal mucosa to the reticuloendothelial system and blood.

Blood leucocyte cytokine production after LPS stimulation at different concentrations of glucose and/or insulin.

BACKGROUND: Previous studies have indicated that alterations in blood glucose and/or insulin levels modify the inflammatory response. The purpose of this study was to elucidate whether increased levels of glucose and/or insulin influence the activation pattern of blood leucocytes and their production of cytokines in vitro. METHODS: Venous blood was obtained from eight healthy male volunteers after an overnight fast. Glucose and/or insulin were added to aliquots of whole blood to increase the blood glucose concentration by 5 or 20 mmol/l and/or the insulin concentration by 6 or 30 nmol/l, respectively, before stimulation with E. coli lipopolysaccharide (LPS) at concentrations of 10, 100 or 1000 ng/ml. The samples were subsequently incubated at 37 degrees C for 6 h before cytokine measurements. After centrifugation the levels of interleukins (IL)-1beta, IL-6, IL-8, IL-10 and tumour necrosis factor (TNF)-alpha were measured in plasma using enzyme-linked immuno-sorbent assays. The results were compared with cytokine levels in parallel control samples to which only identical amounts of LPS were added. RESULTS: The LPS-stimulated production of IL-1beta was significantly reduced by on average 26% in samples to which glucose 20 mmol/l was added; addition of insulin and/or glucose 5 mmol/l had no apparent effect on the IL-1beta production at any LPS concentration. The levels of IL-6, IL-8, IL-10 and TNF-alpha were not manifestly altered by addition of glucose and/or insulin at any LPS concentration. CONCLUSION: A substantial increase in blood glucose concentration changed the IL-1beta production, but not the production of other cytokines, in response to LPS stimulation.

The early effects of intramedullary reaming of the femur on bone mineral density; an experimental study in pigs.

BACKGROUND AND AIMS: Both fracture and fracture treatment affect bone mineral density (BMD). BMD after standard intramedullary reaming of the femoral cavity and after reaming with a reamer-irrigator-aspirator (RIA) system were studied with the hypothesis that the RIA technique would lead to lower BMD levels. MATERIAL AND METHODS: Dual-energy X-ray absorptiometry (DXA) was performed on the third day after operation with standard intramedullary nailing technique (n = 6) or RIA technique (n = 7) in intact femora of young Norwegian landrace pigs. RESULTS AND CONCLUSION: Significantly lower BMD were found in the mid-shaft and total femur after reaming with the RIA technique compared to the non-operated femur. Traditional reaming technique resulted in significantly higher BMD in the distal -femur. INTERPRETATION: The results of this study indicate that standard reaming increased BMD in the distal femur, suggesting compressive effects on trabecular bone. The RIA technique decreased BMD in the femoral diaphysis and total femur, suggesting removal of trabecular bone. A possible clinical impact of the findings remains to be investigated.

Effects of explant size on epithelial outgrowth, thickness, stratification, ultrastructure and phenotype of cultured limbal epithelial cells.

PURPOSE: Transplantation of limbal stem cells is a promising therapy for limbal stem cell deficiency. Limbal cells can be harvested from either a healthy part of the patient's eye or the eye of a donor. Small explants are less likely to inflict injury to the donor site. We investigated the effects of limbal explant size on multiple characteristics known to be important for transplant function. METHODS: Human limbal epithelial cells were expanded from large versus small explants (3 versus 1 mm of the corneal circumference) for 3 weeks and characterized by light microscopy, immunohistochemistry, and transmission electron microscopy. Epithelial thickness, stratification, outgrowth, ultrastructure and phenotype were assessed. RESULTS: Epithelial thickness and stratification were similar between the groups. Outgrowth size correlated positively with explant size (r = 0.37; P = 0.01), whereas fold growth correlated negatively with explant size (r = -0.55; P < 0.0001). Percentage of cells expressing the limbal epithelial cell marker K19 was higher in cells derived from large explants (99.1±1.2%) compared to cells derived from small explants (93.2±13.6%, P = 0.024). The percentage of cells expressing ABCG2, integrin ß1, p63, and p63α that are markers suggestive of an immature phenotype; Keratin 3, Connexin 43, and E-Cadherin that are markers of differentiation; and Ki67 and PCNA that indicate cell proliferation were equal in both groups. Desmosome and hemidesmosome densities were equal between the groups. CONCLUSION: For donor- and culture conditions used in the present study, large explants are preferable to small in terms of outgrowth area. As regards limbal epithelial cell thickness, stratification, mechanical strength, and the attainment of a predominantly immature phenotype, both large and small explants are sufficient.

Effects of the medicinal mushroom Agaricus blazei Murill on immunity, infection and cancer.

Agaricus blazei Murill (AbM) is an edible, medicinal mushroom of Brazilian origin. It is used traditionally against a range of diseases, including cancer and chronic hepatitis, and has been cultivated commercially for the health food market. AbM has recently been shown to have strong immunomodulating properties, which has led to increasing scientific interest. In this article, we review current knowledge as to the immunological properties of AbM, and its possible clinical use in connection with infections and cancer. We also present some novel findings, which point to highly different biological potency between AbM extracts of different source and manufacturing.

Effect of Transportation on Cultured Limbal Epithelial Sheets for Worldwide Treatment of Limbal Stem Cell Deficiency.

Limbal stem cell deficiency can be treated with transplantation of cultured human limbal epithelial cells (LEC). It can be advantageous to produce LEC in centralized labs and thereafter ship them to eye clinics. The present study used transport simulations of LEC to determine if vigorous shaking during transport altered the viability, morphology and phenotype during a 4 day-long storage of LEC with a previously described serum-free storage method. Inserts with LEC cultured on amniotic membranes were sutured to caps inside air-tight containers with generous amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered minimal essential medium (MEM). The containers were distributed among the following testing conditions: 6 hours with full containers, 36 hours with full containers, 36 hours with container three quarters full of medium, and 36 hours with container full of medium containing a shear-protecting agent (Pluronic-F68). Compared to stored, but non-transported controls, no statistically significant changes in viability and immunohistochemical staining were observed. The epithelial sheets remained intact. However, an air-liquid interface in the containers reduced the number of desmosomes and hemi-desmosomes compared to the controls. In conclusion, cultured LEC sheets appear to endure vigorous shaking for at least 36 hours if the container is full.

The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

Cardiac fibrinolytic capacity is markedly increased after brief periods of local myocardial ischemia, but declines following successive periods in anesthetized pigs.

BACKGROUND: Fibrinolysis in blood is mainly reflected by the activities of tissue plasminogen activator (tPA) and of plasminogen activator inhibitor-1 (PAI-1). The effect of myocardial ischemia on their activities in the coronary circulation is, however, not established. OBJECTIVES: With an improved experimental model, we therefore examined the effect of a brief period of myocardial ischemia on their activities. Furthermore, the consequences of repeated periods of ischemia, mimicking the situations in patients with unstable angina, were investigated. METHODS: In six anesthetized pigs, we occluded the distal left anterior descending coronary artery (LAD) four times for 10 min with 40 min intervals and determined the activities of tPA and PAI-1 in arterial and coronary venous blood. By simultaneously recording LAD flow, we could estimate cardiac release of these factors at baseline conditions and during reperfusion. RESULTS: Neither net cardiac release of PAI-1 nor alterations in plasma PAI-1 levels were demonstrated during the experiment. However, a significant net release of tPA activity of 10.4 +/- 3.2 IU mL(-1) (P < 0.005) was recorded during baseline conditions. During reperfusion following the first period of ischemia, the cardiac release of tPA activity increased to a peak of 103 +/- 30-fold baseline release, but declined progressively after repeated periods of ischemia. After the fourth period, tPA release did not exceed an estimated baseline accumulation during ischemia and early reperfusion. CONCLUSIONS: In this porcine model, a substantial local increase in fibrinolytic capacity was observed after brief periods of ischemia, but declined subsequently by repeated periods of ischemia.

Sustained prothrombotic profile after hip replacement surgery: the influence of prolonged prophylaxis with dalteparin.

In a randomized trial on the effect of dalteparin for 5 weeks after HRS we evaluated hemostatic variables in plasma sampled before and 1, 6 and 35 days postoperatively. In 218 patients we found that prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin complexes (TAT), d-dimer and fibrinogen were significantly higher on day 35 as compared with baseline values in the placebo group (P < 0.001 for all). The same pattern was found in the dalteparin group, but with significantly lower values for F1 + 2, TAT and d-dimer. In patients in the placebo group with venographically proven deep vein thrombosis (DVT) on day 35 (33%), significantly higher values were found for F1 + 2, TAT and d-dimer than in patients without DVT. Patients in the highest quartile of d-dimer (>2850 ng mL-1) had an odds ratio for the presence of DVT of 24.0 when compared with patients in the lowest quartile (<1625 ng mL-1). It is concluded that a substantial hypercoagulability is sustained until day 35 after HRS, significantly reduced with prolonged administration of dalteparin.

Effect of cyclic AMP and cyclic GMP on thromboplastin (factor III) synthesis in human monocytes in vitro.

Human monocytes in vitro respond to various agents (immune complexes, lectins, endotoxin, the divalent ionophore A 23187, 12-O-tetradecanoyl-phorbol 13-acetate [TPA], purified protein derivative [PPD] of Bacille Calmette-Guerin) with an increased synthesis of the protein component of thromboplastin. The effect of cyclic AMP and cyclic GMP on this response has been studied. Dibutyryl-cyclic AMP, prostaglandin E1 and the phosphodiesterase inhibitors 3-butyl-1-methyl-xanthine (MIX) and rac-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), separately and in combination have a pronounced inhibitory effect on the response to immune complexes and PPD, and a moderate effect on the response to endotoxin and lectins. The effect on TPA response and on the response to A 23187 was slight. Dibutyryl-cyclic GMP (1 mM) gave a slight inhibition of the TPA and IC response, but had essentially no effect on the response to other inducers. The intracellular cAMP level increased when monocytes were incubated with IC, TPA or A 23187 followed by a decrease to basal levels within 1-2 hr, whereas lectin (PHA) and PPD did not induce such changes. The cAMP response to endotoxin varied. Stimulation with IC induced an increase in monocyte cGMP levels, whereas the other stimulants did not cause such changes.

Lectin stimulation of tissue thromboplastin activity in human monocytes in vitro.

Lectins (phytohaemagglutinin, concanavalin A and wheat germ agglutinin) trigger an increase in tissue thromboplastin activity of human monocytes in vitro. The presence of serum was not necessary and did not enhance the activity. The increase was inhibited by cycloheximide and actinomycin D, suggesting that de novo protein synthesis is involved.

Synthesis of thromboplastin protein by a murine macrophage-like cell line.

The established murine macrophage cell line J-774.1 responds to endotoxin with synthesis of thromboplastin apoprotein. The response develops in the absence of added lymphocytes and there is no increased responsiveness in cocultures of J-774.1 cells and BALB/c lymphocytes. J-774.1 cells therefore do not depend on lymphocyte cooperation for their response to endotoxin.

Adhesion of leukocytes to growing arterial thrombi.

Since the role of leukocytes found present in thrombi and haemostatic plugs is not clearly understood. we have investigated the interaction between leukocytes and growing thrombi in a human ex vivo model of arterial thrombogenesis. At a wall shear rate characteristic of moderately stenosed arteries (2600 s(-1)), granulocytes selectively accumulated at the luminal surface of platelet thrombi. The leukocyte adhesion seemed independent of fibrin formation and was clearly correlated to thrombus growth and platelet activation. In contrast, flow cytometry revealed that the expression of adhesion molecules (CD11a, CD11b, CD11, CD3, CD14, CD62L, HLA-DR and binding of fibrinogen) on the surface of circulating leukocytes passing the thrombi was, on short term conditions (15 min), independent of thrombus growth. The adhered granulocytes probably play a pivotal role in limiting the size of the evolving thrombi, as suggested by our electron micrographs of the arterial thrombi showing lysed and phagocytosed platelets. Thus, granulocytes might play an active role in the acute/semiacute phase of local thromboregulation.