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Aim: Bilirubin overload caused by haemolysis of transfused blood and breakdown of extravasated blood constitutes an important causative factor of jaundice in trauma patients. Intravenous infusions of large amounts of unconjugated bilirubin (UCB) block biliary phospholipid secretion and produce canalicular membrane lesions in pigs, putatively because of enhanced cytotoxicity of bile. Severe intrahepatic cholestasis is the functional end result. The aim of the study was to investigate whether bilirubin ditaurate (BDT) overload, which also inhibits biliary phospholipid secretion, also induces intrahepatic cholestasis., Methods: Six pigs were infused with 2.8 g of BDT for 150 min. Six control pigs were infused with albumin sham solution for 150 min. Bile samples were analysed for bile acid- and phospholipid-secretion rates. Bile and serum samples were analysed for bilirubin concentration. Liver biopsies were obtained for scanning electron microscopic studies (SEM)., Results: Biliary bile acid secretion fell by 7.7% and biliary phospholipid secretion rate fell by 88% after BDT infusion. Thus, infusion of BDT for 150 min did not cause intrahepatic cholestasis. SEM showed some variability in the size of canalicular membrane microvilli, but no evidence of gross destruction., Conclusion: BDT overload markedly lowers biliary phospholipid secretion. In contrast to UCB, BDT does not induce canalicular membrane damage nor cause intrahepatic cholestasis. Sustained, marked inhibition of phospholipid secretion does therefore not adequately explain UCB-induced cholestasis. Accumulation of UCB in the canalicular membrane may be the important factor in the pathogenesis of canalicular membrane lesions and intrahepatic cholestasis during UCB overload.

Background/aims: To assess sterility of cultured human limbal epithelial cells (HLEC) and to investigate the viability, morphology and phenotype of cultured HLEC following 2 and 3 weeks of organ culture storage., Methods: HLEC cultured on amniotic membranes were stored in organ culture medium in a closed container at 23 degrees C. Sterility of storage media was tested using a Bactec 9240 blood culture instrument (Becton Dickinson, Maryland) for incubation and periodic reading. Viability was analysed by calcein-acetoxymethyl ester/ethidium homodimer-1 assay, morphology by light microscopy and cellular phenotype by immunohistochemistry., Results: No microbial contamination was observed after 1 week's storage. Viability of cultured HLEC was 87.9 (SD 6.4)% and 52.7 (13.1)% after 2 and 3 weeks of storage, respectively, compared with 98.8 (2.6)% before storage (p<0.001). The multilayered structure was preserved in 70% of cultures following 2 weeks of storage but lost after 3 weeks. A less differentiated phenotype was maintained., Conclusion: This study is the first to verify the sterility of HLEC cultures prior to transplantation. Although a slight decrease in viability was observed following 2 weeks of storage, the HLEC sheets remain acceptable, whereas 3 week's storage was unsatisfactory.

The mucosal membrane of the oral cavity displays at times classical developmental lesions considered to be variations of normal structures rather than having disease characteristics. Of these lesions leukoedema, Fordyce granules, geographic-, fissured- and hairy tongue, median rhomboid glossitis and lingual varices were studied in 598 patients referred to the School of Dentistry, Tehran, Iran. The prevalence was studied in relation to age, gender, occupation, education, smoking habits, general health, addictions and or drug therapies. Oral developmental lesions were seen in 295 patients (49.3%). Only Fordyce granules (27,9%), fissured tongue (12,9%), leukoedema (12,5%) and hairy tongue (8,9%) had enough cases for statistical analysis. Three of these lesions increased with age but not fissured tongue. All were more common in men. After adjusting for age, the parameters education, occupation and complaints upon referral had little influence on the prevalence of the lesions. Fewer Fordyce granules were seen in oral mucosa of smoking men. Leukoedema and hairy tongue were significantly associated with smoking, leukoedema with diabetes mellitus. We conclude that there was a highly significant association between these oral lesions and age, gender and smoking. Few significant associations were found between oral lesions and general diseases.

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.

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.

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.

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.

Purpose: To examine the endothelium of donor corneas with extended postmortem time for survival and reparative mechanisms in an eye bank organ culture storage system., Methods: We obtained 14 pairs of donor corneas with a postmortem time ranging from 29 to 163 hours. One cornea of a pair was immediately fixed for the study of structural changes postmortem and to serve as a control. The second was stored in organ culture for 3 days and thereafter fixed to be studied for reparative processes. Examination was done with light microscopy and scanning electron microscopy. Immunohistochemical staining with antibodies against proliferating cell nuclear antigen, Ki-67, and n-cadherin was performed to examine for cell proliferation and to characterize the cells., Results: The control corneas showed increasing endothelial cell damage with increasing postmortem time. After 5-7 days postmortem, most cells were structurally damaged. After 3 days in organ culture, all corneas acquired an endothelial covering of the posterior surface, with cells, suggesting proliferation in both scanning preparations and in cross-sections. Positive endothelial cell staining with proliferating cell nuclear antigen was found in all cultured corneas. Ki-67 staining of the endothelium was found in 9 of the cultured corneas., Conclusions: The study showed survival of the corneal endothelium up to 7 days postmortem, and accordingly, the potential clinical use of donor corneas with extended postmortem time. Our results furthermore suggest that repair of the endothelium in donor corneas during organ culture storage occurs also by proliferation and not only by migration and enlargement of existing cells. If we uncover the mechanisms regulating cell proliferation in corneal endothelium, it should be possible to develop better storage methods of corneal transplants to improve quality and supply.

Objective: Previous studies have shown that leucocytes, in particular granulocytes, have an enormous capacity for reactive oxygen species (ROS) production, and that this can be influenced by the physical activity of the individual. Theoretically, endurance-trained athletes could profit by increasing their intake of antioxidants, thus neutralizing increased ROS production. The aim of the present investigation was to study the effect of dietary antioxidant supplementation on leucocyte ROS expression and total plasma antioxidant status (TAS) in endurance-trained athletes over the course of 4 weeks., Methods: Eighteen athletes were recruited for the study. A randomized, double-blinded, placebo-controlled crossover study of 4 weeks of antioxidant supplementation (BiO-Antioxidant 2.1 (400 mg vitamin C and 180 mg vitamin E d(-1)) and BiO-Quinon Q10 (200 mg d(-1) was performed. Flow cytometry was applied to examine the leucocyte expression of ROS using the ROS-sensitive probes dihydroethidium and dihydrorhodamine 123. The Randox Total Antioxidant Status kit was used to measure the plasma TAS of the athletes., Results: After 4 weeks of antioxidant supplementation, we observed no significant differences in ROS levels in granulocytes and monocytes either basally or after in vitro stimulation with phorbol myristate acetate. Plasma TAS did not change significantly during the intervention period., Conclusions: We observed no influence of 4 weeks of dietary antioxidant supplementation on oxidative stress status. Based on these findings, there is no rationale advising athletes to ingest antioxidant supplements in addition to their regular diet if that includes daily recommended doses of vitamins.

Purpose: A previous report has described the use of eye bank storage of cultured human limbal epithelial cells (HLECs) to provide a reliable source of tissue for treating limbal stem cell deficiency. In the present study, conventional organ culture (OC) storage and Optisol-GS (Bausch & Lomb, Irvine, CA) storage of cultured HLECs were compared., Methods: Three-week HLEC cultures were either organ cultured at 31 degrees C or 23 degrees C or stored in Optisol-GS at 5 degrees C in a closed container for 1 week. Morphology was studied by light microscopy and transmission electron microscopy, and phenotypic characterization was assessed by immunohistochemistry. Apoptosis was evaluated by real-time RT-PCR microarray analysis, caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)., Results: The ultrastructure was preserved at 23 degrees C, while storage at 31 degrees C and 5 degrees C was associated with enlarged intercellular spaces, separation of desmosomes, and detachment of epithelial cells. Cultured HLECs remained undifferentiated in all storage conditions. The expression of the antiapoptotic gene BCL2 was prominently upregulated in storage at 23 degrees C and 5 degrees C. Downregulation of BCL2A1, BIRC1, and TNF and upregulation of CARD6 in 23 degrees C and 5 degrees C storage conditions suggests a reduction in nuclear factor-kappaB activity. No significant increase in cleaved caspase-3 and TUNEL staining was observed in response to eye bank storage, and the labeling indices of cleaved caspase-3 (range, 0.0%-4.7%) and TUNEL (range, 0.0%-7.8%) were low., Conclusions: These data indicate that OC storage of cultured HLECs at ambient temperature is superior to OC storage at 31 degrees C and Optisol-GS storage at 5 degrees C and that apoptosis is minimal after eye bank storage of cultured HLECs.

Objective: Insulin and adiponectin exert important effects on the vasculature. We wanted to explore whether the eNOS inhibitor asymmetric dimethylarginine (ADMA) contribute to their effects., Methods: Human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) were incubated with growth medium in the presence or absence of tumor necrosis factor-alpha (TNFalpha), D-glucose, insulin or adiponectin. Further, cells exposed to TNFalpha for 24h were co-stimulated with insulin or adiponectin for additional 24h. Concentrations of ADMA in conditioned media and activity of dimethylarginine dimethylaminohydrolase (DDAH) in cell lysates were determined., Results: The dose-dependent TNFalpha-induced ADMA accumulation was significantly inhibited when co-stimulated with insulin or adiponectin in both cell lines (p<0.01 for all), accompanied by significant increases in DDAH activity in all conditions. Insulin alone resulted in a significant, but inversely dose-dependent accumulation of ADMA as compared to control cells in both cell lines, accompanied by increased DDAH activity. Adiponectin alone tended, dose-dependently to decrease ADMA, but without an increase of DDAH activity., Conclusion: The results indicate that ADMA accumulation in human cultured endothelial cells is influenced by both insulin and adiponectin, and both mediators counteract the TNFalpha-induced accumulation of ADMA through the DDAH pathway.

Purpose: The maximum post-mortem time limit for obtaining donor corneas varies between eye banks. It is not known for how long a time the epithelial cells survive post-mortem, nor is it known if donor corneas with extended post-mortem time are able to regenerate the epithelium. Therefore, we wanted to examine the epithelium in donor corneas for regenerative ability during storage in an eye bank organ culture system., Methods: Twenty-four paired donor corneas with post-mortem time from 28 to 163 hr were obtained. One cornea of a pair was fixed immediately to serve as a control, and the second was cultured in eye bank medium at 32 degrees C for 3 days. Examination of the specimens was performed with light and scanning electron microscopy. Immunohistochemical staining methods with antibodies against K 3, K 19, vimentin and p63 were used to further characterize the cells., Results: The control corneas showed decreasing amounts of epithelial cells with increasing post-mortem time. All the cultured corneas demonstrated rapid regeneration of the epithelium. After 3 days in organ culture, 10 of 12 donor corneas were covered with epithelium., Conclusion: Even up to 7 days post-mortem, viable cells reside in the corneal epithelium. The study demonstrates the hardiness and enormous regenerative potential of peripheral corneal cells. Donor corneas processed in an eye bank organ culture storage system will obtain an intact epithelial layer within a few days.

Aim: To investigate organ culture preservation of cultured limbal epithelial cells in order to enhance the availability of tissue-engineered epithelia that are used to treat patients with limbal stem cell deficiency., Methods: Limbal epithelial cells were cultured for 3 weeks on intact amniotic membrane fastened to a polyester membrane carrier. The cultured epithelia were stored for 1 week at 23 degrees C in organ culture medium. The preserved epithelia were then examined using a colorimetric cell viability assay, light microscopy and immunohistochemistry., Results: The viability of the preserved epithelia was 84% (20%), and no statistically significant difference was found compared with non-preserved epithelia. In general, the cell borders were maintained, the nuclei showed no sign of degeneration, and the original layered structure was preserved. Mild intercellular oedema was occasionally observed. Expression of p63, K19 and vimentin was maintained., Conclusions: Cultured limbal epithelial cells can be preserved in organ culture medium for 1 week at room temperature, while maintaining the original layered structure and undifferentiated phenotype.

We have reported that an extract of the edible officinal mushroom Agaricus blazei Murill (AbM) stimulates synthesis of pro-inflammatory cytokines in human monocytes and vein endothelial cells in vitro and reduces the extent of lethal septicemia in mice with bacterial peritonitis. In the present study on human monocytes and granulocytes in whole blood ex vivo, we studied the dynamic changes of cell adhesion molecules (CD11b, CD62L) and the production of reactive oxygen species (ROS) after stimulation with AbM. The presence of AbM resulted in a similarly increased expression of CD11b in monocytes and granulocytes, although at a lower AbM concentration in monocytes (0.5%) than in granulocytes (2%). Furthermore, there was an AbM-mediated decrease in CD62L expression mirroring the effect on CD11b expression regarding magnitude and dose response. The intracellular production of ROS increased slightly but significantly in granulocytes, but not in monocytes stimulated with AbM. The results suggested that the major effect of AbM on monocytes and granulocytes was the upregulation of CD11b expression, thereby increasing both the phagocytic potential and the ablility to induce diapedesis into inflammatory foci. The rich beta-glucan content of AbM could play a crucial role in this immune response.

Purpose: To compare the histology, phenotype, ultrastructure and barrier function of cultured limbal epithelial cells using two explant culture protocols., Methods: Epithelial cells were cultured for 16 days from limbal explants, positioned with either the stromal side (stromal group) or the epithelial side (epithelial group) on intact amniotic membranes. The cultured epithelium (n = 56) was examined using light microscopy, immunohistochemistry for K3, Cx43, ABCG2 and p63 expression, Western blot analysis of DeltaNp63alpha, transmission electron microscopy, a horseradish peroxidase (HRP) permeability assay and scanning electron microscopy., Results: The epithelial group demonstrated a significantly higher expression of p63-positive cells (85.7 +/- 4.2%) than the stromal group (75.3 +/- 8.9%), and Western blots showed a stronger band of DeltaNp63alpha. K3 and ABCG2 were not detected in either group, whereas Cx43 displayed moderate immunostaining in the suprabasal layer. The number of cell layers, the desmosome number and the undulation length in the epithelial group were not significantly different from those in the stromal group. In both groups, HRP accumulated on the apical surface of the superficial cells, and scanning electron microscopy demonstrated tightly apposed superficial cells., Conclusions: Our findings indicate that limbal explants positioned epithelial side down may give rise to cultured epithelia with higher expression of p63 and DeltaNp63alpha.

Objectives: The aim of this study was to determine the association between exposures in asphalt work and changes in lung function, blood concentrations of interleukin-6 (IL-6), micro-C-reactive protein, and fibrinogen among asphalt workers during a work season., Methods: Blood samples from all asphalt workers (N=140) in Norway's largest road construction and maintenance company were taken in April-May 2005 and again in September-October 2005. Spirometric tests of the asphalt workers and a reference group (heavy construction workers, N=126) were carried out before the asphalt season, and the asphalt workers were tested again at the end of the season. Exposure to total dust, oil mist, polycyclic aromatic hydrocarbons, and gases was measured by personal samplers during the asphalt season., Results: The asphalt workers had a significantly a lower forced expiratory volume in 1 second (FEV(1)) and forced expiratory flow rate of 50% of the forced vital capacity than the reference group at the beginning of the season. The asphalt workers were divided according to their exposure into two groups, asphalt pavers (N=81) and asphalt plant operators and truck drivers (N=54). The screedmen, a group of the asphalt pavers, had a statistically significant lower FVC and FEV(1) after one season of asphalt work than all of the other asphalt workers (P<0.05). The mean plasma concentration of IL-6 increased among the asphalt pavers from 1.55 pg/ml before the season to 2.67 pg/ml at the season's end (P=0.04, adjusted for current smoking)., Conclusions: Exposure in asphalt paving may enhance the risk of lung function decline.

Endothelial dysfunction is reflected by an impaired nitric oxide (NO)-mediated vasodilatation. Insulin resistance may be linked to endothelial dysfunction by several mechanisms, including disturbances in signaling pathways common to both insulin action and NO production. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, may contribute to endothelial dysfunction, and elevated ADMA levels have been associated with both insulin levels and the degree of insulin resistance. The direct link between insulin and ADMA, however, has not yet been established. In the present study, we aimed to investigate the effects of acute hyperinsulinemia on circulating ADMA and L-arginine levels and on forearm blood flow (FBF). Male volunteers, aged 21 to 24 years, with borderline hypertension were included in the study. The participants underwent a 90-minute hyperinsulinemic isoglycemic glucose clamp with insulin levels at the postprandial levels (n=20) or a saline infusion (control) (n=9). Fasting blood samples were drawn at baseline and after 90 minutes. Insulin infusion was accompanied by a reduction in ADMA (0.78 to 0.68 micromol/L, P<.01), which was significantly different (P=.001) from the increase seen in the saline control group (0.69 to 0.79 micromol/L, P<.05). The same profile was obtained for L-arginine with a significantly more pronounced decrease (P<.001) in the insulin clamp group (74 to 61 micromol/L, P<.001) than in the saline control group (59 to 57 micromol/L, P=.95). The FBF level and nitrate/nitrite (NOx) levels were not affected by any of the clamp procedures. Short-term administration of insulin was accompanied by a decrease in both ADMA and L-arginine levels, with no change in FBF, in our population of young men with borderline hypertension. The possible influence of insulin on ADMA levels in a chronic state of insulin resistance can, however, not be deduced from the present investigation.

Introduction: During the Norwegian military ranger-training course, cadets are exposed to prolonged physical exercise combined with sleep-, energy-, and food deficiency. The open-window postexercise hypothesis indicates that after hard physical activity, there is an increased risk of contracting infectious diseases., Purpose: The purpose of the present study was to determine leukocyte reactive oxygen species (ROS) levels, total antioxidant status (TAS), leukocyte expression of the cell adhesion molecules CD62L and CD11b, and plasma levels of soluble adhesion molecule L-selectin before, during, and in the recovery phase of a military ranger-training course., Methods: Ten cadets from the Norwegian Military Academy were recruited to the study. Flow cytometry was used to study the intracellular levels of ROS in leukocytes (basally, as well as after in vitro stimulation with phorbol myristate acetate (PMA)), applying the probes dihydroethidium (DHE) and dihydrorhodamine 123 (DHR) and the leukocyte expression of adhesion molecules CD62L and CD11b. ELISA was used to assess the plasma levels of soluble L-selectin, and TAS in plasma was measured using the ABTS+ reduction assay kit., Results: The basal levels of ROS as well as PMA-stimulated ROS in leukocytes declined gradually during the ranger-training course, being lowest on the last day (P < 0.05). The level of TAS increased (P < 0.01) during the course. A striking decrease (P < 0.001) was observed in leukocyte CD62L expression and was sustained even after 3 d of recovery. The leukocyte expression of CD11b remained unchanged., Conclusion: The ranger-training course leads to a partial exhaustion of the leukocyte ROS-generating machinery and to a nearly total extinguishing of leukocyte CD62L expression. These changes may support the open-window hypothesis indicating reduced ability to combat microbial invasions before total restitution.

Objective: The purpose of the present study was to compare the efficacy of intracoronary and intravenous injection of autologous progenitor cells for homing to the acutely infarcted but reperfused myocardium in pigs., Methods: Myocardial infarction was induced in 11 anesthetized pigs by 60-min balloon inflation in the mid LAD. After balloon deflation, reperfusion was verified and autologous CD31(+) progenitor cells, or bone marrow mononuclear cells, labeled with PKH67, were injected either intracoronarily (n=6) or intravenously (n=3). By autopsy, 4-5 days after induction of infarction, tissue from the heart and other organs was obtained for fluorescence microscopy., Results: In the heart, PKH(+) cells were detected throughout the reperfused infarcted myocardium, and the number of PKH(+) cells was significantly higher after intracoronary than after intravenous injection (3.2+/-0.55 vs. 0.33+/-0.17 cells/high-power field/10(6) cells injected, P=.01). Few PKH(+) cells were detected in the spleen, lung, mesenteric lymph node, and bone marrow. In an additional animal with a coil placed in the mid LAD, progenitor cells were not detected in the infarcted myocardium or in the normal myocardium., Conclusion: Autologous mononuclear and CD31(+) cells from bone marrow accumulated in the infarcted myocardium when injected intracoronarily or intravenously after established reperfusion, and the accumulation of cells was significantly greater after intracoronary injection than after intravenous injection. Accumulation of PKH(+) cells did not appear in the normal myocardium or in the nonreperfused infarcted myocardium. PKH(+) cells were detected in spleen, lung, and bone marrow but to a lesser degree than in the infarcted myocardium.

Background: Increased intramedullary pressure in the femoral cavity causes intravasation of bone marrow content to the circulation which may lead to occlusion of pulmonary vessels and cardiorespiratory dysfunction. A one-step reamer-irrigator-aspirator (RIA) technique has been developed to reduce the intramedullary pressure (IMP) during the reaming procedure. This study was design to compare IMP with a standard reaming technique with IMP during reaming with the RIA system with a hypothesis that the RIA system would involve lower pressures., Material and Method: In a randomised study in 19 Norwegian landrace pigs reamed intramedullary nailing was performed with two different reamer devices. Nine animals were operated with a traditional reamer and 10 animals with RIA. One animal in the RIA group was excluded due to a perioperative femoral fracture, and three animals in the traditional group were excluded due to a perforation of the distal medial femoral cortex. The intramedullary pressure was registrated with a transducer-tipped pressure monitoring catheter during reaming., Results: There was a significantly higher intramedullary pressure (P<0.05) during reaming in the traditional reamer group (mean 188+/-38 mmHg) than in the RIA group (mean 33+/-8 mmHg). Intramedullary pressures recorded before surgery, at the opening of the femoral cavity with an awl, by insertion of a guide wire, at insertion of the intramedullary nail, and 10 min after nail insertion showed no significant differences between the groups., Conclusion: The use of a one-step reamer-irrigator-aspirator technique in the pig femur induced less intramedullary pressure increase than the use of a traditional reamer.

Fat embolism (FE) and fat embolism syndrome (FES) are controversial topics, particularly regarding their clinical relevance and their independency as a clinical syndrome. FE describes the presence of fat globules within the microcirculation. FES on the other hand, is a clinical syndrome characterized by the presence of FE with the manifestation of an identifiable clinical pattern of signs and symptoms. Both experimental and clinical studies have demonstrated the occurrence and the possible harmful effects of FE. For instance, FE involving the lungs can result in adult respiratory distress syndrome (ARDS). The effects of FE are certainly mechanical, leading to capillary congestion, but are also highly likely to be of a proinflammatory and prothrombotic nature. The demarcation of FES against ARDS is mainly demonstrated by the single organ involvement of ARDS, exclusively influencing the lungs, whereas FES has a multiorgan effect with pulmonary, skin and, neurological manifestations. This article is a review of the pathophysiological mechanisms of FE and the clinical occurrence and influence of FE and FES.

Cytokine systems are activated in heart failure, and it is believed that interaction between such systems may be important during progression of this disorder. We have previously shown that failing hearts have increased levels of the interleukin-6 related cytokine leukemia inhibitory factor (LIF) and activin A, a member of the transforming growth factor-beta family. The aim of this study was to examine the effects of activin A on cardiomyocytes and a potential interaction with LIF-mediated changes in cell signaling and growth. Cardiomyocytes were isolated from 1- to 3-day-old Wistar rats, and the cells were treated with LIF, activin A or a combination thereof. Our main findings were: (i) activin A treatment reduced the LIF-mediated increase in cardiomyocyte length, perimeter and sarcomeric organization and was accompanied by a substantially decreased alpha-skeletal actin gene expression. (ii) The activin A-mediated phosphorylation of Smad2 was markedly enhanced by LIF. (iii) Activin A markedly induced SOCS3 gene expression, while LIF potently increased the expression of Smad7 mRNA, representing inhibitors of LIF and activin A signaling pathways, respectively. (iv) Inhibiting activation of the Smad2/3 pathway abolished the effects of activin A on LIF-induced changes in cell length, perimeter and sarcomeric organization. In conclusion, activin A markedly attenuates LIF-induced changes in cardiomyocytes, reflecting a potentially important role for both activin A and the Smad2/3 pathway in regulation of myocardial remodeling.

Neuropeptide Y is released together with norepinephrine from sympathetic nerve terminals during conditions of increased sympathetic activity. Neuropeptide Y is known to inhibit vagal activity, and accordingly, it might increase the risk for ventricular fibrillation during myocardial ischemia-reperfusion, with concomitant sympathetic stimulation. Counteracting the inhibiting effect of neuropeptide Y by the specific neuropeptide Y2 antagonist, BIIE0246, we expected occurrence of ventricular fibrillation in association with repeated periods of myocardial ischemia-reperfusion to decrease. The midleft anterior descending coronary artery was repeatedly occluded in 16 open-chest pigs. Eight pigs received BIIE0246, and the controls received the vehicle only. Ventricular fibrillation developed in 2 animals of the control group, but in 4 pigs receiving BIIE0246. Occurrence of ventricular fibrillation and ventricular tachycardia did not differ significantly between the 2 groups, and in association with repeated periods of regional myocardial ischemia, did not decline in pigs treated by the specific neuropeptide Y2-receptor antagonist BIIE0246.

Objectives: In the present paper the circadian variation and age differences in human leukocyte reactive oxygen species (ROS) levels and plasma total antioxidant status were studied. Different ROS levels could influence disease during ageing and at different times of the day/night., Methods: Unstimulated and stimulated (12-O-tetradecanoyl-phorbol-13-acetate (TPA) blood samples were analysed by flow cytometry using dihydroethidium (DHE) and dihydrorhodamine 123 (DHR) as probes. 60 healthy individuals were divided into five equal groups ((I) 1-2 days post partum, (II) 20 +/- 2 years, (III) 40 +/- 2 years, (IV) 60 +/- 2 years and (V) 80 +/- 2 years) and 6 healthy volunteers were followed with blood samples at 3 hour intervals for 24 hours., Results: We observed a significant peak of ROS levels in both monocytes and granulocytes at 6 pm and 3 am using DHE as probe. DHR showed in principle the same pattern. The monocytes (unstimulated and stimulated) and unstimulated granulocytes of the newborn group showed higher ROS values than all the other groups, whereas the groups of adults did not differ from each other. The plasma total antioxidant status was significantly higher in the newborn group and showed no circadian variation., Conclusions: The present data show that there is a circadian variation of ROS production in leukocytes that might possibly influence the occurrence of cardiovascular incidents like myocardial infarction. No increase in ROS production was found in healthy elderly compared to younger individuals. The increased ROS levels and antioxidant status of newborns might influence neonate immunity and play a part in cell signalling and development.

Purpose: Donor corneas are processed in eye banks and used for transplantation as a standard routine. The maximum time limit post-mortem for harvesting donor tissue varies greatly between eye banks. This study aimed to examine the corneal epithelium for structural changes post-mortem., Methods: A total of 51 corneas harvested between 14 and 163 hours post-mortem were examined using scanning electron and light microscopy., Results: Cell loss occurred through desquamation of flat superficial cells during the first days. In corneas with a post-mortem time of more than 2-3 days, large superficial cell sheets and deeper cells detached, starting centrally. Deep peripheral cells remained. The loss of the superficial cells revealed the 3-dimensional structure of the epithelium and the membrane characteristics of deeper cells., Conclusion: The longer the time post-mortem, the greater the epithelial cell loss. However, a rim of peripheral cells remained, even after 7 days. The superficial cell layer showed signs of strong lateral attachment and broke up in a sheet-like fashion. The intercellular adhesion between deeper cells and adhesion between the basal cells and the basement membrane appeared to be weak post-mortem. The cell membrane structures of the remaining cells were surprisingly well retained. The clinical implication of the study is discussed.

Nitrite and nitrate are the stable end products of the L-arginine-NO pathway, and the sum of nitrite and nitrate (NOx) is a common way to measure nitric oxide (NO) production or secretion. To uncover any genetic influence on NO, we measured NOx in serum samples from monozygotic twin pairs after an overnight fast. Heritability was estimated as intraclass correlation coefficient. We arrived at a heritability estimate of .32 (95% confidence interval 0.17-0.45) for NOx. The numerical heritability estimate was higher for females than for males (0.38 vs. 0.21), and higher for nonsmokers than for smokers (0.36 vs. 0.22). The heritability estimate of NOx was lower than the heritability estimate for other cardiovascular risk factors. This study suggests a low degree of heredity for NOx levels.

Background and Aim: Impaired vasodilatation has been suggested to be caused by inhibition of nitric oxide generation by the recently described asymmetric dimethylarginine (ADMA). In the present study we wanted to explore whether n-3 polyunsaturated fatty acid (PUFA) supplementation and/or diet intervention have beneficial influence on endothelial function assessed as plasma levels of ADMA and L-arginine., Methods: A male population (n = 563, age 70 +/- 6 yrs) with long-standing hyperlipidemia, characterized as high risk individuals in 1970-72, was included, randomly allocated to receive placebo n-3 PUFA capsules (corn oil) and no dietary advice (control group), dietary advice (Mediterranean type), n-3 PUFA capsules, or dietary advice and n-3 PUFA combined and followed for 3 years. Fasting blood samples were drawn at baseline and the end of the study., Results: Compliance with both intervention regimens were demonstrated by changes in serum fatty acids and by recordings from a food frequency questionnaire. No influence of either regimens on ADMA levels were obtained. However, n-3 PUFA supplementation was accompanied by a significant increase in L-arginine levels, different from the decrease observed in the placebo group (p < 0.05). In individuals with low body mass index (<26 kg/m2), the decrease in L-arginine on placebo was strengthened (p = 0.01), and the L-arginine/ADMA ratio was also significantly reduced (p = 0.04)., Conclusion: In this rather large randomized intervention study, ADMA levels were not influenced by n-3 PUFA supplementation or dietary counselling. n-3 PUFA did, however, counteract the age-related reduction in L-arginine seen on placebo, especially in lean individuals, which might be discussed as an improvement of endothelial function.

Objective: Homocysteine measurements may be relevant in geriatric medicine as homocysteine has been identified as an independent risk factor for prevalent disorders such as occlusive arterial vascular disease, cognitive impairment and dementia. The aim of the present study was to study diagnostic correlates of plasma total homocysteine (tHcy) in geriatric in-patients., Material and Methods: Blood samples for the analysis of tHcy and related factors like serum vitamin B12, serum folate, red blood cell folate and clinical data were collected from geriatric patients (n=114) in stable clinical condition., Results: Almost 40% of the patients had tHcy values above 20 micromol/L. tHcy correlated significantly with serum folate, serum vitamin B12, serum creatinine and congestive heart failure, but not with red blood cell folate, cerebrovascular disease, coronary heart disease or cognitive impairment., Conclusions: Hyperhomocysteinaemia seems to be frequent in geriatric patients and might primarily be an indicator of low folate and high creatinine values.

Objective: We hypothesized that umbilical vein serum soluble fms-like tyrosine kinase 1 (sFlt1) concentration was augmented in pre-eclampsia. We also explored a possible association between fetal and maternal concentrations of sFlt1., Study Design: At cesarean delivery, maternal serum samples from pre-eclamptic (n=38) and uncomplicated (n=32) pregnancies were obtained, as well as umbilical vein serum and amniotic fluid samples. ELISA for human sFlt1, vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) were performed., Results: Median sFlt1 concentrations were elevated in pre-eclampsia compared to uncomplicated pregnancy, in umbilical venous serum (246 and 163 pg/mL, P=0.04), in maternal serum (9932 and 3417 pg/mL, P<0.001), as well as in amniotic fluid (51,040 and 33,490 pg/mL, P=0.03). A positive association between the fetal and maternal serum levels of sFlt1 was found in the pre-eclampsia group. Median PlGF concentration in the maternal serum was significantly lower in the pre-eclampsia group compared to the control group (82 pg/mL and 169 pg/mL, P<0.001)., Conclusions: sFlt1 concentration is elevated in the fetal circulation in pre-eclampsia, but at a much lower level than in the maternal circulation. The results of our study do not support a substantial fetal contribution to the elevated circulating maternal sFlt1 protein concentration in pre-eclampsia.

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.

Although increased levels of circulating interleukin (IL)-18 have been demonstrated in patients with cardiovascular diseases, the functional consequences of chronically increased circulating IL-18 with respect to myocardial function have not been defined. Thus we aimed to examine the effects of chronic IL-18 exposure on left ventricular (LV) function in healthy mice. Moreover, to clarify whether IL-18 has direct effects on the cardiomyocyte, we examined effects of IL-18 on cardiomyocytes in vitro. After 7 days of daily intraperitoneal injections of 0.5 microg IL-18 in healthy mice, a 40% (P < 0.05) reduction in the LV maximal positive derivative, a 25% (P < 0.05) reduction in the LV maximal rate of pressure decay, and a 2.8-fold (P < 0.001) increase in the LV end-diastolic pressure were measured, consistent with myocardial dysfunction. Furthermore, we measured a 75% (P < 0.05) reduction in beta-adrenergic responsiveness to isoproterenol. IL-18 induced myocardial hypertrophy, and there was a 2.9-fold increase (P < 0.05) in atrial natriuretic peptide mRNA expression in the LV myocardium. In vitro examinations of isolated adult rat cardiomyocytes being stimulated with IL-18 (0.1 microg/ml) exhibited an increase in peak Ca2+ transients (P < 0.05) and in diastolic Ca2+ concentrations (P < 0.05). In conclusion, this study shows that daily administration of IL-18 in healthy mice causes LV myocardial dysfunction and blunted beta-adrenergic responsiveness to isoproterenol. A direct effect of IL-18 on the cardiomyocyte in vitro was demonstrated, suggesting that IL-18 reduces the responsiveness of the myofilaments to Ca2+. Finally, induction of myocardial hypertrophy by IL-18 indicates a role for this cytokine in myocardial remodeling.

Aim: To determine whether the free fatty acids (FFAs), oleic, linoleic, and palmitic acid, found elevated before 20 weeks of pregnancy in those women who later develop preeclampsia, induced changes in expression of the vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), or E-selectin in cultured human umbilical cord vein endothelial cells (HUVEC), and integrin subunit CD11b, L-selectin or intracellular reactive oxygen species (ROS) in leukocytes., Methods: The VCAM-1, ICAM-1, and E-selectin expression were measured using ELISA in HUVEC after incubation with 100 micromol of either oleic, linoleic, or palmitic acid for 6 hr and 24 hr. The co-reactivity with lipopolysaccharide (LPS), the amount of VCAM-1 mRNA in the cells, and soluble VCAM-1 in the incubation medium were measured as well. Leukocyte adhesion molecules and ROS were measured after incubation with 750 microm of either of the FFAs in a whole blood model using flow cytometry., Results: No effects of the FFAs tested were found on the HUVEC or leukocyte adhesion molecule expression or intracellular ROS. The only exception to this was palmitic acid incubation, which significantly lowered the VCAM-1 expression in HUVEC after 24-hr incubation and also slowed the decay of VCAM-1 expressed after stimulation with LPS., Conclusions: The lack of significant proinflammatory changes of the FFAs tested might indicate that the elevated plasma levels of FFAs seen in preeclampsia most probably are products of the preeclamptic process rather than a causative factor.

Objective: To examine if the decline in post-ischemic hyperemic flow after repeated brief periods of myocardial ischemia is accompanied by augmented cardiac release of the vasoconstrictors endothelin-1 (ET-1) and norepinephrine (NE)., Design: Mid-LAD (left anterior descending coronary artery) was occluded for 10 min with 30 min intervals a total of four times in six anesthetized pigs. Blood from the anterior interventricular coronary vein was drained through a shunt to the right atrium to facilitate blood sampling. Plasma concentrations of ET-1 and NE were repeatedly measured in arterial and coronary venous blood to estimate cardiac vasoconstrictor release., Results: Plasma concentrations of ET-1 and NE remained unaltered, but cardiac release of both vasoconstrictors rose briefly during reperfusion due to the hyperemia. However, release declined progressively after repeated periods of ischemia and reperfusion and amounted to 53% (NE) and 17% (ET-1) of initial release after the fourth period of ischemia., Conclusion: The decline in post-ischemic hyperemia after repeated brief periods of myocardial ischemia is not accompanied by a progressive accentuation of cardiac ET-1 and NE release.

Objective: Pregnancy is characterized by haemostasis activation, and in preeclampsia endothelial dysfunction, platelet and leukocyte activation are further characteristic features. The aim of this study was to investigate to which extent platelets from normotensive pregnant women or those with preeclampsia are circulating as microparticles or platelet-platelet aggregates. We also investigated if platelet-leukocyte multiconjugates were differently present in nonpregnant and pregnant women., Study Design: Using flow cytometry we investigated these parameters in basal samples and after in vitro stimulation with adenosine diphosphate (ADP) or thrombin receptor activation peptide. This was done in samples from 20 matched preeclamptic and normotensive pregnant women and in a group of 12 nonpregnant women., Results: In the basal state we found that women with preeclampsia had a smaller portion of microparticles circulating than the normotensive pregnant women, Upon ADP stimulation both pregnancy groups showed a higher percentage of monocytes and granulocytes with platelets attached and also a higher number of platelets attached to each monocyte and granulocyte than in the group of nonpregnant individuals., Conclusion: This article presents further evidence that changes from the nonpregnant to the pregnant state are associated with hemostasis activation as an integrated part of an inflammatory reaction that is even more pronounced when pregnancy is complicated with preeclampsia.

Background: Calprotectin is a protein found in myelomonocytic cells and plays a role in various physiological functions such as inflammatory processes and antiproliferation of cells, and in the neutrophil defense against bacterial infections. Preeclampsia is characterized by maternal endothelial dysfunction and by insufficient trophoblast invasion into the maternal endometrium (decidua). In addition, preeclampsia is associated with maternal leukocyte activation and we therefore wanted to investigate whether calprotectin levels in plasma from women with preeclampsia differed from the levels in normotensive pregnant and nonpregnant women., Method: Calprotectin measurements were included in a case-control study of 20 preeclamptic women matched with 20 normotensive pregnant women regarding age, pregnancy length, parity and body mass index (BMI). We also measured calprotectin in 12 nonpregnant women. Calprotectin plasma levels were analyzed using an enzyme-linked immunosorbent assay (ELISA)., Results: We discovered significantly elevated plasma calprotectin levels in preeclamptic patients compared to matched normotensive pregnancies: 768 (612-1016) microg/L vs. 445 (276-598) microg/L (medians, 25, 75 percentiles, respectively), p = 0.002., Conclusions: The elevated plasma calprotectin levels demonstrated in the preeclampsia group supports the notion that leukocytes are activated in preeclampsia. The elevated calprotectin level might constitute a part of the innate defense in myelomonocytic cells against microorganisms in pregnancy. We suggest further elucidation of a role for calprotectin in the development of pregnancy disorders such as preeclampsia.

Running leads to biochemical and hematological changes consistent with an inflammatory reaction to tissue injury. We report changes in the plasma concentration of the leukocyte-derived protein calprotectin after long-distance running. Blood samples were collected from runners before and after a marathon, half-marathon, a 30-km cross-country run, a military ranger-training course and short-term maximal physical exercise until exhaustion, VO2max. Leukocyte counts, plasma calprotectin concentration and calprotectin per neutrophilic granulocyte were assayed using a new method. During the marathon, half-marathon, the 30-km run, the ranger-training course and the VO2max exercise, calprotectin levels increased 96.3-fold, 13.3-fold, 20.1-fold, 7.5-fold and 3.4-fold, respectively. These changes may reflect damage to the tissues or vascular endothelium, causing microthrombi with subsequent activation of neutrophils. These cells are known to phagocytose platelets in microthrombi and may contribute to the prevention of clinical thrombosis. The half-life of calprotectin in plasma was about 5 h. The content of calprotectin per neutrophil remained unchanged during exercise at a level similar to that in healthy blood donors: mean: 25 pg/cell, range 18.8-33.6. A reference interval (mean +/- 2 SD) of 18.6-31.4 pg/cell is suggested.

We investigated the levels of asymmetric dimethylarginine (ADMA), an important endogenous inhibitor of nitric oxide (NO), as related to metabolic risk factors known to contribute to atherosclerotic disease. Dimethylarginines were analysed in a cross-sectional study of 563 elderly high-risk men (70 +/- 6 years). ADMA and the l-arginine/ADMA (l-arg/ADMA) ratio were highly significantly correlated with several metabolic risk factors. However, only the association with body mass index (BMI) remained significant after adjustment for inter-related variables. When analyzing the results according to being overweight or not, ADMA levels were independently significantly higher (P = .05) and the L-arg/ADMA ratios were significantly lower (P < .008) in individuals with high BMI (> or =26 kg/m(2), median value) as compared with subjects with low BMI. ADMA levels were furthermore significantly lower (P = .037) and L-arginine and the l-arg/ADMA ratios were significantly higher (P = .004 and P = .001, respectively) in smokers compared with nonsmokers, the latter being independent of other risk factors. The strong relationship found between BMI and plasma levels of ADMA and the l-arg/ADMA ratio indicate a link to endothelial dysfunction in overweight subjects. The beneficial dimethylarginine profile observed in smokers in this elderly population is not easily explainable and should be further investigated.

There still exist many unanswered questions whether physical exercise is beneficial or harmful to the immune system. The 'open-window' post-exercise hypothesis states that athletes are more susceptible to infections after exercise, but there is a need for further elucidation. The aim of the present study was to investigate the effect of long-distance running on leucocyte expression of selected adhesion molecules as well as the plasma levels of soluble leucocyte- and endothelium-derived adhesion molecules. Twenty-seven men participating in Oslo marathon together with 16 entrants (eight men and eight women) in the Oslo half-marathon were recruited to this study. Venous blood was collected before and immediately after the races for analysing the leucocyte expression of CD62L, CD11b and CD14 with the help of flow cytometry, and plasma concentrations of soluble (s) sE-selectin, sL-selectin, sP-selectin, sVCAM-1, sICAM-1 and sCD14 were assessed by means of enzyme-linked immunosorbent assays. A significant increase of leucocyte CD11b expression was observed following both races, compared to the pre-race situation. Monocyte CD14 expression increased only after the marathon race. After both races, CD62L expression was significantly lowered on all leucocyte subsets, whereas the plasma levels of sE-selectin, sP-selectin, sL-selectin, sVCAM-1, sICAM-1 and sCD14 were all increased. Altogether, these changes negatively influence the ability of leucocytes to adhere to and actively transmigrate the endothelium to reach the tissues. Our study thus supports the 'open-window' hypothesis, indicating a reduced capacity to combat infectious agents during the immediate post-exercise period.

Objective: The aim of our study was to compare the expression of leukocyte adhesion molecules, intracellular reactive oxygen species, and vasoactive substances in preeclampsia and matched normotensive pregnancies and to explore differences between pregnancy and the nonpregnant state regarding these parameters., Methods: Flow cytometry was used to analyze the monocyte and granulocyte expression of adhesion molecules from 20 matched pairs of preeclampsia/normotensive pregnancies and 12 nonpregnant subjects. Basal levels of CD11b, CD11c, CD62L, and CD14 were measured. In addition, expression of human lymphocyte antigen-DR, CD4, CD8, and CD4/CD8 ratio were assessed. Basal reactive oxygen species levels, as well as reactivity upon in vitro stimulation with phorbol 12-myristate 13-acetate, were measured in monocytes and granulocytes with the probes dihydroethidium, dichlorofluorescein-diacetate, and dihydrorhodamine-123. Further, the plasma levels of endothelin-1, the nitric oxide metabolites nitrite/nitrate, and total antioxidant status were analyzed., Results: Monocytes expressed significantly higher levels of CD11b and CD14 in preeclamptic patients compared with normotensive pregnant subjects, whereas CD11c was elevated on both monocytes and granulocytes in pregnancy compared with the nonpregnant state. Both monocytes and granulocytes displayed higher basal, as well as phorbol 12-myristate 13-acetate-stimulated, amounts of reactive oxygen species in the preeclampsia group compared with the normotensive group. We also found the endothelin-1 and antioxidant levels significantly elevated in preeclampsia patients compared with normotensive subjects, whereas no differences were seen between the groups regarding nitrite/nitrate levels., Conclusion: These results show that the maternal blood leukocytes are activated in preeclampsia and support the view that oxidative stress is a contributing factor in the pathophysiology of preeclampsia.

Objective: The aims of our study were to determine the basal platelet activation state in women with preeclampsia compared with normotensive pregnant women and nonpregnant women and to investigate the platelet reactivity on in vitro stimulation with adenosine diphosphate or thrombin receptor activation peptide., Study Design: Platelet expression of CD61 (fibrinogen receptor), CD42a (von Willebrand factor receptor), CD62P (P-selectin), CD63 (Glycoprotein 53), and PAC-1 binding (activated fibrinogen receptor) were determined in 20 pairs of women with preeclampsia/normotensive pregnant women and in 12 nonpregnant women, with the use of flow cytometry., Results: Basal platelet expression of CD61, CD42a and CD62P, and adenosine diphosphate-stimulated CD62P expression were increased in women with preeclampsia compared with normotensive pregnant women. Platelets from women with preeclampsia and normotensive pregnant women differed from platelets from nonpregnant women by expressing higher basal CD63 levels and being more responsive to in vitro agonist stimulation, which was demonstrated by increased expression of CD61, CD62P, and CD63., Conclusion: This study supports the notion that platelets are important in the pathophysiologic condition of preeclampsia.

The purpose of this study was to identify essential genes involved in myocardial growth and remodeling following myocardial infarction (MI). Left ventricular noninfarcted tissues from six mice subjected to MI under general anesthesia and from six sham-operated mice were obtained 1 wk after primary surgery and analyzed by means of cDNA filter arrays. Out of a total of 1,176 genes, 641 were consistently expressed, twenty-three were upregulated and thirteen downregulated. Five genes were only expressed following MI. Syndecan-3, a transmembranous heparan sulfate proteoglycan, was found to be upregulated together with a transcriptional activator of syndecans, Wilms tumor protein 1 (WT-1). Northern blotting demonstrated a significant upregulation of syndecan-1, -2, -3, and -4, WT-1, fibronectin, and basic fibroblast growth factor (FGF) receptor 1. Furthermore, Western blot analysis showed statistically significant increases in protein levels for syndecan-3 and -4. In conclusion, we have identified a subset of genes with increased expression in noninfarcted left ventricular tissue following MI, including syndecans 1-4, WT-1, fibronectin, collagen 6A, and FGF receptor 1. Since the syndecans link the cytoskeleton to the extracellular matrix and function as required coreceptors for FGF, we suggest a role for the syndecans in cardiac remodeling following MI.

Background: The present study was undertaken to examine the ability of lipopolysaccharide-containing outer membrane vesicles (OMV-LPS) and purified LPS (P-LPS) from the same meningococcal strain to induce the expression of Toll-like receptors (TLR2 and TLR4) and TNF-alpha production in leukocytes, and further to study the involvement of TLRs, and CD14 in monocyte TNF- alpha production in an ex vivo human whole blood system., Material/methods: OMV-LPS or P-LPS were added to human whole blood and expression of TLR2/4 and production of TNF- alpha in leukocytes were measured by flow cytometry. To study involvement of TLRs and CD14 in monocyte cytokine production we used monoclonal antibodies against TLR2/4 and CD14., Results: OMV-LPS and P-LPS induced surface expression (maximal at 120 min) of TLR2 and TLR4 on granulocytes and monocytes. LPS incorporated in OMV was less potent (weight basis) than P-LPS in inducing monocyte TNF- alpha production. When inducing monocyte TNF-alpha by OMV-LPS, antibodies directed against TLR2 and TLR4 caused 45 and 78% inhibition, respectively. When inducing TNF- alpha by P-LPS, antibodies against TLR2 had no effect, whereas anti-TLR4 antibodies caused 63% inhibition. Antibodies against CD14 inhibited nearly completely the monocyte TNF- alpha response induced by meningococcal LPS irrespective of whether LPS was presented in purified form or incorporated in membrane vesicles., Conclusions: OMV-LPS and P-LPS from the same meningococcal strain induced expression of TLR2/4 on monocytes and granulocytes. Surface receptors TLR2/4 and CD14 are essential for in vitro cellular activation induced by OMV-LPS and P-LPS, but the functional significance of these receptors during meningococcal infections remains elusive.

Objective: Interleukin (IL)-18 has been reported to be an important predictor for mortality in ischemic heart disease. IL-18 has proinflammatory properties, induces cell death and stimulates nitric oxide production. We hypothesized that following myocardial infarction (MI) an increased myocardial IL-18 production occurs, which may be involved in the pathogenesis of post-ischemic heart failure., Methods and Results: Seven days after induction of MI in the mouse, myocardial hypertrophy and pulmonary edema were observed. RNase protection assay of tissue from the non-infarcted left ventricular myocardium revealed an increase in IL-18 (2.0-fold; P<0.001) and IL-1 beta (1.6-fold; P<0.001) mRNA after MI. Enhanced abundance of pro-IL-18 (1.4-fold; P<0.05), IL-18 receptor (3.5-fold; P<0.05) and IL-18 binding proteins (1.6-fold; P<0.05) was also demonstrated, whereas cardiac IL-18 protein decreased by 25% (P<0.05) following MI. However, the concentration of circulating IL-18 was significantly elevated (MI; 90.4+/-11.7 pg/ml, sham; 47.2+/-4.2 pg/ml; P<0.001). After MI, enhanced cardiac activity of the pro-IL-18 processing enzyme, caspase-1, was measured. Additionally, a 3.4-fold increase (P<0.001) in the activity of the IL-18 degrading enzyme, caspase-3, was found in cardiac tissue, which may explain the observed reduction of cardiac IL-18 protein abundance. Finally, IL-18 reduced shortening of electrically stimulated adult cardiomyocytes and left ventricular contractility in vivo., Conclusions: After MI in the mouse, increased production of cardiac IL-18 mRNA and pro-IL-18, as well as circulating IL-18 occurs. Since IL-18 also reduced myocardial contractility, we suggest that IL-18 may be involved in the pathogenesis of contractile dysfunction following MI.

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.

The cysteinyl leukotrienes (cysLTs) and the peptide hormone endothelin (ET)-1 are potent bronchoconstrictor substances, and these mediators are also claimed to be implicated in the development of eosinophilic airway inflammation. In the present study, we have investigated the effect of the cysLT1 receptor antagonist montelukaston the development of an eosinophilic airway inflammation 24 h after intratracheal Sephadex (SDX) provocation in rats. Furthermore, the effect of montelukast treatment on the generation of ET-1 and other pro-inflammatory mediators has been studied. The inflammatory response was significantly reduced in the animals receiving SDX + montelukast compared to animals receiving solely SDX, as evaluated by a decrease in bronchoalveolar lavage fluid total cell count (10.3 +/- 1.2 vs. 18.5 +/- 1.8 x 10(4) ml(-1), P<0.001), number of eosinophils (299.7 +/- 43.8 vs. 577.6 +/- 46.6 x 10(2) ml(-1), P<0.001), and lymphocytes (116.8 +/- 20 vs. 222.0 +/- 34.8 x 10(2) ml(-1), P<0.05), as well as the degree of tissue inflammation (P<0.05). Montelukast also inhibited the increase in the concentration of the pro-inflammatory mediators ET-1 (28.5 +/- 75 vs. 40.9 +/- 7.3 x pg ml(-1), P<0.05) and interferon (IFN)-gamma (4.3 +/- 2.2 vs. 15.6+/-8.7 x pg ml(-1), P<0.05), but not tumor necrosis factor-gamma or interleukin-8. In summary, treatment with the cysLT1 receptor antagonist montelukast reduced the inflammatory response during development of an eosinophilic airway inflammation, possibly by inhibiting the release of pro-inflammatory mediators like ET-1 and IFN-gamma.

Objective: Cardiac glycosides exert their inotropic effect by increasing intracellular calcium. Increased intracellular calcium is a key event in platelet aggregation. In aggregometer studies, digitalis has been found to augment platelet agonist responses. A prothrombotic effect of digitalis might be concealed since heart failure and atrial fibrillation per se predispose to thromboembolism. The present study investigates the effects of digitoxin on platelet function in healthy volunteers., Methods: Twenty healthy, non-smoking volunteers were randomised to receive digitoxin ( n = 10, 0.6 mg day 1, 0.4 mg day 2, then 0.1 mg daily) or placebo ( n = 10) for 10 days. Platelet function was then analysed ex vivo using three-colour whole-blood-flow cytometry, both in non-stimulated mode and after agonist stimulation with 0.1 micromol/l adenosine diphosphate (ADP), 10 micromol/l ADP and 5.0 micromol/l epinephrine (final concentrations). Expression of activated fibrinogen receptor, von Willebrand's factor receptor and P-selectin, formation of platelet-platelet and platelet-leukocyte aggregates and particle size were examined., Results: No significant difference between the placebo and the digitoxin group (digitoxin levels 17-42 nmol/l) was found, neither on a global level nor for any isolated parameter., Conclusions: Theory and in vitro data suggest that digitoxin treatment could activate platelets. No evidence for this was found in healthy volunteers. This observation is strengthened by the unequivocal results for all parameters measured. However, thrombosis-prone patients with heart failure and/or atrial fibrillation may respond differently to digitalis therapy.

Flow cytometry was used to study the expression of leukocyte adhesion molecules CD11a, CD11b, CD11c, CD14, and CD62L (L-selectin) and production of reactive oxygen species (ROS) in an ex vivo human whole-blood system stimulated with lipopolysaccharide-containing outer membrane vesicles (LPS-OMV) from N. meningitidis. Results demonstrated a dose-dependent increase in surface expression of CD11a, CD11b, CD11c and CD14 in granulocytes and monocytes (maximal at 30-120 min) upon OMV-LPS challenge, whereas CD62L expression was heavily downregulated (maximal at 30-120 min). The OMV-associated LPS was almost as potent (on a weight basis) as purified LPS from E. coli in inducing adhesion molecule modulation but the response was delayed. Upon stimulation with OMV-LPS or E. coli-LPS, the production of intracellular ROS increased in both granulocytes and monocytes when dihydroethidium (DHE, mainly reflecting superoxide anion) was used as a probe, whereas peroxynitrite production monitored with dihydrorhodamine 123 (DHR) was not significantly changed. The OMV-mediated modulation of leukocyte adhesion molecule expression and increased ROS production may certainly lead to increased entrapment of leukocytes in the microcirculation and contribute to untoward inflammatory reactions as seen in systemic meningococcal disease.

Unlabelled: A large part of native meningococcal lipopolysaccharide (LPS), i.e., LPS integrated in the outer cell membrane, is released in the form of 'blebs' from surplus outer membrane material. In the present study we investigated the effects of purified outer membrane vesicles (OMVs) on blood platelet-platelet and platelet-leukocyte interactions. Citrated whole blood was stimulated in vitro with equal amounts (on a weight basis) of OMV-integrated LPS, purified LPS (P-LPS) from the same meningococcal strain and purified E. coli-LPS. The samples were analyzed by flow cytometry. Upon OMV stimulation platelet aggregation increased 2.1-fold, platelet degranulation 1.8-fold, (measured as CD62P expression), platelet binding to monocytes 2.6-fold, whereas platelet binding to granulocytes increased 2.8-fold. Also, the fraction of large heteroconjugates, i.e., large CD45-positive cell aggregates increased 15.7-fold compared to control. P-LPS and E. coli-LPS also significantly increased platelet aggregation and heteroconjugate formation but did not influence platelet degranulation and binding of platelets to leukocytes in whole blood. When using platelet-rich plasma (PRP), OMVs increased platelet aggregation 2.1-fold and CD62P expression 1.9-fold. P-LPS and E. coli-LPS also significantly increased platelet aggregation in PRP but did not influence platelet degranulation. None of the LPS preparations induced platelet microvesiculation, either in whole blood or in PRP., Conclusion: Meningococcal-derived OMVs as well as purified meningococcal LPS, contribute to increased platelet-platelet and platelet-leukocyte aggregation and may thus be of great importance in the development of microthrombosis and organ dysfunction related to fulminant meningococcal septicemia.

The objective of our study was to evaluate the effects of N(sigma)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of the nitric oxide (NO) pathway, on cerebral microcirculation during hypoxemia and reoxygenation with 100% oxygen in newborn pigs. Twenty-two pigs were randomized to hypoxemia [inspired fraction of oxygen (FIO(2)) 0.08; 20 min] and reoxygenation (FIO(2) 1.0; 60 min) or normoxia. The hypoxemic animals were further randomized to receive either an intravenous bolus injection of 5 mg/kg L-NAME (n = 8) or a corresponding volume of isotonic saline (n = 8) 30 min before the onset of hypoxemia. The normoxemic group (n = 6) received the same pretreatment with L-NAME. Cerebral hemodynamics were assessed by laser Doppler flowmetry and intracranial pressure monitoring. The cerebral NO concentration was continuously measured using an electrochemical sensor. Pretreatment with L-NAME resulted in a more severe systemic hypotension and reduced cerebral microcirculation during the period of hypoxemia compared with the saline/hypoxemia group. NO synthesis inhibition during reoxygenation with 100% oxygen, however, blunted the increase in NO concentration (p < 0.05) without reduction of cerebral blood flow and cerebral perfusion pressure. In conclusion, in this newborn pig model, pretreatment with a bolus infusion of L-NAME induced severe hypotension and reduced cerebral microcirculation during hypoxemia. However, it appears to have no significant adverse effect on cerebral hemodynamics during the period of reoxygenation with 100% oxygen. This deleterious effect during hypoxemia limits the use of L-NAME as a preventive drug but suggests beneficial effects during reoxygenation with 100% oxygen., (Copyright 2002 S. Karger AG, Basel)

The aim of this study was to investigate intracellular levels of reactive oxygen species (ROS) in circulating leukocytes in populations at risk for atherosclerosis compared with in healthy individuals. The study populations consisted of 27 non-diabetic men (aged 40-69 years) with untreated hypercholesterolemia (HC), 13 individuals (aged 39-56 years) with well-controlled insulin-dependent diabetes mellitus (DM), and 20 healthy individuals (aged 26-61 years) (REF). Citrated whole blood was collected in fasting condition. Using flow cytometric techniques, the resting levels and the response upon phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) stimulation of ROS were measured in circulating monocytes (MO) and granulocytes (GR). The relative mean fluorescence intensity (rMFI) in 10(4) leukocytes of fluorochromes mainly reflecting the levels of peroxynitrite (ONOO-) hydrogen peroxide (H2O2) and superoxide anion (O2-) was recorded. Significantly, higher basal levels of ONOO- in GR from the combined risk population compared with REF were found (1.4 vs. 1.5 rMFI, p<0.05). Upon PMA stimulation, significantly lower levels of O2 in GR in the risk populations compared to REF (119 vs. 90 Si, p<0.001) were observed. In conclusion, increased resting levels of ROS in circulating granulocytes, but reduced response to PMA stimulation could be demonstrated in populations at risk for atherosclerosis compared with in healthy individuals. This might indicate a higher degree of resting oxidative reactions, with partly exhausted cells and less capacity to host defence.