Dietary plant oil supplemented with arachidonic acid and eicosapentaenoic acid affects the fatty acid composition and eicosanoid metabolism of Atlantic salmon (Salmo salar L.) during smoltification.

This study sought to investigate whether a "natural diet" (mimicking the fatty acid composition of freshwater aquatic insects eaten by salmon parr) during the freshwater (FW) life stage of pre-smolt Atlantic salmon (Salmo salar L.) affected red blood cells and gill fatty acid composition as well as eicosanoid metabolism in gill during smolting at different temperatures. Before being transferred to seawater (SW), salmon parr were fed with a modified (MO) diet containing vegetable oils (rapeseed, palm, and linseed oils) supplemented with eicosapentaenoic acid (EPA) and arachidonic acid (ARA) to completely replace the fish oil (FO). Fatty acid composition in red blood cells and gill tissues was determined before SW transfer and six weeks after. Additionally, the expression of genes associated with eicosanoid metabolism and Na+/K+-ATPase (NKA) activity in salmon gill was examined at different temperatures before SW transfer and 24 h after. The results showed the changes in fatty acid composition, including sum monounsaturated fatty acids (MUFAs), docosahexaenoic acid (DHA), ARA, EPA, and sum n-6 polyunsaturated fatty acids (n-6 PUFA) in both red blood cells and gill tissues at the FW stage were consistent with the fatty acid profiles of the supplied MO and FO fish diets; however sum EPA and DHA composition exhibited opposite trends to those of the FO diet. The proportion of ARA, EPA, and n-6 PUFA increased, whereas sum MUFAs and DHA decreased in the red blood cells and gill tissues of MO-fed fish compared to those fed with the FO diet at FW stage. Additionally, 5-lipoxygenase-activating protein (Flap) expression was downregulated in MO-fed fish prior to SW transfer. During the process of SW transfer at different temperatures, the MO diet remarkably suppressed NKAα1a expression in MO-fed fish both at 12 and 16 °C. The MO diet also upregulated phospholipase A2 group IV (PLA2g4) expression in gills at 8, 12, and 16 °C, but suppressed phospholipase A2 group VI (PLA2g6) expression in gills at 12 °C compared to FO-fed fish at 12 °C and MO-fed fish at 8 °C. The MO diet also upregulated Cyclooxygenase 2 (Cox-2) expression at 8 °C compared to FO-fed fish and increased Arachidonate 5-lipoxygenase (5-Lox) expression in MO-fed fish at 16 °C compared to both FO-fed fish at 16 °C and MO-fed fish at 8 °C. Our study also determined that both SW transfer water temperatures and diets during the FW period jointly influenced the mRNA expression of PLA2g4, PLA2g6, and Lpl, whereas 5-Lox was more sensitive to dietary changes. In conclusion, the MO diet affected the fatty acid composition in gill and in red blood cells. When transferred to SW, dietary ARA supplementation could promote the bioavailability for eicosanoid synthesis in gill mainly via PLA2g4 activation, and potentially inhibit the stress and inflammatory response caused by different water temperatures through dietary EPA supplementation.

Tailoring freshwater diets towards boosted immunity and pancreas disease infection robustness in Atlantic salmon post smolts.

The aim of the current study was to investigate how freshwater diets impact on immunity in Atlantic salmon smolts in freshwater, during transfer to seawater and in post smolts during the seawater stage with and without pancreas disease (PD) infection. Three specific freshwater diets were prepared: (i) A diet similar in composition to commercial salmon freshwater diets (Standard diet); (ii) A diet composed of vegetable oils (rapeseed, palm and linseed oils) mimicking the fat composition in aquatic insects - the natural diet of wild salmon in freshwater (Fatty acid diet); (iii) A diet enriched with possible immune modulating amino acids including dl-methionine, l-lysine, l-threonine and taurine (Amino acid diet). After seawater transfer, all fish were fed the same commercial diet. Head kidneys were extracted, and their leukocytes isolated from smolts right before transfer to seawater, from post smolts one and six weeks after transfer to seawater, and from post smolts in seawater after 8 weeks of ongoing PD infection. In addition, to provoke bacterial or virus induced inflammation in vitro, the individual leukocyte suspension from all fish were stimulated by lipopolysaccharide (LPS) or polyinosinic acid: polycytidylic acid (PIC). The transfer of smolts from fresh-to seawater changed the transcription of several types of genes. Particularly in isolates from fish fed the Standard or Fatty acid diet in freshwater, overall gene transcription (IL-1ß, CD83, INF-γ, cox2, cd36, MGAT2, catalase) declined. However, the Amino acid diet stimulated the LPS induced gene transcription of IL-1ß, CD83, Cox2, and INF-γ at this stage. In freshwater smolts, PIC stimulated leukocytes showed higher transcription level of Mx and viperin in the Fatty acid and Amino acid diet groups compared to the Standard diet group. In seawater post smolts, Mx and viperin responded similarly to PIC challenge in all diet groups. Furthermore, leukocytes isolated from PD infected fish, continued responding to PIC, regardless of freshwater diet.

Hydrolyzed fish proteins reduced activation of caspase-3 in H2O2 induced oxidative stressed liver cells isolated from Atlantic salmon (Salmo salar).

Hydrolyzed fish proteins (H-pro) contains high concentrations of free amino acids and low molecular peptides that potentially benefit health. The following study aimed to test whether the water soluble phase of H-pro could reduce apoptosis and inflammation in primary liver cells isolated from Atlantic salmon following H2O2 provoked oxidative stress. Cells were grown as monocultures or co-cultured with head kidney cells to assess possible cross talk in inflammation and metabolism during treatments. Cells were grown in media with or without H-pro for 2 days before being stressed with 200 µM H2O2 then harvested 24 h post exposure. Both treatments were compared to the respective treatments without H2O2 supplementation. Oxidative stressed cells had increased activation of caspase-3, but supplementation with H-pro in the media prior to the oxidative stress reduced caspase-3 activation. In conclusion, free amino acids and low molecular weight peptides from H-pro attenuated oxidative stress, and made cells able to withstand apoptosis after H2O2 provoked oxidative stress.

Inhibition of p38 MAPK during cellular activation modulate gene expression of head kidney leukocytes isolated from Atlantic salmon (Salmo salar) fed soy bean oil or fish oil based diets.

Head kidney leukocytes isolated from Atlantic salmon fed either a diet based on fish oil (FO) or soy bean oil (VO) were used in order to evaluate if different lipid sources could contribute to cellular activation of the salmon innate immune system. A specific inhibitor of p38 MAPK, SB202190, was used to investigate the effect of lipopolysaccharide (LPS) signalling in the head kidney leukocytes. The results show that LPS up regulate IL-1ß, TNF-α, Cox2 expression in leukocytes isolated from fish fed either diet. The p38 MAPK inhibitor, SB202190, reduced the LPS induced expression of these genes in both dietary groups. In LPS stimulated leukocytes isolated from VO fed fish, SB202190 showed a clear dose dependent inhibitory effect on IL-1ß, TNF-α and Cox2 expression. This effect was also observed for Cox2 in leukocytes isolated from FO fed fish. Furthermore, there was a stronger mean induction of Cox2 in LPS stimulated leucocytes isolated from the VO-group compared to LPS stimulated leukocytes isolated from the FO-group. In both dietary groups, LPS stimulation of salmon head kidney leukocytes increased the induction of CD83, a dendrite cell marker, while the inhibitor reduced CD83 expression in the VO fed fish only. The inhibitor also clearly reduced hsp27 expression in VO fed fish. Indicating a p38 MAPK feedback loop, LPS significantly inhibited the expression of p38MAPK itself in both diets, while SB202190 increased p38MAPK expression especially in the VO diet group. hsp70 expression was not affected by any treatment or feed composition. There were also differences in p38MAPK protein phosphorylation comparing treatment groups but no obvious difference comparing the two dietary groups. The results indicate that dietary fatty acids have the ability to modify signalling through p38 MAPK which may have consequences for the fish's ability to handle infections and stress. Signalling through p38MAPK is ligand dependent and affects gene and protein expression differently.

Embryonic stem cells isolated from Atlantic cod (Gadus morhua) and the developmental expression of a stage-specific transcription factor ac-Pou2.

The establishment of embryonic stem cell cultures and the identification of molecular markers for undifferentiated embryonic stem cells (ESC) as well as differentiated cells types will open new opportunities in the study of developmental biology and for developing embryonic in vitro models of the ecologically and economically important fish specie Atlantic cod (Gadus morhua). We report here that cod blastula cells express a Class V POU gene known to be highly expressed in embryonic cell populations of vertebrates. The cod transcript, designated Atlantic cod-Pou2 (ac-Pou2), can be used as a genetic marker for cod blastula cells in vivo and in vitro. Using a quantitative real-time PCR approach, we found that the ac-Pou2 transcript was downregulated before the egg reached the stage of gastrulation, the starting point of extensive cell differentiation. We also demonstrate the culturing of ESC isolated from cod blastula stage eggs. The cod ESC exhibited in vitro characteristics of pluripotency described for both mammalian ESC and fish ES-like cells (medaka, zebrafish, seabream, sea perch and rainbow trout). Cod ESC in culture expressed ac-pou2, differentiated spontaneously and had the ability to form embryoid bodies following retinoic acid treatment. The ESC could also be directed to differentiate.

Novel B and T cell epitopes of chicken ovomucoid (Gal d 1) induce T cell secretion of IL-6, IL-13, and IFN-gamma.

BACKGROUND: Chicken ovomucoid (OM, Gal d 1) has an important role in the pathogenesis of IgE-mediated allergic reactions to hen's egg white. OBJECTIVES: The purpose of this study was to clarify the mechanisms of T cell recognition of ovomucoid using intact OM and chemically modified, characterized and homogeneous solid phase synthetic peptides covering the whole molecule. METHODS: Eighteen overlapping peptides were prepared by solid phase F-moc polyamide peptide synthesis (SPPS), characterized and high-pressure liquid chromatography (HPLC) purified. The peptides, together with intact, denatured and oxidized OM, were used to stimulate patient-derived cell cultures for mapping T cell epitopes. Proliferation responses, T cell phenotype and cytokine secretion using peripheral blood mononuclear cells (PBMC) from eight individuals and T cell lines (TCL) derived from six hen's egg-allergic patients, were examined. In addition, intact, denatured, oxidized and deglycosylated OM, as well as the peptides solely or with their keyhole limpet haemocyanin (KLH) complexes, were tested. For locating IgE and IgG B cell epitopes, seven egg-allergic patient sera and three OM-polyclonal sera were used. Healthy non-allergic individuals were included as controls. RESULTS: Seven peptides were recognized by specific IgE, while OM-specific TCL recognized 10 peptides. Six of the OM peptides were commonly recognized both by patient S-IgE and blood-derived TCL. Among those, one novel epitope, peptide OM 61-74, had the ability to bind IgE. Another peptide, OM 101-114, was recognized by IgE and IgG sera, but not by any of the TCLs. In contrast, the peptides OM 41-56, OM 71-84, OM 131-144 and OM 171-186 were exclusively T cell epitopes with no affinity to specific antibodies. Abundant TCL secretion of IFN-gamma, IL-6, IL-4, IL-13, IL-10 and TNF-alpha in response to OM stimulation indicates the contribution of Th2 as well as Th1/Th0 CD4+ cell subsets. For allergic patients moderate amounts of IFN-gamma, IL-13, and high amounts of IL-6, were secreted in response to TCL stimulation by OM peptides. High amounts of IL-6 were secreted in response to all molecular forms of OM (intact-, modified-OM and the peptides 71-84 and 51-64) when TCLs from two non-allergic donors were used. CONCLUSIONS: One novel B cell epitope (OM 61-74) and 10 T cell epitopes have been identified. The most reactive epitopes of the OM molecule comprise the motifs 1-14 to 71-84, the overlapping peptide-pairs OM 121-134 and OM 131-144 and peptides OM 161-174 and 171-186. Peptides OM 1-14 and 171-186 are the only ones capable of inducing IL-4 secretion. Only one peptide (OM 11-24) induces IL-10 secretion. Those peptides recognized as both T and B cell epitopes or only T cell epitopes, have the potential to induce T cell secretion of moderate to high amounts of IL-13, IFN-gamma and particularly IL-6.

Effects of beta2 adrenoceptor agonists on T-cell subpopulations.

The aim of the present communication is to determine the effects of beta2 adrenoceptor agonists on growth and cytokine secretion using allergen-specific T cells. Four beta2 adrenoceptor agonists were administered at therapeutically relevant doses (salbutamol 1-2 microM; salmeterol 0.03-0.06 microM; terbutaline 0.56-1.12 microM, and fenoterol 0.7-1.4 microM to: a) Cultures of human peripheral mononuclear cells (PBMC) b) Positively selected CD4+ and CD8+ subsets, c) Allergen-specific T-cell lines (TCL). Drug effects on growth kinetics and the secretion of IL-4, IL-5, INF-gamma and IgE following T-cell stimulation were investigated. Comparing the growth inhibitory effect of the 4 beta2 agonists at 2 different concentrations, using 12 PBMC, 10 CD4+ and CD8+ and 10 TCL cultures, the following patterns were observed: PBMC-, CD4+- and CD8+-cultures: salmeterol, followed by salbutamol and fenoterol, was a more potent inhibitor than terbutaline. In long-term TCL-cultures, salmeterol was the most potent drug, followed by fenoterol. No significant differences were observed between salbutamol and terbutaline. TCL secretion of IL-4 and IL-5 (TH2 cytokines) was also significantly inhibited. In one patient, INF-gamma secretion (TH1/THO cytokine) could be enhanced by drug administration. High IgE secretion, from 1% remaining B cells in one of the patients, following PHA+IL-2 stimulation, could be reduced by the drugs. The results showed that the beta2 agonists could influence T-cell growth and function. The changes regarding cell function were individual and related to T-cell phenotypes secreting TH1/THO or TH2 cytokines. These results suggest that administration of beta2 adrenoceptor agonists could be beneficial, not only for bronchodilation, but also for suppressing the underlying inflammatory process dominated by TH2-like cytokine secretion.

[Cost savings with autotransfusion in connection with coronary surgery]. / Kostnadsbesparelse ved autotransfusjon i forbindelse med koronarkirurgi.

The main purpose of this study was to estimate the costs and savings related to a consistent autotransfusion programme. More than 8,000,000 ml of autologous blood or blood-containing fluid were collected from 3,637 consecutive patients undergoing coronary artery bypass and returned to the patients during and after the operation. Economic analyses revealed a price of NOK 13 million (approximately 2 million USD) for this amount of autologous blood, or NOK 3,500 (600 USD) per patient. In the present series the need for bank blood or blood products was modest, since 69 patients (1.9%) received packed red cells, with or without plasma, 76 patients (2.1%) were given plasma transfusions only, and 11 patients (0.3%) received platelets. Thus, 95.7% of the patients were not exposed to any homologous blood products during the stay in hospital. Absence of morbidity related to the low homologous blood transfusion rate was assumed to save costs substantially, although this saving was difficult to calculate in terms of currency. Post-operative complications were few, and the total in-hospital mortality rate was 0.4%.

Facing the era of minimally invasive coronary grafting: current results of conventional bypass grafting for single-vessel disease.

BACKGROUND: The concepts of minimally invasive coronary artery bypass grafting have gained increasing attention and interest from cardiac surgeons. Operations through small incisions are mostly applied to patients with less extensive coronary disease, mostly single-vessel disease. The aim of this study was to identify a baseline level of conventional coronary bypass grafting for this group of patients, particularly with regard to surgical complications and immediate results. METHODS: Of 3,637 consecutive patients undergoing coronary artery bypass grafting during the period 1989 to 1995, 99 patients (2.7%) were identified to have single-vessel disease. The preoperative and hospital data of this subset of patients were analyzed. RESULTS: The left internal mammary artery was grafted in 96% of the patients, either as single graft to the left anterior descending artery or sequentially to the left anterior descending artery and a diagonal branch. Additional vein grafts were placed in 36 patients, and the mean number of distal anastomoses was 1.6 +/- 0.6. Mean ischemic time and cardiopulmonary bypass time were 15.3 +/- 9.6 minutes and 29.0 +/- 12.5 minutes, respectively. The patients were weaned from the ventilator 1.5 +/- 0.8 hours postoperatively, and all patients were out of bed the morning after the operation. No patients required homologous blood or plasma transfusions. The morbidity rate was low, and all patients survived. CONCLUSIONS: For this highly selected group of patients, coronary artery bypass grafting based on median sternotomy, cardiopulmonary bypass, and cardioplegic arrest carries a very high rate of immediate success. Such data may be useful as a baseline when considering the costs and benefits of new surgical procedures.

Heparinized cardiopulmonary bypass and full heparin dose marginally improve clinical performance.

BACKGROUND: The use of completely heparin coated cardiopulmonary bypass circuits in combination with a reduced systemic heparin dose has previously been shown to reduce postoperative bleeding after cardiac operations. However, it has remained unknown whether this effect was related to the improved biocompatibility of the heparin-treated surfaces per se or to the reduced exposure to circulating heparin. Therefore we investigated patients undergoing heparin-coated extracorporeal circulation and full systemic heparinization. METHODS: Two hundred seventeen patients having first-time myocardial revascularization were prospectively randomized either to a group in which a completely ("tip-to-tip") heparin-coated circuit (Duraflo II) was used for perfusion (n = 107) or to a control group (n = 110) in which an uncoated, but otherwise identical, circuit was used. Full systemic heparinization was induced in both groups (activated clotting time, > 480 seconds). The postoperative blood loss, requirements for homologous blood transfusions, clinical performance, and complications were recorded. RESULTS: The amount of postoperative mediastinal drainage was nearly identical in the two groups. The mean 18-hour drainage was 694 +/- 313 mL in the heparin-coated group and 679 +/- 269 mL in the control group (p = not significant). Three patients in the heparin-coated group and 6 patients in the control group received homologous red blood cell transfusions (p = not significant). The incidence of postoperative atrial fibrillation was significantly lower in the heparin-coated group (21.8%) than in the control group (43.1%) (p = 0.002). Otherwise, there were no significant differences in the extubation times, the incidence of perioperative myocardial infarction, the creatinine concentration, the incidence of neurologic dysfunction, the progress in physical rehabilitation, or the hemoglobin concentration at discharge. CONCLUSIONS: The use of completely heparin coated cardiopulmonary bypass circuits and full systemic heparinization in patients undergoing coronary artery bypass procedures did not reduce postoperative bleeding or change clinical performance, except for a significant decrease in the incidence of postoperative atrial fibrillation.

Specific T cell lines for ovalbumin, ovomucoid, lysozyme and two OA synthetic epitopes, generated from egg allergic patients' PBMC.

BACKGROUND: Proteins of hen egg whites are common ingredients in food and difficult to eliminate. Allergens of egg white induce allergic symptoms among relatively high numbers of patients suffering from food allergy. B cell epitopes to hen egg white major allergens have been reported. Considering that IgE antibody formation is mostly T cell dependent, the study of T cell epitopes is essential for both T cell dependent and independent IgE response. OBJECTIVES: Little information on T cell epitopes recognizing food allergens has been reported. T cell responses to hen egg white allergens and two synthetic OA peptides located at amino acid residues No. 105-122 and 323-339 were investigated. METHODS: Peripheral blood mononuclear cells from hen egg allergic patients were investigated. Various allergens of hen egg white were used for stimulation. Primary proliferation responses were detected followed by the generation of long-term cultures which were examined for their specificity, phenotype, cytokine profile and IgE production. The allergen specific T cell lines were mapped using a panel of 13 synthetic peptides of ovalbumin. RESULTS: Human T cells recognizing ovomucoid, lysozyme and ovalbumin epitope 105-122 are reported for the first time. The cell lines were enriched CD4+/CD8+ T cells (CD2+ > 95%). Ovomucoid and ovalbumin induced IgE synthesis by a small fraction of B cells (1%) present in the ovalbumin and ovomucoid specific T cell lines. CONCLUSIONS: Human T cells recognized several egg white allergens and epitopes within the ovalbumin molecule. Specific IgE was produced in cultures stimulated with ovalbumin and ovomucoid. OA peptides 105-122 and 323-339 have no affinity to the specific IgE of the two patients; an observation which could be of particular interest regarding the mechanisms of peptide-based immunotherapy.

Complete heparin-coated cardiopulmonary bypass and low heparin dose reduce complement and granulocyte activation.

Complete heparin-coated extracorporeal circuits, including cardiotomy reservoir, have recently become available for routine cardiac surgery. The effects on complement and granulocyte activation using a heparin-coated circuit in combination with reduced systemic heparinization (activated clotting time (ACT) > 250 s) were studied in 33 patients undergoing elective first time myocardial revascularization. The patients were prospectively randomized either to a heparin-coated group (Group H, n = 17), or to a control group (Group C, n = 16) treated with an identical uncoated circuit and full heparin dose (ACT > 480 s). During cardiopulmonary bypass (CPB) the C3 activation products C3b, iC3b, and C3c (C3bc) and the terminal SC5b-9 complement complex (TCC) increased markedly in both groups compared to baseline, but to a much lesser extent in the heparin-coated group. The maximal increase of C3bc during the operation was a median of 28 arbitrary units (AU)/ml in the heparin-coated group, compared to 45 AU/ml in the control group (P = 0.01). Similarly, in Group H the maximal increase of TCC was significantly lower (median 0.8 AU/ml) than the levels recognized in Group C (median 1.9 AU/ml) (P < 0.0001). The release of the granulocyte activation enzymes lactoferrin and myeloperoxidase also increased during CPB in both groups compared to baseline level. The maximal increase of lactoferrin concentration was a median of 229 micrograms/l in Group H and significantly lower than 647 micrograms/l in the control group (P = 0.0002). As for myeloperoxidase, there were no significant intergroup differences. In conclusion, a complete heparin-coated circuit and low systemic heparinization for CPB in coronary artery surgery were associated with reduced activation of the complement system and less release of lactoferrin. The results indicate improved biocompatibility of this option for extracorporeal circulation.

Complete heparin-coated (CBAS) cardiopulmonary bypass and reduced systemic heparin dose; effects on coagulation and fibrinolysis.

OBJECTIVE: Heparin-coated extracorporeal circuits allow reduced amounts of systemic heparin and protamine. However, the effects on the coagulation and fibrinolytic systems when reducing systemic anticoagulation, have partly remained unknown. METHODS: Thirty-three patients undergoing elective first time myocardial revascularization were prospectively randomized either to have a cardiopulmonary bypass (CPB) circuit completely coated with covalently bound heparin, in combination with reduced systemic heparinization (activated clotting time (ACT) > 250 s (n = 17), or to a control group perfused with identical but uncoated circuits and full heparin dose (ACT > 480 s) (n = 16). Tests indicative of thrombin generation, platelet activation, and fibrinolytic activity were performed intraoperatively and postoperatively. RESULTS: During CPB, the plasma level of prothrombin fragment 1.2 (PF 1.2) increased from median 1.5 (1.1-1.9) nmol/l to 5.4 (3.3-6.6) nmol/l in the heparin-coated group, and was significantly higher (P = 0.01) than the increase from 1.4 (1.2-1.9) nmol/l to 3.2 (2.2-4.3) nmol/l seen in the control group. However, the increase on CPB was modest compared to the major elevation observed after completed surgery and reversal of the anticoagulation. The concentrations reached median 9.7 (6.8-19.5) nmol/l in the heparin-coated group and 13.2 (4.2-18.4) nmol/l in the control group (no significant intergroup difference). A similar pattern was observed for the thrombin-antithrombin (TAT) complex. Regression analysis revealed significant correlation between the levels of the thrombin markers and duration of CPB in both groups (P < 0.05). There was no correlation between ACT or plasma heparin levels on bypass and the PF 1.2 and TAT complex. The platelet release of beta-thromboglobulin increased in both groups during CPB and significantly more in the control group at the end of bypass (P < 0.01), indicating less platelet activation in the heparin-coated group. There were no significant intergroup differences with regard to fibrinolytic activity. Plasma fibrinogen as well as platelet counts were unchanged after the operation, compared to baseline. Except for one patient in the control group sustaining perioperative myocardial infarction, the postoperative course was uneventful in all cases. CONCLUSIONS: Completely heparin-coated CPB can safely be performed in combination with reduced systemic heparinization. The heparin and protamine amounts could be lowered to 35% of normal doses. Indications of more thrombin generation on CPB compared to the uncoated controls were seen, but the levels remained within low ranges in both groups. There was no evidence of thromboembolic episodes or clot formation in the extracorporeal circuits.

Complement and granulocyte activation in two different types of heparinized extracorporeal circuits.

Complement and granulocyte activation were studied in cardiopulmonary bypass circuits completely coated with either end-attached covalent-bonded heparin, the Carmeda BioActive Surface, or with the Duraflo II bonded heparin, in combination with reduced systemic heparinization (activated clotting time > 250 seconds). The control groups were perfused with uncoated circuits and full heparin dose (activated clotting time > 480 seconds). Altogether 67 patients undergoing elective first-time myocardial revascularization were investigated, having extracorporeal perfusion with a Duraflo II coated circuit (n = 17), an identical but uncoated circuit (n = 17), a Carmeda coated circuit (n = 17), or an equivalent uncoated circuit (n = 16). During cardiopulmonary bypass, the C3 activation products C3b, iC3b, and C3c (C3bc) and the terminal SC5b-9 complemented complex increased markedly in all four groups compared with baseline, but significantly less in the two coated groups than in their control groups. Additionally, a significantly lower concentration of C3bc was observed in the Carmeda coated group, with maximal increase of median 28 AU/ml compared with 50 AU/ml in the Duraflo II coated group (p = 0.003). Similarly, in the Carmeda coated group, the maximal increase of terminal complement complex was considerably lower (0.8 AU/ml) than the levels recognized in the Duraflo II coated group (2.4 AU/ml) (p < 0.001). The release of the granulocyte activation myeloperoxidase and lactoferrin increased from the beginning of the operation, with peak levels at the end of bypass. A significant reduction of lactoferrin release was recognized when comparing the coated groups with the control groups. The difference between the two coated groups (Carmeda 228 micrograms/L; Duraflo II 332 micrograms/L; p = 0.05) was marginally significant. For myeloperoxidase, no significant differences were observed between the coated and uncoated groups. In conclusion, both types of heparin-coated circuits reduced complement activation and release of lactoferrin, but the Carmeda circuit proved to be more effective than the Duraflo II equipment.

High and low heparin dose with heparin-coated cardiopulmonary bypass: activation of complement and granulocytes.

BACKGROUND: Cardiopulmonary bypass with heparin-coated circuits allows reduced amounts of systemic heparin. Heparin inhibits activation of the complement cascade experimentally, but the effects of different levels of systemic heparin on activation of complement and granulocytes in patients have remained unknown. METHODS: Fifty-two patients undergoing coronary artery bypass procedures were studied. Cardiopulmonary bypass circuits completely coated with surface-bound heparin were used for one group given low-dose heparin (n = 17) (activated clotting time > 250 seconds), and was compared with a second group having normal high-dose heparin (activated clotting time > 480 seconds) (n = 18). A third control group was perfused with ordinary uncoated circuits and a full heparin dose (n = 17). RESULTS: During cardiopulmonary bypass, the C3 activation products C3b, iC3b, and C3c increased markedly in all three groups compared with baseline, but significantly less in the two heparin-coated groups (high dose, median maximal increase 58 arbitrary units (AU)/mL; low dose, 48 AU/mL) compared with the uncoated control group (74 AU/mL) (p < 0.01). The difference between the two coated groups was not significant. Similarly, the maximal increase in terminal SC5b-9 complement complex was considerably lower in the heparin-coated groups (high dose, 2.5 AU/mL; low dose, 2.6 AU/mL) compared with the level observed in the uncoated control group (5.3 AU/mL) (p < 0.01). The release of the granulocyte activation enzymes myeloperoxidase and lactoferrin increased from the beginning of the operation, with peak levels at the end of cardiopulmonary bypass (p < 0.01). The concentration of lactoferrin was significantly (p < 0.01) reduced in the low heparin dose group compared with the two other groups receiving normal high heparin doses, indicating that circulating heparin is an important granulocyte agonist, acting independently of the presence or absence of heparin-coated surfaces. Also for myeloperoxidase a higher level was observed in the high heparin dose group. CONCLUSIONS: Complement activation was significantly reduced in both heparin-coated groups and was independent of the level of systemic heparinization, whereas granulocyte activation was reduced only in patients who received low doses of systemically administered heparin. The results indicate that a moderate reduction of the systemic heparin dose may be an advantage with regard to improved biocompatibility when using heparin-coated cardiopulmonary bypass circuits.

Effects on coagulation and fibrinolysis with reduced versus full systemic heparinization and heparin-coated cardiopulmonary bypass.

BACKGROUND: Extracorporeal circulation with circuits coated with surface-bound heparin has allowed reduced levels of systemic heparinization. Clinical benefits have included reduced postoperative bleeding and less homologous blood usage. However, the effects on the hemostatic and fibrinolytic systems have remained in part unknown. METHODS AND RESULTS: Indications of thrombin generation, platelet activation, and fibrinolytic activity were investigated in patients undergoing coronary artery bypass surgery. Two groups were perfused with cardiopulmonary bypass (CPB) circuits completely coated with surface-bound heparin: one group with low systemic heparin dose (activated clotting time [ACT] > 250 seconds; n = 17) and a second group having a full heparin dose (ACT > 480 seconds; n = 18). A third control group was perfused with ordinary uncoated circuits and full heparin dose (n = 17). The plasma level of thrombin-antithrombin complex and prothrombin fragment 1.2 increased in all groups during bypass, and somewhat more in both the heparin-coated groups toward the end of CPB, compared with the control group (P < .01). However, the increase during CPB was minimal compared with the major elevation observed 2 hours after surgery in all groups. Platelet release of beta-thromboglobulin increased in all groups (P < .01) during CPB and significantly more in the high-dose group compared with the other two groups (P = .03). Fibrinolytic activities were similar in all groups, and there were no indications of major consumption of coagulation factors. CONCLUSIONS: Reduced systemic heparinization (ACT > 250 seconds) in patients having extracorporeal circulation with completely heparin-coated circuits did not lead to increased thrombogenicity. Thrombin formation remained within low ranges during CPB compared with patients receiving a full heparin dose and with the major elevations observed after surgery.

Completely heparinized cardiopulmonary bypass and reduced systemic heparin: clinical and hemostatic effects.

BACKGROUND: When heparinized circuits are used for cardiopulmonary bypass, the amounts of heparin and protamine administered systemically can be reduced. However, it is not entirely known what effects this reduction in systemic anticoagulation has on clinical performance and on the coagulation and fibrinolytic systems. METHODS: Two hundred three patients undergoing first-time elective myocardial revascularization were prospectively randomized either to a group in which a completely heparin-coated circuit was used for perfusion (group H; n = 101 patients) and in which a reduced heparin dose was given (activated clotting time, > 250 seconds) or to a control group (group C; n = 102 patients) in which an uncoated, but otherwise identical, circuit was used and in which full systemic heparinization was induced (activated clotting time, > 480 seconds). Indicators of thrombin generation, platelet activation, and fibrinolytic activity were studied in a subset of 34 patients. RESULTS: The total amount of postoperative mediastinal drainage was significantly reduced in group H (median, 575 mL) compared with that in group C (median, 635 mL; p = 0.002). Two patients in group C but none in group H received homologous red blood cell transfusions (p = not significant). The loss of hemoglobin in group H was a median of 21 g/L, and this was significantly lower than the 25 g/L noted in the control group (p = 0.006). During cardiopulmonary bypass, the plasma levels of thrombin-antithrombin complex and prothrombin fragment 1.2 increased in both groups. At the end of cardiopulmonary bypass the plasma levels of these markers of thrombin formation were significantly higher in group H, although the increase was modest compared with the major increase observed 2 hours after operation in both groups. There were no significant intergroup differences in the platelet counts, the concentration of beta-thromboglobulin, or the plasma levels of fibrinogen and D-dimer. No differences in perioperative morbidity, the postoperative kidney function, or the intubation time were observed, and there were no hospital deaths. CONCLUSIONS: The combination of complete heparin-coated cardiopulmonary bypass circuits and low systemic heparinization is safe for patients undergoing elective coronary artery bypass procedures and reduces the perioperative blood loss. There was no evidence of increased thrombogenicity, fibrinolytic activity, or consumption of coagulation factors. No clinical or technical side effects were observed.

Conversion of postischemic ventricular fibrillation with intraaortic infusion of potassium chloride.

BACKGROUND: Ventricular fibrillation after declamping of the aorta after cardioplegic arrest is commonly managed by direct-current countershock. However, in coronary artery bypass grafting, placement of the electrodes can cause mechanical damage to the grafts and anastomoses, and the surgical procedure must be interrupted. As an alternative, intraaortic infusion of potassium chloride through the arterial line from the heart-lung machine was investigated. METHODS: In a series of 100 patients with postischemic ventricular fibrillation (group P), 20 mmol of potassium chloride (plus 10 mmol later if necessary) was added to the oxygenator reservoir and perfused through the arterial line into the proximal aorta. The results were compared with those in a matched control group of 100 patients primarily treated with direct-current countershock (group DC). RESULTS: In group P, the ventricular fibrillation was effectively converted to a supraventricular rhythm in 82% of the patients. The remaining 18 patients required significantly (p < 0.005) fewer electric shocks than the patients in group DC. Serum K+ levels were slightly elevated for a short period after the potassium chloride infusion. Otherwise there were no significant differences in regard to incidence of heart block, temporary epicardial pacing, myocardial infarction, or atrial fibrillation between the two groups. CONCLUSIONS: Conversion of postischemic ventricular fibrillation with potassium chloride administered through the arterial line from the heart-lung machine is an effective, gentle, and convenient method. No side effects were noted.

The effect of disodium cromoglycate (DSCG) on in vitro proliferation of CD4+, CD8+, and CD19+ cell populations derived from allergic and healthy donors.

The effect of disodium cromoglycate (DSCG) on in vitro proliferation of CD4+ and CD8+ T cells and CD19+ B cells, positively selected by immunomagnetic separation, was investigated. The cells were obtained from allergic patients with moderate serum IgE levels and mild to moderate atopic dermatitis, and healthy controls. The different cell subfractions were stimulated with mitogens or specific allergens, as well as cell supernatants from the lymphoblastoid B- (RPMI 8866) and T-hybridoma (166 A2) cell lines. Proliferative responses of T- and B-cell subsets stimulated with mitogens together with recombinant interleukin-2 (rIL-2) or accessory cells (AC) could be inhibited by DSCG. In allergic individuals, significant allergen-specific stimulation could be observed in the CD8-depleted peripheral blood mononuclear cell (PBMC) fractions. Isolated CD4+ T cells, without AC or IL-2, could also be stimulated with specific allergen, but the responses were rather low. DSCG inhibited, concentration dependently, all allergen-induced responses. Interestingly, only atopic derived CD4+ and CD8+ T cells were stimulated by soluble low-affinity IgE receptor (Fc epsilon RII/sCD23) and IgE binding factor (IgEBF), including IgE enhancing factor, present in culture supernatants from RPMI 8866 and 166 A2, respectively. These responses were also inhibited by DSCG. This was in contrast to the amplifying effect of DSCG on spontaneously proliferating RPMI 8866 and 166 A2 cells, cultured in fresh cRPMI 1640 medium without sCD23 and IgE enhancing factor. Our results show that DSCG delivers an inhibitory signal or signals to PBMC subpopulations expressing Fc epsilon RII/sCD23, either upregulated by phytohemagglutinin in normal and atopic cells, or by allergens or sCD23 in atopic cells.(ABSTRACT TRUNCATED AT 250 WORDS)