Nutrients and water masses in the Gulf of Maine - Georges Bank region: Variability and importance to blooms of the toxic dinoflagellate Alexandrium fundyense.

We report here the results of ten oceanographic survey cruises carried out in the Gulf of Maine - Georges Bank region of the Northwest Atlantic during the late spring to summer period in 2007, 2008 and 2010, for which we examine and characterize relationships among dissolved inorganic nutrient fields, water mass dynamics and cell densities of the toxic dinoflagellate Alexandrium fundyense. Nutrients are supplied to continental shelf waters of the Gulf of Maine - Georges Bank region by inflows of deep offshore water masses; once in the Gulf they are transported with the residual circulation and mix with surface waters, both in the Gulf and on the Bank. Those fluxes of offshore water masses and their nutrient loads are the major source of nutrients for phytoplankton production in the region, including annual blooms of A. fundyense in the Gulf and on Georges Bank. This much is already known. We suggest here that the locations and magnitude of A. fundyense blooms are controlled in part by variable nutrient fluxes to the interior Gulf of Maine from offshore, and, those interior Gulf of Maine waters are, in turn, the main nutrient source to Georges Bank, which are brought onto the Bank by tidal pumping on the Northern Flank. We present evidence that nitrate is the initial form of nitrogenous nutrient for A. fundyense blooms, but it is quickly depleted to limiting concentrations of less than 0.5 µM, at which time continued growth and maintenance of the population is likely fueled by recycled ammonium. We also show that phosphate may be the limiting nutrient over much of Georges Bank in summer, allowing recycled ammonium concentrations to increase. Our temperature-salinity analyses reveal spatial and temporal (seasonal and interannual) variability in the relative proportions of two deep source waters that enter the Gulf of Maine at depth through the Northeast Channel: Warm Slope Water (WSW) and Labrador Slope Water (LSW). Those two source waters are known to vary in their nutrient loads, with nitrate concentrations about 50% higher in WSW than LSW, for example, and as such the proportions of these two water masses to one another are important determinants of the overall nutrient loads in the interior Gulf. In addition to these deep slope water fluxes, we show evidence here of episodic fluxes of relatively fresh and low-nutrient shelf waters from the Nova Scotian Shelf, which enter the Gulf in pulses at depths between the surface and approximately 150 m, displacing deep slope waters, and consequently they significantly dilute the Gulf's interior waters, reducing nutrient concentrations and, in turn, affect the magnitude of A. fundyense blooms.

Distribution of two alternatively spliced variants of the type II collagen N-propeptide compared with the C-propeptide in bovine chondrocyte pellet cultures.

We have analyzed the distribution of type II collagen N- and C-propeptides in the cell layers and culture medium of bovine articular chondrocyte pellet cultures. Two splice variants of the type II collagen N-propeptide were detected by immunoblotting and immunoassay, using a new anti-peptide antibody, while the C-propeptide was detected using a monoclonal antibody. Type II collagen molecules containing the N-propeptide were detected weakly in cell layers, but not in tissue culture medium of chondrocyte pellet cultures, and both splice variants were observed. Free N-propeptide could not be detected in cell layers or medium. Type II procollagen molecules containing the C-propeptide were detected strongly in cell layers, but not in tissue culture medium, while the free C-propeptide was detected in both cell layers and medium. Since the N- and C-propeptides must be synthesized in a 1:1 molar ratio, we conclude that the N-propeptide is metabolized more quickly than the C-propeptide in this system. Our model can be used to study regulation of procollagen synthesis and propeptidase activity.

Expression of soluble endothelial adhesion molecules in clinical cardiopulmonary bypass.

Soluble endothelial adhesion molecule expression in clinical cardiopulmonary bypass (CPB) was investigated. Neutrophil-mediated endothelial injury plays an important role in CPB-induced organ dysfunction. The adhesion of neutrophil to the endothelium is central to this process. It has been well documented that CPB induces neutrophil activation and changes in neutrophil adhesion molecule expression, but the effect of CPB on endothelial cell activation is not known. This study was designed to measure soluble endothelial adhesion molecules during CPB. We made serial measurements (by specific enzyme-linked immunoabsorbent assay) of plasma levels of the soluble endothelial adhesion molecules, ICAM-1 and E-selectin in patients undergoing routine CPB (n = 7) and in a control group (thoracotomy, n = 3). The results show an initial significant decrease during CPB followed by an increase in plasma E-selectin from 29.3 +/- 5.1 ng/ml (mean +/- SEM) prebypass to 34.0 +/- 5.4 ng/ml at 48 h postbypass. Likewise, plasma ICAM-1 significantly decreased during CPB and then increased from 246.3 +/- 38.0 ng/ml before bypass to 324.8 +/- 25.0 ng/ml and 355.0 +/- 23.0 ng/ml at 24 and 48 h after bypass, respectively. The rise in levels is statistically significant (p < 0.05). This study shows a decrease in circulating ICAM-1 and soluble E-selectin during CPB and an increase in their levels at 48 h after CPB.

Changes in leucocyte counts and soluble intercellular adhesion molecule-1 and E-selectin during cardiopulmonary bypass in children.

A consequence of cardiopulmonary bypass (CPB) in young children is postoperative capillary leak and associated pulmonary dysfunction. Neutrophils sequester in the lungs and may contribute to functional endothelial damage. The endothelial adhesion molecules, E-selectin and intercellular adhesion molecule-1 (ICAM-1), mediate sequential steps in adhesion by binding to leucocyte ligands. Circulating forms of these proteins have been identified. We studied changes in the plasma concentrations of soluble E-selectin and soluble ICAM-1 using fixed phase immunoassays, and associated leucocyte counts in 10 paediatric patients undergoing CPB. Concentrations of soluble L-selectin and soluble ICAM-1 consistently fell during CPB from preoperative levels of 89 +/- 17 ng/ml (mean +/- 2SEM) and 218 + 61 ng/ml, respectively, to 39 +/- 7 ng/ml and 84 +/- 24 ng/ml, respectively at the beginning of maximum hypothermia. The haemodilution that occurred during CPB largely explained this fall, but not the more marked decrease in white cell counts that also occurred over this period (6.7 +/- 1.1 to 1.7 +/- 0.5 x 10(9)/l) which may reflect increased leucocyte sequestration. By 24 h postoperatively, levels of both soluble adhesion molecules approached preoperative concentrations, as did lymphocyte counts. In marked contrast, neutrophil counts rose appreciably towards the end of CPB, and continued to rise to a maximum of 10.9 +/- 3.1 x 10(9)/l during the immediate postoperative period and remained at these elevated levels 24 h later. Major consistent changes in circulating leucocyte numbers which occur early in cardiopulmonary bypass may reflect changes in adhesion to the endothelium and consequent sequestration. Alterations in the levels of soluble adhesion proteins may influence these processes.

Pyoderma gangrenosum in a child with congenital partial deficiency of leucocyte adherence glycoproteins.

Congenital deficiency of beta 2 integrin leucocyte adhesion molecules is a rare immunodeficiency and is often fatal. Neutrophils are unable to bind to ligands on the endothelium, and so cannot leave the circulation during inflammation or infection. When leucocyte adhesion deficiency (LAD) is caused by abnormally low expression of beta 2 integrins, it is termed LAD type 1. We describe a 5-year-old girl with a history of recurrent bacterial infections since early childhood who developed necrotic skin ulcers resembling pyoderma gangrenosum and a persistent circulating neutrophilia. Histologically, the lesions showed deep ulceration with a diffuse lymphohistiocytic infiltrate, but with a relative sparsity of neutrophils. Subsequent investigation revealed a complete absence of CD11a/CD18 beta 2 integrins on the surface of the patient's neutrophils, confirming the diagnosis of LAD type 1. The ulcers responded to treatment with oral prednisolone and colchicine.

Effects of inhibition of complement activation using recombinant soluble complement receptor 1 on neutrophil CD11b/CD18 and L-selectin expression and release of interleukin-8 and elastase in simulated cardiopulmonary bypass.

The inflammatory response to cardiopulmonary bypass includes activation of complement and induction of several neutrophil activation pathways. A recombinant soluble form of complement receptor 1 was used as a specific inhibitor of complement activation in simulated cardiopulmonary bypass circuits. Substantial complement activation was observed in these circuits with progressive accumulation of both plasma C3a and terminal complement complex. Soluble complement receptor 1 resulted in a significant reduction in C3a levels (p < 0.01) but did not inhibit terminal complement complex generation. A marked rise in neutrophil CD11b/CD18 expression, simultaneous loss of L-selectin expression, and a progressive accumulation of plasma elastase-alpha 1-antitrypsin occurred and were not affected by soluble complement receptor. However, generation of interleukin-8 in the circuits was inhibited (p < 0.05) by pretreatment with soluble complement receptor. These data suggest that changes in neutrophil activation seen during cardiopulmonary bypass may not be induced directly by anaphylatoxin generation.

Neutrophil adhesion molecules in term and premature infants: normal or enhanced leucocyte integrins but defective L-selectin expression and shedding.

The functional deficits of neonatal neutrophils are well documented and are thought to contribute to the increased susceptibility of newborn infants to infection. We measured the adhesion molecules L-selectin, CD11a/CD18 and CD11b/CD18 on neutrophils from the cord blood of term (n = 22) and premature (n = 32) infants using a whole blood method with flow cytometry and quantitative bead standards to enumerate cell surface receptors. We also assayed plasma for the shed form of L-selectin (sL-selectin). Our results suggested that L-selectin expression on term infant neutrophils is lower than that on adult neutrophils (unstimulated and stimulated, both P < 0.001), but that stimulated premature infant cell express higher L-selectin than term infants (P < 0.05); it is possible that this deficiency is caused by physiological changes occurring around the normal time of parturition. We observed reduced sL-selectin in term infants (P < 0.001) compared with adults, and even lower concentrations in premature infants (P < 0.001). The sL-selectin concentrations in plasma may be a reflection of granulopoiesis, which may be reduced in premature infants. Our results showed increased resting neonatal neutrophil expression of CD11b/CD18 compared with adults, and the absence of any neonatal deficit of the ability to up-regulate CD11b/CD18 expression on stimulation. These findings are contrary to previous reports. Further studies suggested that the isolation procedures used in previous reports reduces the capability of the cells to respond to a formyl methionine leucine phenylalanine (fMLP) stimulus. This effect is more marked in neonatal neutrophils, suggesting that the previously reported deficiency is in fact due to the isolation techniques used rather than the cells' innate ability to up-regulate CD11b/CD18 expression. The results of our study lead us to propose that the adhesive function of neonatal neutrophils may be less defective than previously thought.

Polymorphonuclear granulocyte expression of CD11a/CD18, CD11b/CD18 and L-selectin in normal individuals.

In this study direct immunofluorescence and flow cytometry with calibration using quantitative bead standards were used to enumerate the cell surface receptors CD11a/CD18, CD11b/CD18 and L-selectin. Holding blood at room temperature and fixation of samples prior to staining induced changes in expression, while immediate staining of polymorphonuclear granulocytes (PMN) in whole blood followed by fixation produced accurate values. The ranges of PMN adhesion molecule expression in 10 normal individuals were CD11a/CD18: 14794-28725, CD11b/CD18: 5300-11939 and L-selectin: 35662-61654 receptors per cell. Differences within individuals over 4 h were also observed. Adhesion molecule expression is used as an index of the adhesive function and state of activation of the cell. The data presented here shows that there is inherent variability in the expression of the PMN adhesion molecules between and within individuals, thus direct comparisons of PMN adhesion molecule expression between patients and "normals" must be interpreted with caution.

Humoral and cellular activation in a simulated extracorporeal circuit.

Endothelial injury consequent upon widespread humoral and cellular activation is probably a major contributor to the phenomenon of cardiopulmonary bypass-induced organ dysfunction. This article reviews some of the mechanisms by which complement and neutrophil activation and interleukin-8 may be involved in this inflammatory response. In a model consisting of a simulated extracorporeal circulation we were able to demonstrate complement activation, profound and specific changes in neutrophil adhesion molecule expression, and interleukin-8 generation. The importance of these changes and their potential interactions are discussed.

Changes in neutrophil CD11b/CD18 and L-selectin expression and release of interleukin 8 and elastase in paediatric cardiopulmonary bypass.

Children undergoing cardiopulmonary bypass (CPB) surgery for congenital heart defects develop an acute post-operative capillary leak which may be due to endothelial injury inflicted by adherent neutrophils (PMN). Direct immunofluorescence and flow cytometry were used to measure CD11a/CD18, CD11b/CD18 and L-selectin (L-s) expression on circulating PMN in CPB circuits containing human blood and in children undergoing CPB. In vitro, a general rise in CD11b/CD18 expression over 2 h contrasted with complete loss of L-s in a small but progressively increasing proportion of PMN. Marked but inconsistent changes in CD11b/CD18 and L-s were observed in vivo, in conjunction with fluctuations in circulating PMN count. Circulating IL-8 was detected starting at rewarming from hypothermia and reperfusion of the heart and lungs with a simultaneous, closely correlated rise in both PMN count and circulating elastase. IL-1 and TNF were not detected. These studies demonstrate changes in the pathways of PMN-endothelial interaction during and after CPB.