Cryoprecipitate reversal of opsonic alpha2-surface binding glycoprotein deficiency in septic surgical and trauma patients.

Human opsonic alpha2-surface binding glyoprotein (alphs2SB-glycoprotein), a molecule having immunologic identity with an amino acid composition similar to cold-insoluble globulin, is concentrated in a cryoprecipitate of plasma. Septic surgical and trauma patients manifesting opsonic alpha2SB-glycoprotein deficiency and associated reticuloendothelial system dysfunction were treated by intravenous infusion of cryoprecipitate. This therapy restored circulating bioreactive and immunoreactive opsonin and improved their septicemia, pulmonary insufficiency, and duration of recovery. Cryoprecipitate infusion may offer a new approach to the treatment of septic injured patients in preventing multiple organ failure; measurement of immuno-reactive serum opsonic alpha2SB-glycoprotein may provide a noninvasive index of reticuloendothelial system function and patient status during servere sepsis that follows trauma.

Mechanism of acute depletion of plasma fibronectin following thermal injury in rats. Appearance of a gelatinlike ligand in plasma.

Plasma fibronectin was depleted within 15 min following sublethal burn, followed by partial recovery at 8 h and complete restoration by 24 h in anesthetized rats. Radiolabeled 75Se-plasma fibronectin, injected intravenously before burn, was rapidly sequestered in burn skin as well as the liver. Fibronectin levels at 2 h postburn as detected by immunoassay vs. 75Se-plasma fibronectin indicated that more fibronectin was in the plasma than detected by electroimmunoassay. Crossed immunoelectrophoretic analysis of fibronectin in early postburn plasma demonstrated a reduced electrophoretic mobility of the fibronectin antigen. Addition of heparin or fibrin, both of which have affinity for fibronectin, to normal plasma was unable to reproduce this altered fibronectin electrophoretic pattern. In contrast, addition of gelatin or native collagen to normal plasma reproduced the abnormal electrophoretic pattern of fibronectin seen in burn plasma. Extracts of burned skin, but not extracts of normal skin, when added to normal plasma, elicited a similar altered electrophoretic pattern for fibronectin. By gel filtration, fibronectin in burn plasma had an apparent molecular weight approximately 40% greater than that observed in normal plasma. These data suggest the release into the blood of a gelatinlike ligand from burned skin, which complexes with plasma fibronectin. Thus, fibronectin deficiency acutely postburn appears mediated by (a) its accumulation at the site of burn injury; (b) its removal from the circulation by the liver; and (c) its presence in the plasma in a form that is less detectable by immunoassay.

Plasma fibronectin synthesis in normal and injured humans as determined by stable isotope incorporation.

In humans, plasma fibronectin decreases early after operative injury, burn, or trauma, followed by a rapid restoration with a secondary decline typically observed if such patients become septic. We determined the rate of plasma fibronectin and plasma fibrinogen synthesis in normal subjects and injured patients using a stable isotope incorporation technique with [15N]glycine. During a constant 14-h infusion of [15N]glycine, the enrichment of [15N]glycine in both the free plasma glycine precursor pool as well as the urinary hippurate pool was determined; the latter used as an estimate of intracellular hepatic precursor enrichment. [15N]Glycine enrichment in both plasma fibronectin and fibrinogen was also quantified. The synthesis rate (Js/V) expressed in micrograms per milliliter of plasma per hour and the fractional synthesis rate (FSR) expressed as percentage of the plasma pool produced per day were determined. In normal subjects, the FSR for plasma fibronectin using 15N enrichment into urinary hippurate was 35.35 +/- 1.46%/d, whereas the Js/V was 4.45 +/- 0.19 micrograms/ml plasma per h. In normal subjects, the FSR for plasma fibronectin using 15N enrichment into free plasma glycine was 14.73 +/- 0.63%/d, whereas the Js/V was 1.98 +/- 0.09 micrograms/ml plasma per h. Early (2-3 d) after burn injury, fibronectin synthesis was increased (Js/V = 5.74 +/- 0.36; P less than 0.05), whereas later after injury, fibronectin synthesis began to decline (Js/V = 3.52 +/- 0.24; P less than 0.05) based on 15N enrichment of urinary hippurate. In contrast, the Js/V and FSR of plasma fibrinogen, a well-documented acute-phase plasma protein, revealed a sustained elevation (P less than 0.05) after injury in both the trauma and burn patients. Thus, plasma fibronectin synthesis is elevated early postinjury, which may contribute to the rapid restoration of its blood level. However, once fibronectin levels have normalized, the synthesis of plasma fibronectin appears to decline.

Von Willebrand factor (VIII R:Ag), fibronectin, and insulin-like growth factors I and II in diabetic retinopathy and nephropathy.

We have measured plasma von Willebrand factor (VWF) as the factor VIII-related antigen, plasma fibronectin, and two of the serum somatomedins, insulin-like growth factor I (IGF I) and IGF II, in 51 diabetic patients and 25 nondiabetic control subjects. VWF was significantly higher in the diabetic group than in the controls (173 +/- 9% SEM versus 101 +/- 9%, P less than 0.001), as has been reported by others. However, within the diabetic group there was no significant difference in VWF between those patients without retinopathy, those with background or proliferative retinopathy, or those with macular edema. There was also no difference in VWF between the diabetic subjects with and those without proteinuria. These results rule against a previously advanced hypothesis that the increase in VWF in patients with diabetes is secondary to microangiopathy. No significant difference was observed in fibronectin, IGF I, or IGF II between the diabetic and control groups, between the diabetic group without retinopathy and the retinopathic subgroups, and between the diabetic subjects with and without proteinuria. In the diabetic patients, there was no correlation between diabetic control as assessed by glycosylated hemoglobin and glycosylated serum protein, and the plasma levels of VWF, fibronectin, IGF I, or IGF II. The results of this study strongly suggest that neither plasma VWF, fibronectin, IGF I, nor IGF II plays an important primary role in the pathogenesis of diabetic microvascular disease, although one or more of these factors might play a permissive role.

Hepatocyte injury during post-operative sepsis: activated neutrophils as potential mediators.

We evaluated the concept that the vascular entrance of both bacterial and nonbacterial particulate material could lead to hepatic parenchymal cell injury, either due to postphagocytic Kupffer cell activity or the margination of activated leukocytes in the liver. Injection of denatured, collagen-coated particles as well as heat-killed bacteria were used as particulate challenges. Hepatic parenchymal cell injury in vivo during postoperative sepsis was evaluated by plasma aspartate aminotransferase (AST) and ornithine carbamyl transferase (OCT) enzyme levels over 3-72 h. AST and OCT levels elevated following either laparotomy plus cecal ligation (mild sepsis) or laparotomy plus cecal ligation with puncture (severe sepsis), with the peak level at 24 h. In addition, the direct intravenous injection of either nonbacterial foreign particles or heat-killed Pseudomonas aeruginosa into normal rats also produced a dose-dependent elevation of AST and OCT. The plasma level of either AST or OCT actually increased 350-400% after injection of the non-bacterial particles. A similar dose related elevation in enzymes followed the intravenous injection of heat-killed Pseudomonas. To differentiate the potential contribution of activated hepatic Kupffer cells versus activated marginated neutrophils to the in vivo hepatic injury, we determined the release of the hepatic specific enzyme OCT by cultured hepatic parenchymal cells when they were exposed to isolated Kupffer cells or isolated PMNs that were activated by exposure to dead bacteria. Bacteria alone when added to cultured hepatocytes did not induce significant OCT release. In contrast, activated PMNs but not Kupffer cells induced a significant (p less than 0.05) release of OCT from parenchymal cells into the culture media. Thus, in vivo transient hepatic parenchymal cell injury with post-operative sepsis may be mediated by the margination of activated PMNs in the liver.

Generation of neutrophil chemotactic activity by phorbol ester-stimulated calf pulmonary artery endothelial cells.

Chemotactic activity for human polymorphonuclear leukocytes (PMNL) was detected in serum-free conditioned media 1 to 4 hr after monolayers of calf pulmonary artery endothelial cells were pretreated with phorbol myristate acetate (PMA). Chemotactic activity was increased in conditioned media following pretreatment with either PMA or the less lipophilic active phorbol ester, 4-beta-phorbol-12,13-dibutyrate (P(Bu)2) in a dose-dependent manner. Chemotactic activity of conditioned media from PMA-treated endothelial cells was confirmed by checkerboard analysis. The chemotactic activity in conditioned media from PMA-pretreated endothelial cells was completely inhibited by pretreating endothelial cells with either cycloheximide, actinomycin D, or the lipooxygenase inhibitor, diethylcarbamazine. Furthermore, the chemotactic activity was heat-stable, inhibited by trypsin treatment, and present in both aqueous and lipid phases after ether extraction. The data demonstrate that pulmonary artery endothelial cells exposed to active phorbol esters release potent chemotactic factor(s) for PMNL. These findings suggest a role for activators of protein kinase C in mediating endothelial cell release of chemotactic factor(s) that may be important in the directed migration of circulating leukocytes to sites of vascular injury.

Fibronectin-enhanced attachment of gelatin-coated erythrocytes to isolated hepatic Kupffer cells.

The phagocytic process is a combination of a sequence of events which includes a recognition attachment phase and a subsequent internalization phase. The present study was designed to investigate the effect of plasma fibronectin on the attachment and ingestion of gelatinized sheep erythrocytes to isolated rat Kupffer cells in a monolayer assay. Kupffer cells were isolated by sequential collagenase-pronase digestion followed by metrizamide density gradient centrifugation and subsequent adherence to plastic. Classification as Kupffer cells was confirmed by the presence of functional Fc receptors, a positive peroxidase reaction, and phagocytic activity. Purified plasma fibronectin as well as rat serum containing fibronectin promoted attachment of gelatinized fixed sheep erythrocytes to Kupffer cells in a dose-dependent manner, whereas fibronectin-deficient serum did not. Heparin did not enhance the fibronectin-mediated attachment or ingestion of gelatinized sheep erythrocytes at lower particle doses, whereas at higher particle doses heparin augmented the response. These results indicate that fibronectin can enhance the binding and ingestion of foreign gelatin-coated particulates by Kupffer cells.

Proteolysis of gelatin-bound fibronectin by activated leukocytes: a role for leukocyte elastase.

Fragmentation of subendothelial matrix-bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonuclear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 125I rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125I-labelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125I-fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked in a dose-dependent manner by the addition of both methoxysuccinyl-alanine-alanine-valine chloromethyl ketone (AAPVCK), a leukocyte elastase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non-specific serine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large molecular weight fragments (60-200 kD) were not labelled, but the smaller molecular weight fragments (less than 45 kD), derived from the artificial matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to proteolytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of activated blood leukocytes.

Hepatic removal of 125I-DLT gelatin after burn injury: a model of soluble collagenous debris that interacts with plasma fibronectin.

The decline of plasma fibronectin after surgery, trauma, and burn, as well as during severe sepsis after injury, appears to limit hepatic Kupffer cell phagocytic activity. Intravenous infusion of gelatin-coated particles to simulate blood-borne particulate collagenous tissue debris in the circulation after injury also depletes plasma fibronectin. We used soluble gelatin conjugated with 125I-labeled dilactitol tyramine (DLT-gelatin) as a model of soluble collagenous tissue debris. We studied its blood clearance as well as organ localization in normal and postburn rats. Fibronectin-deficient plasma harvested early after burn exhibited limited ability to support in vitro phagocytic uptake of the gelatinized microparticles by Kupffer cells in liver tissue from normal rats. However, Kupffer cells in liver tissue from normal and postburn rats phagocytized the test particles at a normal rate when incubated in normal plasma. The DLT-gelatin ligand bound to fibronectin in a dose-dependent manner as verified by its capture with anti-fibronectin coated plastic wells when coincubated with purified fibronectin. By gel filtration chromatography, the binding of fibronectin with the DLT-gelatin ligand was readily detected, resulting in the formation of a high-molecular-weight complex. In normal animals the plasma clearance and liver localization of 125I-DLT-gelatin was competitively inhibited by infusion of excess nonradioactive gelatin. The blood clearance and liver localization of the soluble gelatin ligand were also impaired after burn injury during periods of fibronectin deficiency similarly to the pattern observed with gelatin-coated microparticles. By autoradiography, the cellular site for the uptake of the 125I-DLT-gelatin was primarily but not exclusively hepatic Kupffer cells; 125I-DLT-asialofetuin and 125I-DLT-ovalbumin were removed by hepatocytes and sinusoidal endothelial cells, respectively. Thus, gelatin conjugated with 125I-DLT can be used to simulate blood-borne soluble collagenous tissue debris after burn. It rapidly binds to plasma fibronectin before its hepatic Kupffer cell removal, and its blood clearance is markedly delayed after burn injury during periods of plasma fibronectin deficiency.

High-affinity binding of fibronectin to cultured Kupffer cells.

Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative "fibronectin receptors" per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

Plasma fibronectin (opsonic glycoprotein): its synthesis by vascular endothelial cells and role in cardiopulmonary integrity after trauma as related to reticuloendothelial function.

Progressive multiple organ failure in association with septic complications in the surgical, trauma and burn patient is of major clinical importance. Reticuloendothelial system host defense mechanisms are abnormal following severe trauma and burn. Failure in systemic host defense is, in part, mediated by a deficiency in a circulating opsonic alpha 2 surface binding (SB) glycoprotein. This opsonic deficiency and reticuloendothelial host defense failure appears etiologic in the genesis of organ failure with sepsis. Opsonic alpha 2SB glycoprotein is identical to cold-insoluble globulin or plasma fibronectin. Plasma fibronectin is antigenically related to cell surface fibronectin which appears to be synthesized by both fibroblasts and vascular endothelial cells. Although these two proteins are antigenically related, they may or may not be identical with respect to biochemical properties and function. Cell surface fibronectin appears to be an adhesive glycoprotein mediating cell-cell interaction and cell adhesion to a substratum. Plasma fibronectin is a more soluble form which mediates reticuloendothelial or macrophage clearance of particulates such as fibrin microaggregates, collagenous debris, perhaps other bacterial or nonbacterial particulates. Since opsonic glycoprotein is identical to cold-insoluble globulin which can be readily concentrated in plasma cryoprecipitate, it has been shown that cryoprecipitate infusion can reverse opsonic deficiency in the injured patient with sepsis. Reversal of opsonic deficiency by cryoprecipitate infusion results in a marked improvement in cardiopulmonary function which includes a decline in the pulmonary shunt, a decrease in the physiologic dead space, an increase in limb blood flow, an increase in reactive hyperemia of the peripheral circulation and an increase in limb oxygen consumption. This cardiopulmonary response is paralleled by a decline in the septic state and normalization of other hematologic parameters. These studies suggest an important homeostatic role for fibronectins in organ and microvascular integrity, especially in the septic injured patient. Cell surface fibronectin which participates in cell adhesion may, in part, modulate microvascular integrity, vascular permeability and would repair. In contrast, the more soluble plasma fibronectin or opsonic alpha 2SB glycoprotein may mediate reticuloendothelial clearance of blood-borne particulates to prevent pulmonary and peripheral vascular microembolization and organ injury. Thus, reversal of opsonic deficiency may be an effective modality of therapy in the septic injured patient with organ failure.

Reversal of opsonic deficiency in surgical, trauma, and burn patients by infusion of purified human plasma fibronectin. Correlation with experimental observations.

Plasma fibronectin deficiency has been documented in critically ill surgical, trauma, and burn patients. Human plasma fibronectin was isolated by gelatin-Sepharose affinity chromatography and evaluated with respect to its opsonic activity following pasteurization, its in vivo clearance kinetics, and its short-term influence on cardiovascular hemodynamics in postoperative septic sheep. Six patients with low plasma fibronectin levels were also evaluated with respect to temporal changes of immunoreactive fibronectin and opsonic activity following infusion of fibronectin at a dose calculated to elevate the plasma fibronectin level to 400 micrograms/ml. With utilization of three different in vitro radioisotopic phagocytic assays, i.e., liver slice assay, peritoneal macrophage monolayer assay, and Kupffer cell monolayer assay, retention of opsonic activity by fibronectin following pasteurization was documented. The normal biphasic kinetics associated with plasma clearance of fibronectin were also not altered by pasteurization. In postoperative septic sheep with hemodynamic instability, intravenous infusion of 500 mg of purified human fibronectin initiated no abnormal hemodynamic response. Indeed, as compared with placebo, the infusion of fibronectin into the postoperative septic sheep resulted in a more stable systemic vascular resistance and pulmonary vascular resistance with a higher arterial pressure. It also elevated immunoreactive fibronectin levels (p less than 0.05) and increased opsonic activity (p less than 0.05). Surgical, trauma, and burn patients (ages 18 to 80 years) with low plasma fibronectin levels (160 to 236 micrograms/ml) manifested no disturbance in cardiovascular, respiratory, or hematologic parameters following fibronectin infusion (590 to 988 mg per patient), but did display an early increase of opsonic activity. This standardized, pasteurized, and opsonically active preparation of purified human plasma fibronectin (5.0 mg/ml after reconstitution) has utility for future randomized clinical trials in injured patients with sepsis.

Neutrophil-mediated lung localization of bacteria: a mechanism for pulmonary injury.

The reticuloendothelial system (RES) is thought to ensure organ integrity following trauma, burn, and sepsis by removing potentially embolic particulate matter and blood-borne bacteria from the circulation. Blockade of the RES with foreign colloids is known to result in a consumptive depletion of opsonic fibronectin, which modulates reticuloendothelial function, and an increase in lung localization of test particles. We investigated the role of neutrophils as a contributing factor in the increased localization of blood-borne bacteria in the lung after blockade. RE blockade induced by gelatin-coated colloid particle injection resulted in an acute (15-minute) increase in the number of 51Cr-labeled neutrophils localized in the lung, with return to control levels at 60 minutes after blockade. Fibronectin administration following blockade resulted in a significant (P less than 0.05) prolonged retention of neutrophils in the lung up to 2 hours after blockade. A parallel increase (P less than 0.05) in lung localization of heat-killed 14C-labeled Pseudomonas aeruginosa following colloid-induced RE blockade was observed, and fibronectin further increased the number of bacteria localized in the lung. Experimentally induced neutropenia abrogated the effect of colloid injection on lung localization of bacteria. It is concluded that a particulate load results in simultaneous RE blockade and neutrophil margination in the lung, both of which contribute to the increase in lung localization of bacteria. A mechanism for neutrophil-mediated pulmonary injury related to RE dysfunction following trauma is proposed.

Fibronectin and the critically ill patient: current status.

In summary, deficiency of plasma fibronectin has now been documented in a variety of clinical entities. Persistently low fibronectin may have prognostic value, and in certain patients may provide a clue to occult sepsis and potential organ failure. The clinical benefit of infusion of fibronectin-rich cryoprecipitate or purified human plasma fibronectin has yet to be determined in well-controlled randomized clinical trials. However, if such results become available then infusion of plasma fibronectin may provide a valuable therapeutic modality in the care of the critically-ill patient.

Cardiovascular hemodynamics after opsonic alpha-2-surface binding glycoprotein therapy in injured patients.

Depression of reticuloendothelial (RE) phagocytic function has been clearly documented following trauma and operation. This phagocytic failure is mediated in part by depletion of an opsonic glycoprotein. Depletion of this opsonic protein may result in prolonged blood retention of potentially harmful particulates that may interfere with the microcirculation and may possibly result in altered organ function. Isolation and identification of this opsonic protein has led to the finding of the identity between opsonic glycoprotein and cold insoluble globulin (CIg) or so-called plasma fibronectin. Since CIg is concentrated in cryoprecipitate, this blood component was used as a readily available source of opsonic protein for replacement studies. Nine patients were studied following a 1-hour infusion of cryoprecipitate obtained from 10 units of plasma and suspended in a volume of 250 ml. Both the pulmonary shunt fraction and the fraction of dead space ventilation decreased significantly (P = 0.02) after cryoprecipitate administration. Limb blood flow (P = 0.001), limb oxygen consumption (P = 0.001), and reactive hyperemia of the limb (P = 0.05) increased significantly following cryoprecipitate infusion. Cardiac output, total oxygen consumption did not change consistently. The data demonstrate that the infusion of cryoprecipitate resulted in improved pulmonary and microcirculatory function--possibly due to opsonic glycoprotein replacement.

Plasma fibronectin therapy and lung protein clearance with bacteremia after surgery.

Plasma fibronectin modulates macrophage phagocytic function and can also incorporate into the insoluble tissue pool of fibronectin where it influences endothelial cell adhesion and tissue integrity. We studied the effect of postoperative bacteremia on lung protein clearance in relation to plasma fibronectin levels using the unanesthetized sheep lung lymph fistula model and the effect of infusion of purified human plasma fibronectin on lung protein clearance. Sheep received live Pseudomonas aeruginosa (5 X 10(8) iv) at a time of normal plasma fibronectin (590 +/- 37 micrograms/ml) or 5 days later at a time corresponding to elevation of plasma fibronectin (921 +/- 114 micrograms/ml). After the first bacterial challenge, there was a 22% decrease (P less than 0.05) in plasma fibronectin. Lung lymph flow (QL) initially increased 308% (P less than 0.05) by 2 h (0 h = 4.7 +/- 1.1 ml/h; 2 h = 14.4 +/- 3.5 ml/h), and the total protein lymph-to-plasma concentration ratio (L/P) declined. This was followed by a sustained second phase response over 3-12 h which was characterized by a 202-393% elevation in QL (P less than 0.05), an increase in the L/P ratio, and a 240-480% (P less than 0.05) increase in lung transvascular protein clearance (TVPC = QL X L/P). Sheep with elevated fibronectin levels also manifested the early (2 h) elevation in QL (P less than 0.05) coupled with a decline in L/P ratio after the second bacterial challenge, but the second-phase increase in TVPC was markedly attenuated. Intravenous infusion of 500 mg of human plasma fibronectin into normal sheep to elevate the fibronectin level comparable to that in the hyperfibronectinemic sheep also attenuated (P less than 0.05) the second-phase (3-12 h) increase in lung protein clearance with sepsis. Thus elevation of plasma fibronectin during postoperative Gram-negative bacteremia may protect the lung vascular barrier. This response may be mediated by either fibronectin's opsonic support of phagocytic function or its influence on lung endothelial cell adhesion.

Simultaneous measurement of fluid and protein permeability in isolated rabbit lungs during edema.

Fluid conductance and protein permeability have been studied in isolated perfused lung models of pulmonary edema. However, previous studies have not investigated changes of both fluid conductance and protein permeability in the same isolated lung preparation after injury. Arachidonic acid (AA) metabolites are involved in the inflammatory processes that lead to the development of pulmonary edema. The hemodynamic effects of AA have been well established; however, controversy exists concerning the ability of AA to alter the permeability of the pulmonary microvasculature to fluid and protein. The purpose of this study was to simultaneously determine whether transvascular fluid conductance and protein permeability are increased in isolated perfused rabbit lungs with pulmonary edema induced by AA. Indomethacin (80 microM) was added to the perfusate to inhibit the hemodynamic effects of AA and produce a pressure-independent model of pulmonary edema. Fluid conductance was assessed by determination of the capillary filtration coefficient (Kf), and protein permeability was evaluated by measurement of 125I-albumin clearance. The injection of AA (3 mg/200 ml of perfusate) into the pulmonary arterial catheter resulted in an increase in lung weight over the remaining 30-min experimental period. Kf (microliter.s-1 x cmH2O-1 x g dry lung-1) was increased (P < 0.05) in AA-treated lungs at 10 and 30 min post-AA injection when compared with control lungs and baseline values (determined 10 min before AA injection). Albumin clearance was also greater (P < 0.05) in lungs that received AA. 125I-albumin clearance was measured at different rates of fluid flux produced by elevation of venous pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoproterenol decreases protein permeability in edematous isolated rabbit lungs: estimation of PS and sigma.

The objective of the present study was to determine whether the ability of the beta-adrenergic agonist isoproterenol to attenuate pulmonary edema occurs via a permeability and/or hemodynamic mechanism. In isolated perfused rabbit lungs, the restrictive property of the vascular barrier to the movement of fluid and protein was assessed by measurements of the capillary filtration coefficient (Kf) and the transvascular clearance of 125I-labeled albumin, respectively. Regression analysis of albumin clearance vs. transvascular fluid flux was performed to estimate the permeability-surface area product (PS) and the reflection coefficient (sigma) by use of the linear or nonlinear flux equation. Arterial, capillary, and venous pressures and resistances, weight gain, and the wet-to-dry weight ratio were also assessed. Isoproterenol (8 ng.ml-1.min-1) attenuated the arachidonic acid (4 mg)-induced increases in fluid flux, wet-to-dry weight ratio, albumin clearance, and PS and the decrease in sigma. Isoproterenol had no effect on the increase in Kf, and there was no correlation between capillary pressure and fluid flux in any of the four groups. Regression analysis revealed that the non-linear flux equation provided estimates of PS and sigma that more accurately described the statistical differences in albumin clearance among the groups studied than the linear flux equation. These findings demonstrate that isoproterenol attenuated the increased transvascular flux of albumin in edematous lungs by modifying the protein permeability of the vascular barrier.

Plasma fibronectin levels during cardiopulmonary bypass.

Plasma fibronectin, also called cold-insoluble globulin, is a cryoprecipitable glycoprotein with both opsonic and adhesive activities. It binds to collagen, actin, and heparin and can form soluble as well as cryoprecipitable complexes in the cold. Fibronectin augments particulate phagocytosis by the reticuloendothelial system and can influence lung vascular permeability. Plasma fibronectin deficiency is temporally associated with respiratory failure in septic surgical, trauma, and burn patients. We measured plasma fibronectin and albumin levels in nine adults undergoing elective cardiopulmonary bypass to determine whether dilution alone could account for the changes in plasma fibronectin. Plasma fibronectin concentration decreased 17% with the surgical trauma of opening of the chest and placement of the vascular cannulas. On heparinization and initiation of cardiopulmonary bypass, plasma fibronectin fell an additional 48% (P less than 0.001), whereas albumin concentration (corrected for albumin in the pump prime) fell only 25% (P less than 0.001), emphasizing that dilution was not the only mechanism contributing to the decline in plasma fibronectin. Fibronectin levels began to increase after discontinuation of cardiopulmonary bypass and in association with diuresis, but unexpectedly they remained subnormal until 4 days postoperation. Thus the decline in fibronectin concentration with cardiopulmonary bypass may be due to dilution as well as opsonic consumption and possible complexing with heparin in the cold.

Correction of serum opsonic defects after burn and sepsis by opsonic fibronectin administration.

Opsonic fibronectin modulates reticuloendothelial (RE) uptake of nonbacterial particulates, as well as some bacterial strains, and may thus play an important role in host defense against sepsis after burn injury. We evaluated the relationship between burn injury, sepsis, and opsonic fibronectin levels in rats, as well as the ability to reverse the acute opsonic deficiency after burn injury by administration of purified opsonic fibronectin. Burn injury resulted in an acute (within one hour) depletion of opsonic fibronectin (from 341 +/- 30 to 98 +/- 7 mg/L) that was correctable by administration of purified opsonic fibronectin when accompanied by moderate sepsis, while burn injury plus severe sepsis (level, 168 +/- 30 mg/L) limited attempted restoration of normal opsonic levels (level, 121 +/- 18 mg/L). The in vitro serum opsonic deficit was partially correctable (from 2.2% to 6.7% of the injected dose per 100 mg), while in vivo RE functional deficits could not be corrected. We conclude that the acute postburn deficiency in opsonic fibronectin is amenable to repletion therapy; however, many additional factors may contribute to acute RE failure after burn injury.