Biomedical subjects
Publications and source records attributed to E Cho.
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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.
Fibronectin is found in plasma as well as in association with connective tissue and cell surfaces. Depletion of plasma fibronectin is often observed in septic trauma and burned patients, while experimental rats often manifest hyperfibronectinemia with sepsis. Since Factor XIII may influence the rate of clearance and deposition of plasma fibronectin into tissues, we evaluated the temporal changes in plasma fibronectin and plasma Factor XIII following bacteremia and RE blockade in rats in an attempt to understand the mechanism leading to elevation of fibronectin levels in bacteremic rats, which is distinct from that observed with RE blockade. Clearance of exogenously administered fibronectin after bacteremia was also determined. Rats received either saline, Pseudomonas aeruginosa (1 X 10(9) organisms), gelatinized RE test lipid emulsion (50 mg/100 gm B.W.), or emulsion followed by Pseudomonas. Plasma fibronectin and Factor XIII were determined at 0, 2, 24, and 48 hours post-blockade or bacteremia. At 24 and 48 hr following bacteremia alone or bacteremia after RE blockade, there was a significant elevation (p less than 0.05) of plasma fibronectin and a concomitant decrease (p less than 0.05) of plasma factor XIII activity. Extractable tissue fibronectin from liver and spleen was also increased at 24 and 48 hours following R.E. blockade plus bacteremia. In addition, the plasma clearance of human fibronectin was significantly prolonged (p less than 0.05) following bacterial challenge. Infusion of activated Factor XIII (20 units/rat) during a period of hyperfibronectinemia (908.0 +/- 55.1 micrograms/ml) resulted in a significant (p less than 0.05) decrease in plasma fibronectin (548.5 +/- 49.9 micrograms/ml) within 30 min. Thus Factor XIII deficiency in rats with bacteremia may contribute to the elevation in plasma fibronectin by altering kinetics associated with the clearance of fibronectin from the blood.
Fibronectin is a glycoprotein found in a soluble form in plasma and in an insoluble form in many tissues. We evaluated the influence of postoperative intraperitoneal sepsis on the clearance, tissue distribution, and synthesis of plasma fibronectin in rats (300-400 g). Experimental sepsis was induced by cecal ligation following laparotomy, whereas control animals underwent laparotomy (5 cm) alone. At 24 and 48 h after laparotomy, plasma fibronectin levels were normal. After laparotomy plus cecal ligation, plasma fibronectin increased by 47% at 24 h and remained elevated (52% above 0 time) at 48 h. At 24 h postsurgery the disappearance and tissue distribution of 75Se-plasma fibronectin and 75Se-plasma albumin was evaluated. Tissue distribution was quantified at 2 and 24 h after intravenous injection of both tracer proteins in separate groups. Both fibronectin and albumin demonstrated an initial distribution between vascular and extravascular sites and then a progressive decrease in plasma. In control (laparotomy) rats the half-life (t1/2) for plasma clearance of 75Se-plasma fibronectin was 25.33 +/- 2.53 h compared with 13.21 +/- 0.78 h in the septic rats. Septic rats manifested decreased sequestration of 75Se-fibronectin at the area of surgical incision (laparotomy), increased sequestration at the focus of intraperitoneal infection, and increased uptake in the nonviable portion of the cecum. The synthetic rate for plasma fibronectin in laparotomized control rats was 3.03 +/- 0.29 mg X 100 g-1 X 24 h-1, whereas after laparotomy plus cecal ligation the synthetic rate increased to 4.58 +/- 0.35 mg X 100 g-1 X 24 h-1. In contrast the synthetic rate for albumin decreased from 84.70 +/- 1.66 mg X 100 g-1 X 24 h-1 in controls to 52.38 +/- 1.77 mg X 100 g-1 X 24 h-1 in the septic animals. Thus intraperitoneal sepsis in the rat will enhance the vascular clearance, alter the distribution, and increase the synthetic rate for plasma fibronectin.
Reticuloendothelial (RE) phagocytic function and plasma fibronectin are altered early after trauma and during septic shock. Since fibronectin-coated particles will tend to aggregate if not efficiently phagocytized, we hypothesized that elevated fibronectin levels during hepatic and/or splenic RE depression could potentiate the lung deposition of blood-borne foreign microparticles. To evaluate this concept, we measured plasma fibronectin, hepatic RE function, and tissue deposition of blood-borne colloids in rats after they were injected with nonbacterial and bacterial particulates. Rats were injected intravenously with gelatin-coated colloids (50 mg/100 gm) to simulate blood-borne collagenous tissue debris after trauma, or with live Pseudomonas aeruginosa (1 X 10(9)/rat) to simulate bacterial entrance into the blood with sepsis, or with both to simulate sepsis after trauma. Phagocytic function was evaluated by liver and spleen uptake of gelatinized 125I RE test emulsion. Fibronectin was quantified by electroimmunoassay. There was an acute 60-80% decrease in plasma fibronectin 2 hr following either colloid or colloid coupled with bacterial infusion. Bacterial infusion alone elicited only a mild 20% decrease in fibronectin by 2 hr. By 24 hr, restoration of fibronectin levels was observed in all groups with hyperfibronectinemia observed in animals challenged with Pseudomonas. Following colloid alone, liver uptake of the RE test particle was acutely depressed at 2 hr in association with an acute depletion of fibronectin, but at 24 hr the RE depression persisted even with normalization of fibronectin. In contrast, with only bacteremia, the rebound elevation of fibronectin was associated with increased hepatic RE function. In rats given both colloid and Pseudomonas, the hyperfibronectinemia (60-100% above controls) at 24 hr coexisted with inadequate liver phagocytic uptake ability. This resulted in a significant 20-fold (P less than 0.05) increment in lung localization of the blood-borne test microparticles. Thus, hyperfibronectinemia without a parallel increase in liver phagocytic ingestive ability may actually enhance lung localization of blood-borne microparticles, which have a high affinity for fibronectin.
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Plasma fibronectin modulates reticuloendothelial (RE) phagocytosis of cellular and tissue debris, fibrin microaggregates, and gelatin-coated particulates. An antigenically related, but more insoluble form of fibronectin is found in various tissues and suspected to play a role in vascular permeability, cell adhesion, and wound healing. The current study developed a fluorescent immunoassay which could be utilized for the quantification of tissue fibronectin following its extraction from tissues. Additionally, the changes in tissue fibronectin induced by the intravenous injection of gelatin-coated colloids in rats (300-350 g), which acutely depletes the plasma fibronectin level, were also investigated. Injection of gelatinized RE test lipid emulsion (50 mg/100 g) depleted the plasma fibronectin at 2 hr (80-85% depletion) followed by rebound restoration within 24 hr as quantified by either electroimmunoassay or fluorescent immunoassay. RES system clearance of the test particles from the blood resulted in an acute elevation in fibronectin extractable from the liver with a normalization by 48 hr. In contrast, assay of tissue fibronectin following a single extraction revealed a decrease in lung extractable fibronectin within 2 hr following RE blockade which persisted for 24-48 hr. Extractable fibronectin in spleen and renal tissue was unaltered by RE blockade. This microfluorescent immunoassay may provide a sensitive method to quantify fibronectin in small aliquots of tissue. Increased hepatic fibronectin most likely reflects interiorization of plasma fibronectin during Kupffer cell clearance of the test particles. Decreased lung extractable fibronectin may alter lung vascular sensitivity to a subsequent septic and/or intravascular coagulation stress. Thus, similar to the labile nature of plasma fibronectin, the concentration of fibronectin in various tissues, can undergo dynamic alterations.
Plasma fibronectin is an opsonic glycoprotein which augments reticuloendothelial phagocytic clearance of nonbacterial particulates. We evaluated the influence of intravenous infusion of plasma cryoprecipitate on circulating immunoreactive fibronectin and associated opsonic activity at 0.5, 2.0, 4.0, 10, and 21 hr postinfusion in septic (n = 8) and nonseptic (n = 6) surgical and/or trauma patients with documented plasma fibronectin deficiency. The study was a randomized, double-blind, crossover clinical protocol in which fibronectin-poor (0.116 +/- 0.025 mg/ml) cryoprecipitate extracted plasma (placebo) was compared to fibronectin-rich (2.139 +/- 0.161 mg/ml) plasma cryoprecipitate. Septic injured patients (149.37 +/- 17.11 micrograms/ml) had lower (p less than 0.05) plasma fibronectin levels than nonseptic injured patients (212.17 +/- 7.14 micrograms/ml) and both were less (p less than 0.05) than normal (330 +/- 30 micrograms/ml). As tested in vitro with a peritoneal macrophage monolayer assay, cryoprecipitate manifested opsonic activity related to its fibronectin concentration. Intravenous infusion of fibronectin rich cryoprecipitate reversed both the immunoreactive fibronectin and opsonic deficiency, while infusion of the placebo at a comparable total protein load did not reverse either deficient parameter. Reversal of fibronectin deficiency was more sustained in nonseptic injured patients as compared to septic injured patients. Thus, reversal of opsonic deficiency in septic and nonseptic injured patients is observed after infusion of plasma cryoprecipitate and not with infusion of fibronectin deficient plasma at comparable protein loads. Also, cryoprecipitate extracted plasma may serve as an appropriate control solution for randomized studies evaluating the therapeutic value of fibronectin-rich plasma cryoprecipitate.
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Circulating fibronectin, also known as opsonic alpha 2 surface binding glycoprotein or cold-insoluble globulin, modulates phagocytosis of tissue debris, fibrin microaggregates, and gelatin-coated colloids by the reticuloendothelial (RE) system. Opsonically active fibronectin has an actin binding site and a demonstrated in vitro affinity for actin. Since actin potentially released into blood and tissue fluids following tissue injury could complex with fibronectin, the present study evaluated the effect of actin on plasma opsonic activity and Kupffer cell phagocytosis. Intravenous injection of actin did not acutely decrease plasma immunoreactive fibronectin levels although fibronectin levels increased at 6, 12, and 24 hr postinjection. However, intravenous actin injection did depress RE phagocytic activity in vivo as measured by decreased blood clearance of test colloid and impaired hepatic uptake of colloid particles as well as retention of the particles in the circulation. In vitro, preincubation of plasma with actin depressed the opsonic activity of plasma with respect to its ability to support phagocytosis, but such treatment of plasma did not alter the detection of fibronectin by immunoassay. Utilizing purified fibronectin with demonstrated opsonic activity, it was also observed that actin interaction with fibronectin would block its biological ability to enhance phagocytosis. This effect appeared to be mediated at the humoral level, since no direct depressant effect of actin on Kupffer cell function was observed. Thus, actin, if released into the blood following injury, may contribute to bioassayable opsonic fibronectin deficiency and phagocytic dysfunction, but this disturbance would remain undetectable by immunoassay of fibronectin levels.
Fibronectin is an important non-immune opsonic protein influencing phagocytic clearance of blood-borne nonbacterial particulates which may arise in association with septic shock, tissue injury, and intravascular coagulation. In the present study, serum fibronectin was measured by both electroimmunoassay as well as rapid immunoturbidimetric assay in healthy children (n = 114) ranging in age from 1 month to 15 years in order to delineate the temporal alterations in fibronectin with age. Normal adult serum fibronectin concentrations are typically 220 micrograms/ml +/- 20 micrograms/ml. Serum concentration is 35-40% lower than normal plasma concentration due to the binding of fibronectin to fibrin during clot formation. Children between 1-12 months of age had significantly (P less than 0.05) lower serum fibronectin levels than children between the ages of 1-15 years. Progressive elevation in fibronectin levels was observed within the last 8 months of the first year of age. Fibronectin levels in children older than 1 year of age remained constant up to 15 years and were within the lower limit of the normal adult concentration. No significant (P greater than 0.05) difference in serum fibronectin was observed between male and female children at all age groups. Fibronectin levels thus, increase during the first year of age and normal levels of this blood protein in the infant are less than the normal range for adults.
The opsonic activity of plasma fibronectin is important in modulating the reticuloendothelial system (RES) phagocytic removal of a variety of endogenous and exogenous particulate material from the vascular compartment. Purification of plasma-opsonic fibronectin by affinity chromatography with gelatin-Sepharose revealed that although in vitro hepatic Kupffer cell phagocytosis was absolutely dependent upon the presence of fibronectin, the purified fibronectin evaluated in concentrations similar to that found in plasma (350-450 micrograms/ml) supported phagocytosis at a level two- to threefold less than that observed in whole plasma. In contrast, the combination of purified fibronectin with small aliquots of opsonically inactive fibronectin-free plasma restored normal opsonic activity as assessed by liver slice bioassay and enhanced fibronectin-mediated attachment of gelatinized particulate to isolated Kupffer cells in vitro. Evidence is presented in this study that there exists in plasma a macromolecular species that amplifies the opsonic activity of fibronectin in a dose-related manner. This amplification or cofactor activity is nondialysable and has a molecular weight greater than 12,000. Inactivation of the amplification activity present in affinity-absorbed plasma can be achieved by heating the fibronectin-free plasma at 60 degrees C for 20 min, supporting the hypothesis that the cofactor is a protein. The amplification response is dose related, suggesting that the mechanism of its action is stoichiometric rather than catalytic. Evidence is presented that partial purification of the cofactor can be achieved by (NH4)2SO4 precipitation at 4 degrees C. Purification of this cofactor will provide an opportunity to evaluate its role in the altered opsonic states known to exist after trauma, burn, and sepsis.
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