Effects of anti-C5a antibodies on the adult respiratory distress syndrome in septic primates.

In vitro and in vivo studies have suggested that human complement component C5a plays a key role in neutrophil injury in the adult respiratory distress syndrome (ARDS). First, using leukocyte aggregometry, we demonstrated that the addition of a recently developed rabbit anti-human polyclonal antibody to C5a des arg to endotoxin-activated plasma prevented leukocyte aggregation in vitro. We then administered the anti-C5a des arg antibody to septic primates (Macaca fascicularis). Three groups of primates, control, septic, and anti-C5a antibody treated septic, were studied (n = 4 in each group). A 30-min infusion of Escherichia coli (1 X 10(10)/kg) resulted in severe sepsis and ARDS. Primates were killed 4 h after completion of the E. coli infusion. Septic animals not treated with anti-C5a antibody had 75% mortality (3/4), decreased oxygenation, severe pulmonary edema, and profound hypotension. Septic primates treated with anti-C5a antibodies did not die and did not develop decreased oxygenation (P less than 0.05) or increased extravascular lung water (P less than 0.05). They also had a marked recovery in their mean arterial blood pressure (P less than 0.05). This study demonstrates that treatment with rabbit anti-human C5a des arg antibodies attenuates ARDS and some of the systemic manifestations of sepsis in nonhuman primates.

Manufacture of antithymocyte globulin (ATGAM) for clinical trials.

Methods are described for preparing large amounts of horse anti-human thymocyte globulin (ATG, ATGAM; The Upjohn Company) for clinical use. These methods have been used since 1968 to provide material for clinical trials. Characteristics of 40 lots of ATG are summarized.

Cyclophosphamide is neuroprotective in a gerbil model of transient severe focal cerebral ischemia: correlation with effects of tirilazad mesylate (U-74006F).

Using a gerbil model of severe, temporary focal ischemia (3 h unilateral carotid occlusion), preliminary experiments identified an involvement of neutrophils in the reperfusion injury to the ischemic hemisphere. The present experiments were designed to (1) quantitate the temporal accumulation of neutrophils in the gerbil model, (2) determine if cyclophosphamide-induced neutropenia provided cytoprotection to the ischemic hemisphere, and (3) attempt to correlate the cytoprotective efficacy of tirilazad mesylate with possible effects on postischemic neutrophil accumulation. Following 3 h of unilateral carotid occlusion, animals were collected at increasing times of reperfusion and the CA1 region of the hippocampus and the lateral cortex were assessed for postischemic neuronal damage using a semiquantitative index (N.D.I.) of 0 (no damage) to 4 (>75% neuronal loss). The extent of neutrophil accumulation was determined by counting intensely cytochrome oxidase-positive cells. Minimal neuronal death was evident after 2 h of reperfusion, mean N.D.I. = 0.36. However, between 2 and 4 h of reperfusion, neuronal death did not increase. By 6 h of reperfusion, the neuronal death began to proceed at an accelerated rate, N.D.I. = 0.78. By 12 h, the N.D.I. reached 3.20. The accelerated neuronal death coincided with parenchymal invasion of neutrophils. Cyclophosphamide administration delayed neuronal death in the hippocampus, but exhibited a more sustained protective effect in the lateral cortex. Administration of tirilazad mesylate also resulted in a significant reduction in neutrophil accumulation and significant neuronal protection in both brain areas. Thus, in this gerbil model of transient, but prolonged focal cerebral ischemia, neutrophils appear to play an active role in the reperfusion injury to brain tissue. Our experiments confirm the previously demonstrated neuroprotective efficacy of tirilazad mesylate in this model and provide evidence for a similar protective effect of cyclophosphamide. Although other effects of this antioxidant are also thought to contribute to the overall efficacy, the data are consistent with the hypothesis that one mechanism by which tirilazad acts involves limiting the ability of neutrophils to participate in the reperfusion phase of ischemic cerebral injury.

Complement activation and clearance in acute illness and injury: evidence for C5a as a cell-directed mediator of the adult respiratory distress syndrome in man.

The appearance of the adult respiratory distress syndrome (ARDS) during the course of acute illness is believed to result, in part, from intrapulmonary neutrophil sequestration and degranulation induced by circulating inflammatory mediators. To evaluate the role of complement-neutrophil interactions in the pathogenesis of ARDS in man, 34 patients suffering from intra-abdominal sepsis (seven), multisystem trauma (15), or acute pancreatitis (12) were serially studied with regard to neutrophil migratory responses to C5a and F-Met-Leu-Phe, lysosomal content of beta-glucuronidase and lysozyme, and simultaneously obtained plasma levels of immunoreactive C3adesArg and C5adesArg. Nineteen patients developed ARDS. In these patients, plasma C3adesArg levels obtained within 72 hours of admission to the hospital were elevated to 305 +/- 35 ng/ml compared with 145 +/- 16 ng/ml for patients who did not develop ARDS (p less than 0.0005). C5adesArg levels were not elevated in either group. In vitro studies showed that neutrophils from normal persons were able to clear all of the C5a/C5adesArg generated in up to 5% zymosan-activated serum, while no clearance of C3adesArg was identified. Patient migratory responses could be divided into three groups based on their initial (less than 72 hour) samples: (1) hyperresponsive to both N = formyl-methionyl-leucyl-phenylalanine (FMLP) and C5a, (2) specifically deactivated to C5a, and (3) deactivated to both C5a and FMLP. Patients in the latter two groups developed ARDS. Enzyme content of neutrophils from patients who developed ARDS showed a substantial fall in beta-glucuronidase and lysozyme levels. The finding of elevated plasma C3a levels and deactivation of migratory response to C5a support the contention that complement activation had occurred in these patients and that their neutrophils had been exposed to C5a/C5adesArg in vivo. The finding of nonspecific migratory dysfunction associated with lysozymal enzyme loss, a circumstance not reproducible in vitro by C5a exposure, suggests that other stimuli produced degranulation of neutrophils made hyperresponsive by prior exposure to C5a.

Complement-induced expression of cryptic receptors on the neutrophil surface: a mechanism for regulation of acute inflammation in trauma.

We explored the hypothesis that identified changes in neutrophil function in patients with acute injury result from in vivo exposure to C5a. To evaluate this hypothesis, we performed a battery of tests on 26 trauma patients (14 with blunt injury, 12 with penetrating injury). Measured were plasma levels of the complement activation products C3a and C5a; neutrophil chemotaxis to C5a and N-formyl-methionyl-leucyl-phenylalanine (FMLP); neutrophil receptors for FMLP and C3b; and superoxide response to FMLP and serum-opsonized zymosan. Patient responses measured within 48 hours of admission were divided into two groups based on neutrophil migratory response to C5a. Patients unresponsive to C5a (but responsive to FMLP) showed elevated plasma C3a levels (248 +/- 6 ng/ml) compared with patients with normal C5a migratory response (104 +/- 8 ng/ml). FMLP receptor number was markedly increased in the chemotactically deactivated group (group I: 155,680 +/- 100; group II: 51,200 +/- 200) and receptor affinity was diminished. Binding activity of C3b increased in the C5a-unresponsive cells to 126% that of controls versus 94% for normally responsive patient cells. Superoxide production was found to be significantly increased in patient cells with increased receptor numbers. These results support the concept that a subgroup of trauma patients manifest plasma and neutrophil changes compatible with complement activation. The neutrophil changes identified demonstrate a state of cellular activation. The clinical significance of these results may reside in a risk of pulmonary microvascular injury if activated cells are marginated and then subsequently stimulated.

Differential prostacyclin production by human umbilical vasculature.

Using the avidin-biotin immunohistochemical technique with rabbit antihuman 6-keto-prostaglandin (PG)-F1 alpha (6KPGF), we studied the distribution of the stable prostacyclin metabolite, 6KPGF, in 14 formaldehyde-fixed human umbilical cords. All umbilical veins demonstrated intense endothelial cell staining. None of the arteries stained. To corroborate the immunohistochemical findings, three fresh umbilical cords were dissected to separate arteries from veins and then were incubated in oxygenated tissue baths containing Ringer's lactate (37 degrees C) for 30 minutes. Cumulative 6KPGF production as measured by radioimmunoassay of tissue effluents was markedly different between arteries and veins with the umbilical vein producing the largest quantity of 6KPGF. Thus, immunohistochemistry and ex vivo capacitance studies suggest that there is a differential distribution of 6KPGF in human umbilical arteries and veins.

Effects of complement activation in the isolated heart. Role of the terminal complement components.

The mechanisms of the complement-mediated myocardial injury associated with ischemia and reperfusion have not been elucidated fully. Complement activation may directly mediate injury through actions of the anaphylatoxins C3a and C5a or generation of the membrane attack complex C5b-9. A model was developed to examine the direct effects of complement activation on heart function, assess myocardial tissue damage, and determine which complement components mediate tissue injury. Isolated rabbit hearts were perfused with Krebs-Henseleit buffer by using a modified Langendorff apparatus. Human plasma was added to the perfusate as a source of complement. Rabbit tissue activates human complement. Treatment with 6% normal plasma resulted in complement activation as assessed by the generation of Bb, C3a, C5a, and SC5b-9. Functional changes in cardiac performance became apparent 7-15 minutes after plasma addition and developed fully over the next 20-30 minutes. The effects were dependent on the complement titer and included 1) an increase in the end-diastolic pressure, 2) a decrease in the developed pressure, 3) an increase in the coronary perfusion pressure, and 4) an increase in lymphatic fluid formation. These effects were not elicited when an inhibitor of complement activation (FUT-175) was present or when heat-inactivated plasma was used. The effects of complement activation on myocardial function could not be reproduced by treatment with recombinant human C5a, zymosan-activated plasma, or plasma selectively depleted of C8. Myocardial tissue accumulated sodium and calcium and lost potassium as a result of complement activation. Activation caused the release of creatine kinase from myocytes and an increase in the radiolabeled albumin space of the hearts. The data demonstrate that complement activation caused decrements in myocardial function and increased the coronary perfusion pressure and lymphatic fluid flow rate. The effects were not mediated by the anaphylatoxins but were dependent on the distal complement component C8, suggesting that C5b-9 was responsible for the physiological changes. Complement activation directly mediated tissue injury in a manner consistent with plasmalemmal disruption as a result of C5b-9 formation. The data suggest that the C5b-9 complex, which is known to form under conditions of ischemia, may contribute directly to myocardial cell injury.

Sulfhydryl compounds, captopril, and MPG inhibit complement-mediated myocardial injury.

Factors including complement activation, neutrophil infiltration, and oxygen-derived free radicals have been implicated in the pathogenesis of myocardial tissue injury during ischemia and reperfusion. Certain sulfhydryl-containing compounds have been shown to inhibit complement activation. The sulfhydryl compounds captopril and N-(2-mercaptopropionyl)-glycine (MPG) are antioxidant compounds that previously have been shown to protect the myocardium from ischemia and reperfusion-induced damage. In this study, captopril (an angiotensin-converting-enzyme inhibitor; ACEI) and MPG, and the non-sulfhydryl compound enalaprilat (also an ACEI) were tested for their ability to protect the isolated perfused rabbit heart against complement-induced injury. Both captopril and MPG protected hearts against complement-mediated increases in left ventricular end-diastolic pressure and increases in coronary arterial perfusion pressure in a concentration-dependent manner, whereas enalaprilat was not protective. The ability of these compounds to inhibit complement activation also was tested using an in vitro complement-mediated red blood cell hemolysis assay. These findings offer additional insight as to the mechanism whereby captopril, MPG, and possibly other sulfhydryl compounds, may be acting to provide cytoprotection during myocardial ischemia and reperfusion.

Immunohistochemical localization of 6-keto-PGF1-alpha in canine coronary vasculature.

The localization of the prostacyclin metabolite, 6-keto-PGF1-alpha, in canine coronary vasculature was accomplished using immunohistochemical techniques (avidin-biotin method of immunoperoxidase staining). Six-keto-PGF1-alpha was localized to the intimal endothelial cell layer of epicardial and intramyocardial arteries and veins. No specific staining was seen in the the media or adventitia of canine coronary vasculature, or in capillaries, or myocardial fibers. To our knowledge these studies represent the first immunohistochemical demonstration of the endothelial cell localization of the prostacyclin metabolite, 6-keto-PGF1-alpha. The described technique allows the cellular localization of prostaglandin metabolites in histologic sections.

Plasma catecholamines in acute magnesium deficiency in weanling rats.

A controlled study was conducted to quantitate plasma catecholamines in magnesium-deficient weanling rats experiencing the seizure-shock episode. Eighty-four male Sprague-Dawley rats each weighing 35.6 +/- 0.3 g (mean +/- SEM) were fed purified diets to which was added 150 mg magnesium/100 g (Mg-150) or no magnesium (Mg-0). Studies were conducted between d 5 and 8. Plasma and bone magnesium and calcium were measured by atomic absorption spectrophotometry, and plasma catecholamines by radioenzymatic assay using 3H. Compared with Mg-150 rats, the Mg-0 rats showed reduced weight gain (P less than 0.001); reduced plasma magnesium (P less than 0.001) and reduced bone magnesium (P less than 0.001) with no corresponding changes in calcium concentration; and a 25% mortality by d 8. Pair-feeding and 80-dB noise provoked no changes in plasma catecholamines in Mg-150 rats, but both strychnine-induced seizures in Mg-150 rats and seizures induced by 80-dB noise in Mg-0 rats were accompanied by massive increases in plasma catecholamines. In contrast, 80-dB noise in Mg-0 provoked a massive increase in plasma catecholamines (P less than 0.001). However, gross pulmonary pathology developed only in Mg-0-shocked rats, not Mg-150-shocked animals. The study provides no evidence for a role of catecholamines in the pathogenesis of Mg-0 shock. The weanling rat displayed the ability to release massive quantities of three catecholamines during the final stages of acute magnesium deficiency and to normalize the plasma catecholamine levels within 16 h after seizure shock.

Soluble complement receptor type 1 prevents human complement-mediated damage of the rabbit isolated heart.

The purpose of this study was to determine if recombinant human soluble CR1 (sCR1) could prevent tissue damage associated with the activation of human complement. Directly mediated human complement-dependent myocardial injury was induced in the rabbit isolated heart perfused with a Krebs-Henseleit buffer containing 6% human plasma. There were three study groups: 1) 6% heat-inactivated human plasma (control); 2) 6% normal human plasma (NHP); or 3) 6% normal human plasma + 20 nM sCR1 (NHP + sCR1). Recorded functional parameters of the control group remained stable throughout the duration of the 70-min protocol. Complement activation in hearts perfused with 6% NHP increased the diastolic pressure; decreased developed pressure; and increased coronary perfusion pressure. These alterations were accompanied by a decrease in the maximum positive and negative dP/dt. Complement activation also increased cardiac muscle lymphatic fluid flow rate. The changes were greatest between 20 and 40 min, but persisted for the duration of the protocol. sCR1 (20 nM) in the perfusate containing 6% NHP prevented the complement-mediated alterations in the systolic, developed, and coronary perfusion pressures. sCR1 prevented the decrement in the positive and negative dP/dt, and the increase in the lymphatic fluid flow rate. Values for each of these parameters in hearts perfused with 6% NHP + sCR1 were not altered from those of controls at any time point in the protocol. Ultrastructural changes were present in tissues perfused with 6% NHP along with immunohistochemical evidence for presence of the terminal C5b-9 complex. sCR1 prevented the ultrastructural changes and the formation of the terminal complex. sCR1 offers significant protection against the cytolytic effects resulting from activation of the human complement system.

The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis to detect renal damage in Sprague-Dawley rats treated with gentamicin sulfate.

Renal toxicity is a common manifestation to the exposure of laboratory animals and humans to a wide range of xenobiotics. Traditional methods for evaluating renal damage by clinical chemistry such as blood urea nitrogen (BUN) and serum creatinine are not sensitive to early, mild changes. The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to measure the molecular weight spectrum of urinary proteins allows for an evaluation of the functional changes associated with renal damage. The ability of the kidney to filter and reabsorb proteins is related to the functional ability of glomeruli and the proximal tubules. Gentamicin sulfate produces injury to the S-1 and S-2 segments of the proximal tubule in laboratory animals and humans. While severe damage to the tubules is associated with increased BUN, serum creatinine, and N-acetyl-beta-glucosiminadase (NAG), mild injury is not detected by these means. The evaluation of urinary proteins by SDS-PAGE demonstrated renal toxicity at a dose of 6 mg/kg after 2 days of sc treatment. The NAG: creatinine ratio was shown to be elevated after 2 days of treatment at 63 mg/kg. The use of SDS-PAGE as described in this paper provides a sensitive method for detecting renal injury.

Active E-rosette formation in women taking oral contraceptives.

PIP: On the assumption that the number of E-rosettable lymphocytes (active T lymphocytes) is an index of cell-mediated immunity, rosette assays were performed at early cycle and at midcycle for 6 women taking oral contraceptives (OCs) for 1-4 years. OC subjects at midcycle had 21.4% active rosette-forming lymphocytes as compared with 14.1% in controls (p less than .05). The 2 youngest subjects had higher values during the early cycle. These results imply the possibility of hormonal regulation of human T-cell activity.^ieng

Inhibition of C1q functions by RHP, a protein elevated in sera from patients with rheumatoid arthritis.

We have previously shown that serum levels of C1q, unbound to C1r X C1s, are elevated in rheumatoid arthritis. We have also shown that RHP, a newly described serum protein which affects the C1q-anti C1q precipitin reaction, is also present at elevated levels in rheumatoid arthritis. We now show that RHP inhibits the hemolytic activity of C1q, disaggregates C1, and inhibits the ability of C1q bound to latex beads or to aggregated IgG to enhance the oxidative metabolism of neutrophils.

Bioluminescent enzyme immunoassay for estriol. Use of reversibly inactivated bacterial luciferase as label.

A bioluminescent enzyme immunoassay using estriol labeled with reversibly inactivated bacterial luciferase is described. An estriol derivative bearing an alkylthiolsulfonate is linked to the cysteinyl thiols of luciferase by formation of mixed disulfide linkages; thus, luciferase becomes inactive. After immunoassay, the inactive luciferase of the label bound to the immunoprecipitate is reactivated by incubation with dithiothreitol and the luciferase activity then is quantitated by a 20-s reaction performed with an automated luminometer (LKB 1251). Under the defined conditions, the labels are stable for at least 14 days as tested at 4 degrees C. A standard curve with a wide linear range from 50 to 6000 pg is demonstrated. This unique technology discussed here, therefore, offers exciting possibilities as a sensitive and rapid enzyme immunoassay for estriol.