A sensitive enzyme immunoassay for the quantitation of human CTLA4Ig fusion protein in mouse serum: pharmacokinetic application to optimizing cell line selection.

A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantitation of human CTLA4Ig in mouse serum was validated. The EIA method employed a technique in which a monoclonal anti-CTLA4 antibody was adsorbed onto 96-well polystyrene microtiter plates and used to capture the CTLA4Ig in mouse serum samples. The captured CTLA4Ig was then detected using a goat anti-human IgGFc antiserum conjugated to the enzyme horseradish peroxidase. The validation included assessments of method accuracy and precision, range of reliable response, lower limit of quantitation (LLQ), inter-analyst robustness, storage stability in mouse serum and assay specificity. The results indicate that this validated assay is precise, accurate, and reproducible. This EIA has a range of reliable response in 10% mouse serum of 0.14-4.58 ng ml-1 resulting in a 100% serum equivalent curve of 1.4-45.8 ng ml-1. Assessment of individual standard curve variations indicated a reproducible response with R2 values of > or = 0.995. The LLQ was established at 1.4 ng ml-1. The accuracy and precision estimates, based on the quality control values, were within 3.8% and 5.2% respectively, for CTLA4Ig. Stability of CTLA4Ig was established in mouse serum for 5 days at both 4 degrees C and room temperature, for 2 months at -70 degrees C and through five freeze-thaw cycles. This validated assay was successfully employed in the assessment of pharmacokinetic characteristics of CTLA4Ig in mice and to aid in the selection of an optimal CTLA4Ig-producing cell line.

A pharmacokinetic study of intravenous CTLA4Ig, a novel immunosuppressive agent, in mice.

Three skin-intact mice in each group received a single 0.07-, 0.29-, or 0.57-mg dose of CTLA4Ig intravenously (i.v.). Three skin-grafted mice received a single 0.29-mg dose i.v. and another three skin-grafted mice received a 0.29-mg dose once daily for 7 days (the dose was administered via the tail vein). Serial blood samples (0.15 mL) were obtained by retro-orbital bleeds up to 240 h after all single doses and up to 360 h after the last multiple dose. Serum samples were analyzed for CTLA4Ig by a validated enzyme immunoassay method. The concentration data were subjected to noncompartmental pharmacokinetic analysis. Both Cmax and AUCinf values increased in a dose-proportional manner in skin-intact mice. The CLT values were dose independent. The MRT, t1/2, and Vdss values at the 0.07-mg dose level were significantly lower than those obtained for both the 0.29- and 0.57-mg dose levels; however, the respective values between the 0.29- and 0.57-mg dose levels were not significantly different. No significant differences were found in the pharmacokinetic parameters between the skin-intact and skin-grafted mice.

Differential distribution of insulin-like growth factors and their binding proteins within bone: relationship to bone mineral density.

To evaluate the possibility that insulin-like growth factors (IGFs) and their binding proteins (BPs) in bone play a role in regulating cortical bone formation in growing animals, we compared changes in IGF and IGF BP levels with changes in bone mineral density (BMD) at three different regions (proximal, middle, and distal) along the rabbit femoral shaft. BMD measured by dual-energy x-ray absorptiometry decreased progressively from proximal to distal regions of the shaft, from 0.449 +/- 0.005 to 0.354 +/- 0.002 g/cm2 (mean +/- SEM; n = 9), respectively; total protein concentrations also decreased toward the distal region. We extracted the IGFs and their BPs from bone by demineralization in 10% EDTA and 4 M guanidine-HCl (pH 4.5). The IGFs were then separated from their BPs by size exclusion HPLC. The pH of the extraction buffer profoundly influenced the recoveries of the IGFs and, to a lesser extent, the total protein; at least 100% more IGFs were recovered at acid (4.5) pH than at neutral (7.5) or basic (10.5) pH. The levels of IGF-I decreased markedly from proximal to distal regions, from 273 +/- 27 to 100 +/- 38 ng human IGF-I equivalent/g bone (or 103 +/- 10 to 52 +/- 11 ng human IGF-I equivalent/mg protein), respectively. IGF-II was uniformly distributed (385 +/- 17 ng human IGF-II equivalent/g bone; mean of all three regions). Levels of the predominant 28-32 kD IGF BP doublet increased by about 100% from proximal to distal segments, regardless of whether the data were expressed per unit mass or protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation of bone mineral density from age-related decreases in insulin-like growth factor-I and its binding proteins in the male rat.

We evaluated the possibility that age-related decreases in circulating and/or bone-associated insulin-like growth factor-I (IGF-I) and its binding proteins (BPs) were associated with the development of osteopenia in 8-, 16-, and 24-month-old specific pathogen-free Brown Norway/Fischer 344 male rats. We measured bone mineral densities (BMD) of femurs by dual-energy x-ray absorptiometry. IGFs and IGFBPs were extracted from bone and separated by molecular exclusion HPLC before quantitation by specific radioligand assays. BMD did not change significantly between 8 and 24 months of age. IGF-I levels decreased by about 30% between 8 and 24 months in both serum and bone. Similarly, both circulating and bone-derived IGFBPs also declined (30% and 60%, respectively) with age. Thus, maintenance of femoral BMD throughout most of the adult rat life span was dissociated from the age-related decline in circulating and bone-associated IGF-I and IGFBPs.

Heterogeneity of human bone.

Matched samples of bone from the lumbar spine and tibia were obtained at autopsy from three adult males who had no known evidence of metabolic bone disease at the time of their demise. The soluble noncollagenous bone proteins were quantitatively extracted from these samples and assayed for the relative content of two bone-associated proteins, osteocalcin and osteonectin. When compared to trabecular bone, cortical bone had higher levels of osteocalcin and much lower levels of osteonectin. When concentration is expressed per gram of dried bone, the osteocalcin excess in cortical bone ranged from 30- to 32-fold, and the osteonectin excess in trabecular bone ranged from 21- to 47-fold. These differences were significant (P less than 0.01) using analysis of variance. We conclude that the human skeleton is not homogeneous with regard to these biochemical markers and that cortical and trabecular bone are biochemically quite distinct. This implies that these two types of bone may be subject to distinct regulatory mechanisms and that global assessments of skeletal function and bone quality based upon soluble markers should be applied with caution. The data also imply that a differential assessment of skeletal performance may be possible using biochemical serum markers.

Simultaneous measurement of total and IgA-conjugated alpha 1-microglobulin by a combined immunoenzyme/immunoradiometric assay technique.

alpha 1-Microglobulin (alpha 1m), a glycoprotein (Mr = 30,000) found in serum and urine, is also present in serum conjugated to monomeric IgA (alpha 1-m-IgA). We have developed a simultaneous enzyme-linked immunoenzyme/immunoradiometric assay that involves three different monoclonal antibodies. Assay of serial dilutions of serum and urine demonstrated parallelism. Normal mean concentrations in serum (n = 75) were: total alpha 1m, 2.33 mumol/L; alpha 1m-IgA, 1.24 mumol/L; unconjugated (free) alpha 1m, 1.09 mumol/L; molar ratio (alpha 1m-IgA/total alpha 1m), 0.53. The mean concentration of alpha 1m in eight urine specimens from normal individuals was 0.19 mumol/L, with no detectable alpha 1m-IgA. A low urinary pH does not significantly affect assay results, unlike assays of beta 2-microglobulin. In patients with myeloma-related renal disease, total and free alpha 1m values for serum correlated well with values for creatinine and beta 2-microglobulin in serum.

Defenses of the hamster lung against human neutrophil and porcine pancreatic elastase.

Instillation of human neutrophil elastase (HNE) into hamster lungs produces milder emphysema but more pulmonary hemorrhage than an equivalent amount of porcine pancreatic elastase (PPE), whether equivalence is determined by elastolytic units or moles. We undertook a study of the mechanisms of these differences. 125I-HNE or 3H-PPE were instilled intratracheally into hamsters. The partitioning of radioactivity between bronchoalveolar lavage fluid (BAL) and lung tissue was similar for HNE and PPE as were the half-lives, 45 and 51 min, respectively, for uncomplexed, enzymatically active HNE and PPE. In BAL there was preferential binding and inactivation of HNE by the hamsters' alpha-1-protease inhibitor (a-1-PI) whereas PPE was preferentially bound by alpha-2-macroglobulin (a-2-M). This was also observed in vitro when HNE and PPE were incubated with plasma from untreated hamsters. Nevertheless, when the sum of the elastase binding capacity of a-1-PI and a-2-M was considered, hamster plasma had similar binding capacities for HNE and PPE. It is known that the enzymatic activity of elastases is inhibited by formation of a stable complex with a-1-PI. On the other hand, elastases bound to a-2-M are protected against a-1-PI inhibition but can free themselves by proteolysis and exhibit elastolytic activity. Preferential inactivation of HNE by a-1-PI may be one mechanism that accounts for the lesser emphysema-inducing potency of HNE than of PPE.

Elastin in a neonatal rat smooth muscle cell culture has greatly decreased susceptibility to proteolysis by human neutrophil elastase. An in vitro model of elastolytic injury.

A neonatal rat aorta smooth muscle cell culture system with a unique elastin-rich extracellular matrix was used as a model substrate for elastases. To study the susceptibility to solubilization of insoluble elastin, cultures were incubated in the presence of human neutrophil elastase (HNE) or porcine pancreatic elastase (PPE) and in the absence of serum for periods up to 45 min. Both the incubation media and cell layers were then assessed for elastin and collagen markers, total protein, and lactate dehydrogenase (LDH). Although HNE and PPE exhibited comparable activity against elastin purified from the cell layer, HNE exhibited a 6.7- to 25-fold reduction in its elastin solubilizing activity using intact cell layers as compared with the purified elastin, whereas PPE exhibited only a 1.5- to 2.5-fold reduction. This effect could not satisfactorily be explained as preferential inhibition of HNE activity in the culture system, because the amount of protein solubilized by HNE was 59% that of PPE. The mean elastin content of PPE-solubilized protein was 110% that of the elastin content of the corresponding cell layer; the value for HNE-solubilized protein was only 16%. Thus, the amount of elastin per microgram of solubilized protein for HNE was 15% that for PPE. Possible explanations for the greatly diminished elastolytic activity of HNE in the culture system include the preference of HNE for other substrates in the cell layer, the inability of HNE to penetrate sufficiently into the cell layer, and the presence of sulfated glycosaminoglycans in the vicinity of the elastin that act in an inhibitory fashion. Although there was extensive proteolytic damage to the extracellular matrix, LDH and DNA measurements indicated that little loss of cells or cell viability occurred. The observed differences in elastolytic activity of HNE and PPE in the culture system parallel the relative emphysema-inducing potency of the elastases in the hamster model of elastase-induced emphysema.

Effect of varying the time interval between intratracheal administration of eglin-c and human neutrophil elastase on prevention of emphysema and secretory cell metaplasia in hamsters. With observations on the fate of eglin-c and the effect of repeated instillations.

Eglin-c is a naturally occurring polypeptide of 70 amino acids with a molecular mass of 8,100 daltons. It is a strong inhibitor of human neutrophil elastase (HNE) and cathepsin-G, and, when given intratracheally to hamsters 1 h before human neutrophil elastase, it can prevent or ameliorate the emphysema produced by HNE. The present experiments were designed to determine the duration of the effectiveness of eglin-c, prepared by DNA technology from Escherichia coli, in preventing the emphysema and secretory cell metaplasia induced by HNE. Eglin-c (2,000 micrograms in 0.5 ml saline) was effective in ameliorating emphysema, as determined histologically and physiologically, when it was given intratracheally to hamsters 1, 2, 4, and 8 h before the intratracheal instillation of 300 micrograms of HNE. Eglin-c ameliorated bronchial secretory cell metaplasia when given 1 h before HNE but not when the time intervals were 2 h or longer. The clearance of [3H]eglin-c from the lungs was assessed. Four h after intratracheal instillation of 446 micrograms of [3H]eglin-c, 33% of the tritium was found in the lung tissue and bronchoalveolar lavage fluid; 83% of the radioactivity in the lavage fluid supernatant was associated with functionally active eglin-c. No evidence of bronchopulmonary toxicity was seen in hamsters given 4 intratracheal instillations of 2,000 micrograms of eglin-c at 1-wk intervals.

The association of an elastase with amyloid fibrils.

The fibrils of all systemic forms of amyloid (primary, AL; secondary, AA; and hereditary, AF) that had been isolated by the water extraction procedure demonstrated elastolytic enzyme activity when examined in a specific assay using tritiated elastin. The source of this fibril-bound enzyme activity was consistent with human neutrophil elastase (HNE), since it was readily extracted by high salt solutions and inhibited by an elastase-specific chloromethyl ketone inhibitor, human alpha-1-protease inhibitor or by an antibody specific for HNE. The presence of an elastase on the amyloid fibril may suggest physiologic mechanisms of amyloid precursor protein degradation.

Eglin-c, a polypeptide derived from the medicinal leech, prevents human neutrophil elastase-induced emphysema and bronchial secretory cell metaplasia in the hamster.

Eglin-c (Eg-c), a polypeptide with a molecular mass of 8,100 daltons, was purified from the medicinal leech Hirudo medicinalis. The Eg-c was tritiated by reductive methylation for in vitro studies. Incubation of 2.1 X 10(-10) moles of human neutrophil elastase (HNE) with 3H-elastin in the presence of 8.2 X 10(-10) moles of 3H-Eg-c inhibited 98.7% of the elastolytic activity of the enzyme. Using Sephadex G 100 chromatography and 1.7 moles of 3H-Eg-c per mole of HNE, a 34,000-dalton complex (3H-Eg-c-HNE) was observed. The stability of the complex formed between 3H-Eg-c and HNE that had been inactivated with succinyl-ala2-pro-val CH2Cl was much less than that of the 3H-Eg-c-HNE complex. In vivo studies were carried out in weight-matched groups of anesthetized golden Syrian hamsters given 100, 300, 500, or 2,000 micrograms of Eg-c in 0.5 ml saline intratracheally 1 h before 300 micrograms HNE was administered intratracheally. Control animals received saline followed by HNE or 2 doses of saline 1 h apart. Eight weeks later, lung statics and dynamics were measured in anesthetized animals, followed by histologic study of lung parenchyma and the mucosa of the large intrapulmonary airways. There were no deaths, and final mean body weights were similar in all groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of combined human neutrophil cathepsin G and elastase on induction of secretory cell metaplasia and emphysema in hamsters, with in vitro observations on elastolysis by these enzymes.

To determine whether purified human neutrophil cathepsin G (Cat-G) can act by itself or in concert with purified human neutrophil elastase (HNE) in the induction of emphysema and bronchial secretory cell metaplasia (SCM), we gave golden Syrian hamsters 100 micrograms of HNE alone or in combination with either 100 or 200 micrograms of Cat-G. Other groups of animals received intratracheal doses of up to 600 micrograms of Cat-G alone. The severity of emphysema was determined from measurements of lung volumes, compliance, forced expiratory flow, and the mean linear intercept. The severity of SCM in the main airways was graded on sections stained by the alcian blue and periodic acid-Schiff reaction. The Cat-G was a weak inducer of SCM; significant SCM was produced by 400 and 600 micrograms but not by 100 or 200 micrograms or 200 micrograms of Cat-G. The Cat-G (100 and 200 micrograms) did not potentiate the SCM induced by 100 micrograms of HNE. The Cat-G alone did not produce emphysema, and neither 100 nor 200 micrograms of Cat-G potentiated the mild emphysema induced by 100 micrograms of HNE. These results were not consonant with a report that Cat-G and HNE were synergistic in solubilizing human lung elastin. We therefore measured the ability of Cat-G and HNE to solubilize several radiolabeled elastins. The combination of Cat-G and HNE did not solubilize significantly more hamster lung elastin (23%) than the sum of their individual activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Myeloperoxidase-induced inactivation of alpha 1-antiprotease in hamsters.

While numerous studies have demonstrated that the myeloperoxidase system found in neutrophils can oxidize and functionally inactivate alpha 1-proteinase inhibitor in vitro, there is little direct evidence that this phenomenon is relevant in vivo. Using incubation with tritiated porcine pancreatic elastase followed by column chromatography to quantitate binding, we demonstrated recovery of microgram amounts of functional alpha 1-protease inhibitor from bronchoalveolar lavage of hamster lungs. When exposed to the myeloperoxidase system in vitro, hamster alpha 1-protease inhibitor was 97% inactivated. Functional alpha 1-protease inhibitor recovered by bronchoalveolar lavage 20 minutes after hamsters were given intratracheal injections with myeloperoxidase and either hydrogen peroxide or glucose plus glucose oxidase was only half that recovered from control animals. These studies suggest that the myeloperoxidase system is effective in oxidizing alpha 1-protease inhibitor in vivo. They support the concept that oxidation of alpha 1-protease inhibitor by myeloperoxidase from neutrophil granules in the presence of H2O2 and halide may produce elastase-antielastase imbalance in vivo and contribute to the development of acute lung injury and emphysema in humans.

Serum antielastase and neutrophil elastase levels in PiM phenotype cigarette smokers with airflow obstruction.

In order to assess blood factors which might explain why some cigarette smokers develop airflow obstruction while others do not, we compared two groups of PiM phenotype volunteers matched for age, sex and total pack-years of cigarette smoking; one group had airflow obstruction and the other did not. Functional levels of alpha-2-macroglobulin (alpha-2-M) and alpha-1-protease inhibitor (alpha-1-PI) were separately assessed by a protease binding procedure. Neutrophils were isolated from blood by counterflow centrifugation, and their elastase content was assayed with 3H-elastin-SDS (sodium dodecyl sulfate). The obstructed and nonobstructed groups were not different with respect to functional or immunoreactive levels of alpha-1-PI and alpha-2-M or elastase levels in their neutrophils. We do not find imbalances of circulating elastase or antielastase levels in PiM phenotype smokers with airflow obstruction.

Emphysema and bronchial secretory cell metaplasia induced in hamsters by human neutrophil products.

Purified human neutrophil elastase (HNE) and a crude extract of human neutrophils (EXT) were administered intratracheally to hamsters, and their effects were determined 21 and 56 days later by measurement of lung statics and dynamics and by light microscopy and morphometry of the lungs. A dose of 450 micrograms of HNE (HNE 450) produced focal emphysema of moderate severity. The combination of HNE 450 and EXT (HNE 450 + EXT) produced no more severe emphysema than HNE 450 alone; EXT alone did not produce emphysema. The HNE 450, HNE 450 + EXT, and EXT all produced persistent secretory cell metaplasia in large intrapulmonary airways. In a second experiment, 40 micrograms of HNE (elastolytic activity equivalent to that in EXT), designated HNE 40, did not produce secretory cell metaplasia. Neither EXT nor EXT treated with the elastase inhibitor suc-ala-ala-pro-val chloromethyl ketone (EXT + CMK), which had a residual elastolytic activity equivalent to 15 micrograms of HNE, produced emphysema; both preparations caused bronchial secretory cell metaplasia 21 days after treatment. Values of maximal expiratory flow, measured 21 days after treatment, were reduced at points between 20 and 50% of vital capacity for HNE 450 and between 30 and 80% of vital capacity for HNE 450 + EXT; maximal expiratory flows were not significantly different from saline controls for HNE 40, EXT, or EXT + CMK.(ABSTRACT TRUNCATED AT 250 WORDS)

Human neutrophil elastase does not bind to alpha 1-protease inhibitor that has been exposed to activated human neutrophils.

The effect of leukocyte-derived oxidants on the elastase-inhibitory capacity of alpha 1-protease inhibitor was examined in an in vitro system using cells and purified proteins from human sources. The exposure of alpha 1-protease inhibitor to the myeloperoxidase-hydrogen peroxide-halide system resulted in a nearly complete loss of its ability to bind and inactivate purified human neutrophil elastase. A similar loss of binding to and inactivation of human neutrophil elastase was observed on exposure of alpha 1-protease inhibitor to human neutrophils in the presence of a halide and the neutrophil-activating agent, phorbol myristate acetate. This loss of elastase binding activity was abrogated by the addition of azide or catalase but not superoxide dismutase or heated catalase. The data suggest oxidative inactivation of alpha 1-protease inhibitor by secreted myeloperoxidase and hydrogen peroxide. Thus, the reported effects of leukocyte oxidants, especially the myeloperoxidase system, on alpha 1-protease inhibitor have been confirmed using the most pathophysiologically relevant protease, human neutrophil elastase, as the test enzyme. The role of the neutrophil in the pathogenesis of emphysema may, therefore, include the secretion of both elastase and oxidants that impair antielastase defenses.

Functional alpha 1-protease inhibitor in the lower respiratory tract of cigarette smokers is not decreased.

Cigarette smoking is the major risk factor for the development of pulmonary emphysema, a disorder that may result from an imbalance between the elastase and antielastase levels in the lungs. Decreased functional alpha 1-protease inhibitor, an inhibitor of neutrophil elastase, might render smokers susceptible to elastase-catalyzed destruction of pulmonary elastic fibers and the development of emphysema. Binding and inactivation of isotopically labeled porcine pancreatic elastase and human neutrophil elastase by alpha 1-protease inhibitor were measured in fluid obtained by bronchoalveolar lavage of volunteers. The inhibition of elastase-catalyzed solubilization of elastin and a tripeptide substrate were also determined. The mean level of functional alpha 1-protease inhibitor in the bronchoalveolar lavage fluid of smokers was found to be equal to or greater than that of nonsmokers, contradicting reports by other investigators. Increased elastase derived from pulmonary neutrophils, rather than decreased functional alpha 1-protease inhibitor, appears to be the main factor in the genesis of emphysema in smokers.

The fate of neutrophil elastase incorporated by human alveolar macrophages.

Although protease-antiprotease imbalance is widely thought to contribute to the genesis of emphysema, the involvement of the alveolar macrophage in this process is poorly defined. We have quantified the uptake of radioiodinated human neutrophil elastase by human alveolar macrophages in monolayer culture and assessed the enzyme's fate during periods as long as 48 h after uptake using molecular sieve chromatography. Approximately half of the radiolabel eluted with enzymatically inactive material of molecular sizes corresponding to either degraded or alpha1-protease-inhibitor-bound elastase. The remainder of the radiolabel eluted at 29,000 daltons, in fractions containing enzyme that solubilized particulate elastin, and likely represented intact neutrophil elastase. Macrophages from smokers and nonsmokers showed similar characteristics of incorporation and disposition of neutrophil elastase. Lysates of uncultured alveolar macrophages from smokers and nonsmokers contained (mean +/- SE, n = 4) 7.4 +/- 0.8 and 3.3 +/- 1.4 ng of neutrophil elastase activity per 10(6) cells, respectively (not significant). However, as smokers have 11-fold more cells obtainable by lavage, the total lavaged elastase loads in alveolar macrophages were 561.7 +/- 72.3 and 21.3 +/- 6.8 (p less than 0.01) in smokers and nonsmokers, respectively. We conclude that the alveolar macrophage may clear and inactivate neutrophil elastase in the lung. In addition, by conserving some ingested elastase in an enzymatically active form, the macrophage may serve as an elastase reservoir. In areas of high macrophage density, as around the respiratory bronchioles of smokers, the macrophage could release a portion of the incorporated elastase and damage lung elastin.