Establishment of a sandwich ELISA for human megsin, a kidney-specific serine protease inhibitor.

BACKGROUND: We previously identified a novel serine protease inhibitor (serpin), megsin, which is predominantly expressed in the kidney. Megsin expression is up-regulated in human and experimental renal diseases associated with mesangial proliferation and expansion, suggesting that urinary megsin may be a novel diagnostic marker for some renal diseases. METHODS: We established a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for megsin and measured urinary megsin of patients with various renal diseases. RESULTS: Megsin ELISA specifically detected megsin but not other serpins. The detection limit was 0.04 ng/ml, which allowed detection of urinary megsin in 3.6% of healthy individuals. The antigenic epitope in the urine detected by the ELISA was confirmed as megsin protein by time-of-flight mass spectrometry. Among patients with rapidly progressive glomerulonephritis (n = 18), 55.6% were urinary megsin-positive, while 24.1% in IgA nephropathy (n = 112) and 15.1% in chronic non-IgA glomerulonephritis (n = 245) were urinary megsin-positive, respectively. Among patients with chronic renal failure due to unknown causes (n = 74), 18.9% were positive for urinary megsin. In diabetic patients with or without nephropathy (n = 1073), 12.3% were urinary megsin-positive, while positivity of urinary megsin in patients with non-renal diseases (n = 768) was equivalent (3.3%) to that of healthy individuals. Of note, when urinary megsin-positive patients with diabetic nephropathy (n = 71) were classified into four stages by their proteinuria and estimated glomerular filtration rate, urinary megsin excretion increased as the stage progressed up to stage 3A, suggesting correlation of that with mesangial expansion level. Urinary megsin decreased in the advanced stage, probably reflecting development of glomerulosclerosis. CONCLUSION: We established a high-sensitive megsin ELISA, which detects urinary megsin in some patients with renal diseases and in only a few healthy subjects. Megsin ELISA may be a novel diagnostic tool for renal diseases.

Absorption, metabolism, degradation and urinary excretion of rosmarinic acid after intake of Perilla frutescens extract in humans.

BACKGROUND: Rosmarinic acid (RA) is a natural polyphenolic substance contained in many Lamiaceae herbs such as Perilla frutescens. Previous studies have shown RA has antioxidative and anti-inflammatory activity. However, little is known on the absorption, metabolism, degradation and excretion of RA. AIM OF THE STUDY: The aim of this study in healthy humans was to determine the absorption, metabolism, and urinary excretion of RA after a single intake of perilla extract (PE). METHOD: Six healthy men (mean age 37.2 +/- 6.2 y and mean body mass index 22.0 +/- 1.9 kg/m(2)) were enrolled in the study that was a crossover design involving single intakes of PE containing 200 mg RA and placebo with a 10 day interval between treatments. Blood samples were collected before intake and at designated time intervals, while urine samples were collected over the periods 0-6 h, 6-24 h and 24-48 h after intake. RA and its related metabolites in plasma and urine were measured by LC-MS. RESULTS: RA, methylated RA (methyl-RA), caffeic acid (CAA), ferulic acid (FA) and a trace of m-coumaric acid (COA) were detected in the urine after intake of PE. In plasma, RA, methyl-RA and FA were detected, with maximum levels obtained 0.5, 2 and 0.5 h after intake of PE, respectively. The majority of these components in both plasma and urine were present as conjugated forms (glucuronide and/or sulfated). The proportion of RA and its related metabolites excreted in the urine was 6.3 +/- 2.2% of the total dose, with approximately 75% of these components being excreted within 6 h after intake of PE. CONCLUSIONS: RA contained in PE was absorbed, conjugated and methylated following intake, with a small proportion of RA being degraded into various components, such as conjugated forms of CAA, FA and COA. These metabolites were then rapidly excreted in the urine.

The trimannosyl cores of N-glycans are important for the procoagulant protease-inhibitory activity of urinary protein C inhibitor.

We investigated the relationship between the procoagulant protease-inhibitory activity and the N-glycan structures in urinary protein C inhibitor (uPCI) by sequential exoglycosidase digestions based on the N-glycan structures elucidated in this report. uPCI was glycosylated on the three potential N-glycosylation sites, asparagines 230, 243 and 319 (N230, N243 and N319) in the molecule and had four biantennary complex type sugar chains. The inhibitory activities of uPCI toward thrombin and plasma kallikrein were little changed by the sequential removal of N-acetylneuraminic acid and galactose residues from the termini and N-acetylglucosamine residues from the branches of the N-glycans. However, the inhibitory activities were markedly decreased by further removing alpha-mannose residues from the trimannosyl cores of the N-glycans. These results suggest that the trimannosyl cores of N-glycans are important for uPCI to inhibit the procoagulant protease.

Excretion of bikunin and its fragments in the urine of patients with renal stones.

PURPOSE: Bikunin is a Kunitz-type protease inhibitor found in serum and urine. It has been implicated in urinary stone formation. This study was designed to investigate the role of urinary bikunin in stone formation. MATERIALS AND METHODS: Urinary concentrations of bikunin were measured in 18 male formers of urinary stones 28 to 74 years old and in 77 healthy controls, including 39 males and 38 females, without urological abnormality. A sensitive competitive solid phase enzyme immunoassay was established for urinary bikunin. Bikunin was also qualitatively assessed by Western blot analysis. RESULTS: The mean urinary bikunin-to-creatinine ratio plus or minus standard deviation in stone formers was significantly elevated compared with that in healthy male and female controls (52.9 +/- 46.0 microg./mg. creatinine versus 28.0 +/- 30.4 and 26.5 +/- 21.7, p = 0.005 and 0.006, respectively). By Western blot analysis all urine samples contained authentic 40 kDa. bikunin species. However, a significantly higher proportion of patients was found to have aberrant 25 kDa. bikunin species compared with controls (10 of 18 or 55.6% versus 15 of 77 or 19.5%, p = 0.002). Experiments on de-glycosylation with chondroitinase ABC, amino acid sequencing of the aberrant bikunin species and calcium oxalate crystal growth inhibition assay demonstrated that the 25 kDa. bikunin fragment was identical to de-glycosylated bikunin and less inhibitory on calcium oxalate crystal growth. CONCLUSIONS: If urinary bikunin is important in the pathogenesis of urolithiasis, its effect is probably attributable to the concentration and degree of glycosylation.

Changes of urinary alpha1-microglobulin in the assessment of prognosis in renal transplant recipients.

BACKGROUND: After transplantation, even if the graft starts functioning immediately, there are morphological and functional changes in tubular structures. In addition, acute allograft rejection causes damage in the tubular epithelium, tubular basement membrane, and intertubular connective tissue. It also affects the functional capacity of proximal tubular cells resulting in impaired reabsorption and thus increased urinary excretion of low molecular weight proteins. METHODS: We present a double-antibody radioimmunoassay for determination of the concentration of alpha1-microglobulin (alpha1 M) in urine. It was used to measure urinary excretion of alpha1 M approximately once a week during the first 1-6 posttransplant weeks in 136 consecutive patients: 30 patients developing acute rejection (75 24-hr urine samples) and 106 patients with stable graft function (223 24-hr urine samples). The results are expressed as alpha1 M/creatinine ratios. RESULTS: Approximately 8 days after transplantation the mean (+/-SD) urinary alpha1 M/creatinine ratio of all patients was 17.0+/-14.8 mg/mmol, being about the same both in patients with uncomplicated posttransplantation course (16.3+/-14.0 mg/mmol) and in those who later developed rejection (19.3+/-15.1 mg/mmol), but about 60-fold higher than in healthy controls (0.27+/-0.15 mg/mmol). At that time, when all patients were included there was a correlation (r=0.3465, P<0.001) between alpha1 M/creatinine ratio and duration of cold ischemia. Thereafter, during the second week alpha1 M/creatinine ratio decreased in 89% of patients with stable graft function, but only in 14% of patients who later developed rejection (P<0.001). On the contrary, a significant increase (P<0.01) of alpha1 M/creatinine ratio was observed 4 to 1 day before rejection in all 15 patients, who had urines collected at that time. At the end of the follow-up period, alpha1 M/creatinine ratio in patients with rejection was 3-fold compared with the nonrejecting patients, and 100-fold compared with the healthy controls. CONCLUSION: These results show that cadaveric transplantation results in impaired low molecular weight protein reabsorption, the degree of dysfunction relating to the duration of cold ischemia, and suggest that during the posttransplant weeks decreasing alpha1 M/creatinine ratio in consecutively collected urine samples indicates improved tubular function and in most cases rules out development of acute rejection.

Effect of urinary protein C inhibitor on lipopolysaccharide-induced disseminated intravascular coagulation in rats.

The effect of urinary protein C inhibitor (uPCI) on disseminated intravascular coagulation (DIC) was investigated using an experimental DIC in rats. uPCI (0.5 and 1.0 mg/kg) was continuously administrated into the left femoral vein of the rats with lipopolysaccharide (50 mg/kg)-induced DIC. In all doses, uPCI significantly prevented the drastic changes in the parameters such as fibrinogen concentration, activated partial thromboplastin time (APTT), prothrombin time (PT), fibrin/fibrinogen degradation products (FDP) level, aspartate amino-transferase (AST) level and alanine aminotransferase (ALT) level. Furthermore, uPCI significantly inhibited the increase in the levels of plasma kallikrein and thrombin which act not only as the procoagulant proteases but also as the chemotactic factors to neutrophils and monocytes. These results show that uPCI may prevent hypercoagulation, the induction of secondary fibrinolysis and organ failure in the DIC model. Therefore, uPCI may be a useful agent for the clinical treatment of DIC.

Urinary transforming growth factor-beta1 and alpha1-microglobulin in children and adolescents with type 1 diabetes.

OBJECTIVE: Transforming growth factor (TGF)-beta1 is an important mediator in the pathogenesis of diabetic nephropathy. Urinary TGF-beta1 reflects TGF-beta1 production in the kidney, and alpha1-microglobulin tubular dysfunction. These 2 markers were studied in the early phases of type 1 diabetes. RESEARCH DESIGN AND METHODS: There were 113 type 1 diabetic children and adolescents (mean +/- SD: age 14.1 +/- 2.9 years, and diabetes duration 7.4 +/- 2.9 years, HbA1c 9.3 +/- 1.5%) and 39 healthy subjects (age 13.8 +/- 2.8 years) who participated in the study. Of the diabetic patients, 105 were normoalbuminuric (2-3 consecutive overnight urinary albumin excretion rates [AERs] <20 microg/min) and 8 had microalbuminuria (at least 2 AERs 20-200 microg/min). Overnight urinary TGF-beta1 and alpha1-microglobulin levels were measured and the results expressed as the ratio to urinary creatinine concentration. RESULTS: Data are medians (range). Diabetic patients had higher urinary TGF-beta1 levels than those of control subjects: 0.9 ng/mg (0.05-122.3) vs. 0.3 ng/mg (0.05-2.2) creatinine, respectively (P = 0.003). Urinary TGF-beta1 levels correlated with urinary glucose (r = 0.2, P = 0.03) and alpha1-microglobulin (r = 0.2, P = 0.02) levels, but not with HbA1c, AER, age, or duration of diabetes. In 43 patients with urinary TGF-beta1 above the control levels, urinary TGF-beta1 levels correlated with urinary glucose (r = 0.6, P < 0.001) and alpha1-microglobulin (r = 0.6, P < 0.001) levels. Diabetic patients had higher urinary alpha1-microglobulin levels than those of control subjects: 4.8 microg/mg (0.6-48.8) vs. 2.7 microg/mg (0.8-11.6) creatinine, respectively (P < 0.001). Alpha1-microglobulin levels correlated with AER (r = 0.2, P = 0.02), HbA1c (r = 0.3, P = 0.001), urinary glucose (r = 0.5, P < 0.001), and urinary TGF-beta1 levels. CONCLUSIONS: An early rise in urinary TGF-beta1 levels was observed in young type 1 diabetic patients. Urinary TGF-beta1 is associated with 2 interrelated tubular markers, alpha1-microglobulin and urinary glucose.

Differential expression of urinary inter-alpha-trypsin inhibitor trimers and dimers in normal compared to active calcium oxalate stone forming men.

PURPOSE: We determine if the immunoreactive profile of urinary inter-alpha-trypsin inhibitor can be used to distinguish between normal individuals and individuals with calcium oxalate stone disease. MATERIALS AND METHODS: Urinary proteins were dialyzed against water (15 kDa. molecular weight cutoff), lyophilized and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6% acrylamide, reducing conditions) followed by Western blot. Inter-alpha-trypsin immunoreactive proteins were detected by enhanced chemiluminescence. Stone formation was confirmed to be active radiologically or passed as stone or gravel within 12 months of the sample. Stone composition was confirmed crystallographically. Normal individuals had no personal or familial history of urolithiasis and matched stone forming patients regarding race (white) and age (23 to 71 years old). Urine from a total of 101 individuals was analyzed. RESULTS: The intact inter-alpha-trypsin trimer (approximately 220 to 240 kDa.) and heavy chain (HC) 2-bikunin/HC1-bikunin dimers (approximately 115 to 130 kDa.) were detected more often in stone forming men (23 of 26 [89%] and 26 of 26 [100%], respectively) than in normal individuals (6 of 26 [23%] and 5 of 26 [19%], respectively, p < 0.0001). In those normal individuals who expressed inter-alpha-trypsin trimer and HC-bikunins the relative intensities were 5.3+/-1.4% and 16.3+/-17.1% of the stone forming controls, respectively. The identity of high molecular weight-inter-alpha-trypsin immunoreactive bands was confirmed using antibodies against the individual subunits (HC1, HC2, HC3, bikunin). In contrast to men high molecular weight-inter-alpha-trypsin's were readily detected in normal and stone forming women with equal frequency (inter-alpha-trypsin-trimer p=0.1337, HC-bikunins p=0.2836): inter-alpha-trypsin-trimer 17 of 18 [94%] and 9 of 13 [77%]; HC-bikunins 17 of 18 [94%] and 10 of 13 [85%]). Inter-alpha-trypsin-trimer and HC-bikunins, respectively, were detected in 2 and 5 of 10 patients with chronic renal disease. Expression was not related to hematuria or proteinuria. CONCLUSIONS: Immunoreactive profiles of urinary proteins may be able to be developed into a useful diagnostic tool to identify active stone formation, although a separate panel may be required for men and women. It is possible that these differences may provide clues as to why the incidence of stone disease is higher in men than women.

Inter-alpha-inhibitor in urine and calcium oxalate urinary crystals.

OBJECTIVE: To determine which chains of inter-alpha-inhibitor (I alpha I) are present in urine and whether they are also found in calcium oxalate (CaOx) crystals generated in human urine. MATERIALS AND METHODS: Fresh urine specimens were collected from five women and five men with no previous history of stone disease. An aliquot of each urine was retained for analysis, the remainder treated with a standard load of oxalate and the CaOx crystals precipitated from each specimen demineralized with ethylenediamine tetracetic acid. The resulting organic extracts from crystals and their corresponding urine samples were subjected to sodium dodecyl sulphate gel electrophoresis analysis and Western blotting using a commercial polyclonal antibody to I alpha I. RESULTS: Heavy chain 1 (H1) and 2 (H2) of I alpha I were commonly found in every urine sample, and in the CaOx crystals precipitated from those urine samples. Several protein bands were visible in urine samples from both sexes in the molecular mass range 25-70 kDa, which may be bikunin or its fragments. As well as H1 and H2, the crystals from both sexes contained a protein band at approximately 33 kDa. In many cases there appeared to be no direct relationship between the proteins detected in the crystals and the urine samples from which they were derived, which probably reflects the well known instability of I alpha I and the occurrence of a range of bikunin fragments in urine. CONCLUSION: These results show for the first time that H1 and H2 are present in human urine and urinary CaOx crystals, that the bikunin chain of I alpha I is not the only part of the molecule capable of participating in CaOx crystallization in urine, and in theory at least, in the regulation of crystallization events in stone formation. It is also apparent that significant fragmentation of I alpha I occurs both in vivo and in vitro, and this must be considered in any study attempting to elucidate the influence of this protein in the formation of CaOx stones.

Biodistribution and pharmacokinetics of 131I-labeled LEX 032, a recombinant hybrid of antichymotrypsin.

Pharmacokinetic and biodistribution studies were conducted in rats on a novel serine protease inhibitor, LEX 032, that was radiolabeled with 131I by the Bolton-Hunter reagent. LEX 032, a genetically engineered recombinant human nonglycosylated serpin, has been shown to have antiinflammatory properties in a number of animal models of inflammation and reperfusion injury. When 131I-LEX 032 was injected intravenously, a rapid whole body clearance of radioactivity was seen. Blood clearance followed a similar pattern. Forty-eight hours postinjection, 2.00 +/- 0.65 of the administered dose remained in the body. Greater than 59% of the radio-activity was excreted in the urine within the first 24 hr. Little radioactivity was found in the feces. With the exception of the thyroid, no significant organ-related uptake was noted. Radioactivity in the liver peaked at 20 min postinjection, with 1.00 +/- 0.13% administered dose/g and approximately 10% administered dose in the whole liver. At 1 hr, uptake in the kidney (9.30 +/- 1.52% administered dose/g) was the highest among all tissues, except for the thyroid. Gamma camera images were consistent with the biodistribution pattern. Pharmacokinetics and biodistribution were not affected by the dose of LEX 032 and were quite different from those of glycosylated wild type antichymotrypsin. These data indicate that LEX 032 exhibits the pharmacokinetics expected of a nonglycosylated 45 kDa protein.

Acid-stable serine proteinase inhibitors in the urine of Alzheimer disease subjects.

A comparative study of the levels of acid-stable proteinase inhibitors (kallikrein and trypsin inhibitors) in the urine of healthy and Alzheimer subjects, of both sexes, has been performed. A preliminary characterization of the purified inhibitors indicates that the urinary antitryptic activity is accounted for by the presence of the well known Urinary Trypsin Inhibitor (UTI) while an apparently new molecule appears to be responsible for the antikallikrein activity. The urinary levels of kallikrein inhibitors are very similar in healthy and sick subjects while the levels of trypsin inhibitors appear significatively increased in Alzheimer subjects of both sexes. The data presented here support the hypothesis that unpaired proteolytic processes could be involved in the pathogenesis of Alzheimer's disease and suggest that the levels of urinary acid-stable inhibitors may prove to be useful markers of the disease.

Aprotinin prevents the development of the trauma-induced multiple organ failure in a chronic sheep model.

Trauma-induced multiple organ failure in sheep was prevented by aprotinin therapy. Multiple organ failure was induced in 16 female merino sheep by initial haemorrhagic shock and intramedullary femoral nailing (day 0), and 12 hourly injections of 0.75 micrograms/kg Escherichia coli endotoxin +0.7 ml/kg zymosan-activated plasma (days 1-5). In addition, the aprotinin group (n = 6) received simultaneous injections of 5 mg/kg (35 695 KIU/kg) aprotinin, whereas ten animals did not receive aprotinin and served as the control group (n = 10). Organ functions were monitored for a total of 11 days by measuring haemodynamic, cardio-respiratory and biochemical quantities of blood, urine and epithelial lining fluid. During the subsequent eleven day period, aprotinin induced a significant (p < 0.05) reduction of the pathological changes (development of multiple organ failure) seen in the control group. Thus, aprotinin prevented an alteration of cardiac function (cardiac index for control/aprotinin groups at day 1: 6.5/6.2, and at day 10: 10.47/7.0 1/min x m2), an impairment of lung function (mean pulmonary arterial pressure at day 1: 2.26/1.86, and at day 10: 3.83/2.13 kPa; epithelial lining fluid/plasma ratio of albumin concentrations as a direct marker of lung capillary permeability damage at day 0: 0.18/0.16, and at day 10: 0.45/0.15), a deterioration of liver function (plasma sorbitol dehydrogenase at day 0: 7.9/7.6, and at day 10: 29.6/7.4 U/1), but not of renal function (creatinine clearance at day 1: 91.4/66.1, and at day 10: 53.1/59.2 ml/min). Urinary aprotinin excretion increased up to day 3, then decreased rapidly despite further aprotinin administration. As a non-specific marker of cell damage, plasma lactate dehydrogenase indicated an aprotinin-induced organ protection (day 0: 501/409, and at day 10: 719/329 U/1). The neutrophil count and the measured chemiluminescence of neutrophils from the blood and epithelial lining fluid showed that aprotinin reduced the in vivo neutrophil activation, the alveolar neutrophil invasion, the production of inflammatory mediators, and the production of reactive oxygen metabolites during the passage of the capillary-interstitial-alveolar space by neutrophils.

Antiprotease activity in urine of patients with inflammatory skin disorders.

Polymorphonuclear leukocytes contain well-defined proteolytic enzymes in their azurophilic granules that can be released into tissues during inflammation, producing a localized excess of proteases that causes a protease-antiprotease imbalance with subsequent tissue destruction. The antiproteolytic compounds of the epidermis, such as the protease inhibitors elafin and antileukoprotease, are thought to counteract the proteolytic tissue damage. We investigated the urine of patients suffering from inflammatory skin conditions (e.g., erysipelas, psoriasis) for the presence of urinary antiprotease activities. Purification of elastase-inhibitory activities from pooled urine samples by cation exchange high-performance liquid chromatography and preparative and analytical reverse-phase high-performance liquid chromatography yielded two different types of inhibitors. One was a cationic, acid-stable, and elastase-specific inhibitor of M(r) 6,000 by size-exclusion high-performance liquid chromatography. N-terminal amino acid sequence analysis of the first 28 residues showed identity with elafin, an elastase-specific inhibitor recently isolated from psoriatic scales. The second anti-protease activity was due to two forms of urinary bikunin, the inhibitory subunit of inter-alpha-inhibitor. Both bikunin fragments, with M(r) 4,000 and 16,000, were identified by N-terminal amino acid sequence analysis of the first 10 residues and were characterized by an antiproteolytic profile against human leukocyte elastase, cathepsin G, and trypsin. Urinary protease inhibitors may serve as diagnostic markers of inflammatory diseases.

Levels of skin-derived antileukoproteinase (SKALP)/elafin in serum correlate with disease activity during treatment of severe psoriasis with cyclosporin A.

The epidermal serine proteinase inhibitor SKALP (also known as elafin), directed against human leukocyte elastase and proteinase 3, is strongly induced in suprabasal keratinocytes during inflammation. The presence of SKALP/elafin in urine has been demonstrated for several inflammatory skin disorders, such as psoriasis, erythroderma, and erysipelas. In this study we investigated whether SKALP/elafin levels in serum and urine of psoriatic patients can be used as a marker for disease activity during treatment. Patients with severe chronic disabling psoriasis were treated for 16 weeks with cyclosporin A, which resulted in a marked clinical improvement as measured with the PASI score. SKALP/elafin levels both in serum and urine were determined with an enzyme-linked immunosorbent assay (ELISA). Measurements were performed at the start of the cyclosporin A treatment, and after regular intervals up to 16 weeks. The results indicate that 1) SKALP/elafin determination in serum rather than in urine is the preferred method, because the decrease in serum SKALP levels during therapy is more pronounced and correlated better with the clinical course of the patients; 2) SKALP/elafin levels in serum decreased during cyclosporin A treatment (p < 0.05); and 3) SKALP/elafin levels in serum correlate with the PASI score (p < 0.01). We conclude that SKALP/elafin measurement in serum of patients with severe psoriasis provides a tool for monitoring disease activity.

Vitronectin modulates glycosaminoglycan dependent reactions of protein C inhibitor.

Protein C inhibitor (PCI), a glycosaminoglycan (GAG) dependent serine protease inhibitor, inhibits its target proteases by forming SDS-stable 1:1 complexes. GAGs alter target enzyme specificity of PCI in such a way that e.g. urokinase (uPA) is the preferred target enzyme in the presence of GAGs while in their absence preferentially tissue kallikrein (TK) complexes are formed. The effect of the GAG-binding adhesive glycoprotein vitronectin (Vn) on the GAG-stimulated inhibition of uPA by PCI was studied using an amidolytic assay. In the presence of heparin, Vn protected uPA from inhibition by PCI in a dose-dependent manner with respect to both, Vn- and heparin-concentration. Vn also was active when heparin was replaced by low-molecular weight heparin or heparan sulfate, respectively. In the absence of GAGs, Vn had no effect on the inhibition of uPA by PCI. In a similar system, Vn was far less effective in modifying the inhibitory function of heparin on the inhibition of TK by PCI. When equimolar concentrations of radiolabelled uPA and TK were incubated with PCI in the presence of heparin, only complexes of PCI with uPA were detectable. Addition of Vn reduced this complex formation, whereas, in contrast, complexes of PCI and TK appeared. These results indicate that Vn modulates both, the activity and specificity of PCI and suggest different structural heparin-requirements for the PCI/uPA versus PCI/TK interaction.

Intravenous recombinant secretory leukoprotease inhibitor augments antineutrophil elastase defense.

Secretory leukoprotease inhibitor (SLPI), a 12-kDa serine antiprotease, normally protects the upper airway epithelial surface from attack by neutrophil elastase (NE). In the context that a variety of inflammatory lung diseases are characterized by large neutrophil burdens with resultant high levels of NE in the lung, recombinant SLPI (rSLPI), a molecule identical to natural SLPI, may be an effective means to augment the anti-NE protective screen of the lung. To determine whether intravenous rSLPI will augment respiratory tract and epithelial surface levels of SLPI and anti-NE capacity, rSLPI was administered intravenously to sheep and SLPI levels were quantified in plasma, lung lymph (as a measure of lung interstitial levels), lung epithelial lining fluid (ELF), and urine. rSLPI (1 g) was administered over 10 min, and after 30 min plasma levels of SLPI were 8 microM and decreased with a half-life of 1.8 h. Lymph SLPI levels paralleled the plasma levels: 4 h after infusion the lymph-to-plasma ratio was 0.8. ELF SLPI levels paralleled the lymph levels: 4 h after infusion the ELF-to-lymph ratio was 0.3. Western analysis demonstrated intact SLPI in lymph and ELF, and functional analysis showed increases in lymph and ELF anti-NE capacity that paralleled the levels of SLPI. As might be expected from a protein with a molecular mass of 12 kDa, urine excretion was high, with 20% of the SLPI excreted over 5 h. However, if the rate of infusion was slowed, SLPI excretion decreased significantly, with a 3-h infusion associated with 9% excretion and a 12-h infusion associated with less than 0.2% excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of skin-derived antileukoproteinase (SKALP) in urine of psoriatic patients.

Recently we described a new elastase inhibitor (skin-derived antileukoproteinase, SKALP) that is expressed in psoriatic epidermis and cultured keratinocytes, but is virtually absent in normal skin. In this study we investigated whether SKALP activity could be measured in urine of psoriatic patients and healthy controls. We found that urine of psoriatic patients contained considerable amounts of anti-elastase activity, whereas this activity in urine from normals was significantly lower. The properties of the urinary anti-elastase activity in psoriatic patients were indistinguishable from that of epidermal SKALP. It was found to be a cationic, heat-stable protein with an apparent molecular weight of 11 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and a K(i) of approximately 2 x 10(-11) M. In addition, in Western blotting partially purified inhibitor from urine was found to react with a polyclonal anti-SKALP serum. SKALP in urine was either present in a free form or in a latent form, most likely complexed with elastase. We speculate that SKALP in urine of psoriatic patients is derived from the epidermis, and that it might serve as a marker for disease activity.

Inter-alpha-trypsin inhibitor-related immunoreactivity in human tissues and body fluids.

The distribution of inter-alpha-trypsin inhibitor (ITI) and related inhibitors was investigated in normal human tissues and body fluids by using an enzyme-linked immunosorbent assay (ELISA) and a streptavidin-biotin-peroxidase immunohistochemical technique. ITI-related immunoreactivity was localized in different cell types of various organs, such as liver, kidney, testis, gross intestine, cutis and brain. Specific immunoreactivity was also detected in serum, urine and bronchial mucus. This widespread, but not ubiquitous pattern of localization suggests that, in addition to the well known plasmatic role, ITI and/or ITI-related inhibitors may play a number of different physiological roles in various human tissues.

R-[N-acetyl]eglin c:poly(oxyethylene) conjugates: preparation, plasma persistence, and urinary excretion.

In this paper, we describe the preparation, purification, and characterization of conjugates of R-[N-acetyl]eglin c (Eglin c) with poly(oxyethylene) (POE; Eglin c:POE). The plasma profile and urinary excretion of the conjugates has been determined after iv administration in mice. The modification of Eglin c with POE does not significantly impair the ability of Eglin c to bind elastase as measured by an in vitro assay. In the best example, 79% of theoretical activity was retained by the conjugate. The in vivo results clearly show that the amount of Eglin c:POE in plasma after iv administration is much higher than comparative doses of unconjugated Eglin c. The time course of the plasma concentration of the conjugate matches closely that of the corresponding free polymer. Consequently, we can expect that higher plasma concentration could be achieved, if and when required, by selecting polymers of appropriate size.