In vitro interaction of porcine serum and colostrum protease inhibitors with pancreatic trypsin, chymotrypsin and elastase.

The partition of 125I-labelled pancreatic trypsin, chymotrypsin and elastase between the inhibitors, alpha 2-macroglobulin f and s, alpha 1-protease inhibitor, alpha 2-antitrypsin, inter-alpha-trypsin inhibitor and the specific sow colostrum protease inhibitor, was studied in vitro by gradually increasing the concentration of these proteases in blood serum from adult and newborn pigs. As revealed by immunoelectrophoresis in combination with autoradiography, differences were noted in the abilities of the various protease inhibitors to interact with and to form complexes with the three proteases, resulting in changes in location, height and numbers of precipitates. Among the serum inhibitors, alpha 2-macroglobulins showed the highest relative affinity to all three proteases, while alpha 1-protease inhibitor showed a high relative affinity only for chymotrypsin. Serum alpha 2-antitrypsin complexed only with trypsin, with a low relative affinity. alpha 2-Antitrypsin also interacted with chymotrypsin and elastase, but without forming complexes. When complexes of sow colostrum protease inhibitor and trypsin were added to the serum from neonatal pigs, these complexes remained stable. The results obtained from these in vitro studies, indicating differences in the relative affinities of the inhibitors to the various proteases, give some information about the role of the inhibitors in vivo, both in adult and in neonatal pigs.

Lung to blood passage of different-sized molecules during lung inflammation in the rat.

The passage of different-sized marker molecules over the lower respiratory tract into the blood circulation during pulmonary inflammation induced by dextran, endotoxin [i.e., lipopolysaccharide from Escherichia coli (LPS)], or ferritin was assessed in the rat. Bovine immunoglobulin G (BIgG, mol wt = 150,000 Da), bovine serum albumin (BSA, mol wt = 67,000 Da), and the nonapeptide 1-deaminocysteine-8-D-arginine vasopressin (dDAVP, mol wt = 1,067 Da) were used as permeability markers after intratracheal instillation. The pathophysiological indexes of a proceeding lung inflammation were increased total cell number, changed leukocyte proportions and increased total protein content obtained in bronchoalveolar lavage, and lung edema formation shown as an increased lung wet-dry weight difference. Intratracheal instillation of dextran induced a moderate neutrophil invasion into the lungs but had no effect on the passage of the different markers over the lungs (BIgG 1.8 +/- 0.6%, BSA 3.5 +/- 1.2%, dDAVP 26.1 +/- 20.7%) compared with control rats instilled with the markers alone (1.8 +/- 0.4%, 4.1 +/- 1.3%, 20.0 +/- 3.8%, respectively). Endotoxin administration resulted in markedly higher lavage cell counts and lung edema concomitantly with an increased lung passage of the markers (3.2 +/- 0.9%, 22.0 +/- 6.1%, 33.3 +/- 12.0%, respectively; P less than 0.01-P less than 0.001). The highest marker passage was obtained when the inflammation was most severe, i.e., after ferritin administration (17.6 +/- 2.3%, 60.0 +/- 6.7%, 41.6 +/- 6.9%, respectively; P less than 0.001), which resulted in markedly elevated lavage cell numbers and protein content as well as edema formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar epithelial clearance of protein.

Substantial progress has been made in understanding the rate, the pathways, and the mechanisms regulating alveolar protein removal from the uninjured lung. Whole animal studies and cellular studies have demonstrated that the majority of alveolar epithelial protein clearance occurs by passive nondegradative diffusional pathways. Some evidence, however, has been recently presented that alveolar epithelial cells express an albumin-binding receptor as well as a polymeric immunoglobulin receptor, both of which might be important for alveolar epithelial clearance of protein. However, the contribution of these receptors requires further studies. Little is known about alveolar clearance of protein during pathological conditions; further studies are required to determine the roles of the different cell types in the lung for removal of protein from the alveolar spaces of the lung. Alveolar macrophages are likely to play an important role in the degradation and removal of insoluble protein from the distal air spaces after acute lung injury. In conclusion, the present data suggest that most proteins and peptides deposited on the epithelial surfaces in the distal air spaces are cleared as intact molecules, predominantly via paracellular routes. The contribution of pinocytic processes appear to be of minor importance for translocation of bulk quantities of proteins or peptides across the alveolar epithelium.

Development of porcine pancreatic hydrolases and their isoenzymes from the fetal period to adulthood.

The appearance and activity of various porcine pancreatic hydrolases were studied during fetal and postnatal development. Quantitatively, the enzyme activities in activated pancreas homogenates were low but increased during the second half of the fetal period, using the substrates Bz-Arg-pNA for measuring anodal and cathodal trypsin, Suc-Phe-pNA (chymotrypsin A and C, and elastase II) and Suc-(Ala)3-pNA (elastase I and protease E). Postnatally, after an initial decrease during the first week, the enzyme activities increased markedly, especially from 10-14 weeks to 6 months. The individual hydrolases were identified after electrophoretic separation in agarose gel and staining with various substrates either directly in the gel or after transfer to nitrocellulose membranes (enzymoblotting). During the fetal period, chymotrypsin A and B, elastase II, carboxypeptidase A, and amylase appeared at approximately 65 days and anodal trypsin, at approximately 76 days. After birth, new proteinases appeared after the first week including chymotrypsin C, cathodal trypsin, and protease E, whereas elastase I was found from 5 weeks after birth. Concomitantly, unidentified "fetal proteinase(s)" with caseinolytic, Ac-Phe-beta NE and CBZ-Ala-beta NE activities began to diminish and disappeared 10-14 weeks after birth. This study showed a marked increase in the overall pancreatic enzyme activities, as well as an age-dependent expression of the variety of pancreatic hydrolases during porcine ontogeny.

Stimulation of endocrine, but not exocrine, pancreatic secretion during 2-deoxy-D-glucose-induced neuroglycopenia in the conscious pig.

The effects of autonomic nervous activation, initiated by 2-deoxy-D-glucose (2-DG)-induced neuroglycopenia, or endocrine and exocrine pancreatic secretion were investigated in the conscious pig. Pigs were surgically fitted with permanent pancreatic duct and duodenal reentrant cannulas, allowing long-term sampling of pancreatic juice, and a jugular vein catheter for blood sampling and infusion of 2-DG. 2-DG was administered as a 5-min intravenous infusion at three dose levels to conscious pigs. 2-DG (400 mg/kg) was found to elevate plasma glucagon and insulin levels (p < 0.01). In contrast, exocrine pancreatic secretion, measured as volume, total protein output, and output of trypsin activity was not affected by 2-DG at the dose levels of 75, 200, and 400 mg/kg. Secretin (440 pmol/kg/h), however, stimulated pancreatic exocrine output of fluid (p < 0.01), protein (p < 0.01), and trypsin (p < 0.05). It is concluded that autonomic nervous activation by 2-DG-induced neuroglycopenia, in the conscious pig under basal conditions, elevates the plasma levels of glucagon and insulin but does not affect exocrine pancreatic secretion. 2-DG-induced neuroglycopenia is, thus, a suitable model for studying autonomic neural influences on the porcine endocrine pancreas.

Functional structure of the glomerular filtration barrier and the proximal tubuli in the developing foetal and neonatal pig kidney.

1. The detailed anatomy of the structures forming the glomerular filtration barrier and the proximal tubules was studied during development of foetal and neonatal pigs. 2. In the glomerular filtration barrier during ontogenetic development an increased number of fenestrae was noted in the endothelium. The condensation decreased in the glomerular basement membrane and an increase in the widths of the pedicles was observed. 3. The proximal tubular cells revealed a highly specialised organisation with an enlarged cell surface (microvilli) towards the lumen. In the apical part of the cell a zone containing vesicles probably pinched off from the base of the microvilli was observed. At the base of the cell elongated mitochondria and electron dense bodies were found. 4. An increase in the number of vesicles was observed in the proximal tubular cells from piglets receiving colostrum and exhibiting proteinuria. Judging from these results it seems probable that the nephrons are able to function already in the metanephros of the pig foetuses of 4 cm CR-length. 5. The results imply that there was no structural evidence of an immature glomerular filtration barrier being the cause of neonatal proteinuria. On the other hand an enhanced involvement of the cells of the proximal tubules was noted and this is interpreted to be due to the absorption of proteins from the primary urine after onset of colostrum ingestion by the newborn piglet.

Mortality and morbidity among patients who undergo combined valve and coronary artery bypass surgery: early and late results.

AIM: To describe mortality and morbidity early and late after combined valve surgery and coronary artery bypass grafting (CABG) as compared with CABG alone. PATIENTS AND METHODS: All patients from western Sweden in whom CABG in combination with valve surgery or CABG alone was carried out in 1988-1991. RESULTS: Among 2116 patients who underwent CABG, 35 (2%) had this combined with mitral valve surgery and 134 (6%) had this combined with aortic valve surgery, whereas the remaining 92% underwent CABG alone. Patients who underwent combined valve surgery and CABG were older, included more women and had a higher prevalence of previous congestive heart failure and renal dysfunction but on the other hand a less severe coronary artery disease. Among patients who underwent mitral valve surgery in combination with CABG the mortality over the subsequent 5 years was 45%). The corresponding figure for patients who underwent aortic valve surgery in combination with CABG was 24%. Both were higher than for CABG alone (14%; P < 0.0001 and P = 0.003, respectively). In a stepwise multiple regression model mitral valve surgery in combination with CABG was found to be an independent significant predictor for death but aortic valve surgery in combination with CABG was not. Among patients who underwent mitral valve surgery in combination with CABG and were discharged alive from hospital 77% were rehospitalized during the 2 years following the operation as compared with 48% among patients who underwent aortic valve surgery in combination with CABG and 43% among patients with CABG alone. Multiple regression identified mitral valve surgery in combination with CABG as a significant independent predictor for rehospitalization but not aortic valve plus CABG. CONCLUSION: Among patients who either underwent CABG in combination with mitral valve surgery or aortic valve surgery or CABG alone, mitral valve surgery in combination with CABG was independently associated with death and rehospitalization, but the combination of aortic valve surgery and CABG was not.

Intestinal macromolecular transmission in underprivileged and unaffected newborn pigs: implication for survival of underprivileged pigs.

Passive immunisation of the newborn pig depends upon intestinal macromolecular transmission to the blood, which ceases within 18 to 36 hours after birth in fed pigs. Macromolecular transmission in newborn, unsuckled pigs was tested by gavage feeding bovine serum albumin and fluorescein isothiocyanate-labelled dextran (molecular weight 70 kDa) together with sow colostrum. Serum levels measured four hours after feeding showed that underweight pigs under 1.0 kg, both apparently normal and weak, had a greater capacity for transmission than did apparently normal pigs over 1.0 kg (unaffected). Weak pigs 1.0 kg, or more, had a transmission equivalent to that of unaffected pigs, indicating that they may be otherwise normal animals affected by birth. Splayleg pigs and their clinically unaffected littermates also showed a greater capacity for transmission than unaffected pigs; splayleg apparently involves the entire litter, although the expression of the syndrome varies. The capacity for transmission in the unaffected pig increased with litter size and decreased with an increase in weight and age after birth. In addition, it was shown that transmission was greater in pigs with higher glucose, insulin and alpha-fetoprotein concentrations and lower haemoglobin, cortisol and alpha 2-macroglobulin f concentrations at birth. It was concluded that immature pigs have a greater capacity for intestinal macromolecular transmission at birth than unaffected pigs over 1.0 kg. Since the capacity for macromolecular transmission decreases with age, the probability of their survival depends very much on the husbandry practices in the herd.

The pattern of the circadian rhythm of pancreatic secretion in fed pigs.

The pattern of the circadian rhythm of pancreatic secretion was studied in four 6- to 7-wk-old intact male pigs that were kept in metabolic cages under 12 h light:12 h dark cycles and fed three times a day at 0800, 1500, and 2200. Three 24-h collections of pancreatic juice and blood were begun at 0800 every 2nd day during 5 d. A biphasic pattern of the exocrine pancreas was detected. Secretions during the first phase (postprandial peak) contained large amounts of protein and enzymes, and secretions appeared immediately after feed ingestion. Secretions during the second phase (between meals) had lesser amounts (P < .001) of protein and enzymes, but were of longer duration (P < .001). The output of protein and trypsin activity were generally correlated (P < .001) with each other, but the correlation of either with the volume outflow were small. However, these variables were correlated (P < .01) with the volume outflow at night during the second phase. Plasma concentrations of insulin and glucose increased after each meal, but the increase was significant (P < .05) only for insulin. Insulin and glucose concentrations were correlated (P < .01) with each other after the meals at 0800 and 1500. The results indicated a biphasic pattern of the exocrine pancreas and a monophasic pattern of the endocrine pancreas in pigs. The regulation of these patterns seems to vary during the 24-h period. Furthermore, feed enhanced secretion, but it did not seem to be the only factor controlling the circadian pattern.

Blood serum characteristics of newborn pigs: comparison of unaffected pigs with pigs belonging to five mortality groups.

The blood serum levels of glucose, hemoglobin, insulin, cortisol, albumin, alpha-fetoprotein, alpha 2-macroglobulin f and s, alpha 2-antitrypsin inhibitor and alpha 1-protease inhibitor were determined at birth in 5 clinically and morphologically identified mortality groups of pigs. These were compared with the levels observed in unaffected, apparently normal newborn unsuckled pigs. The blood serum profile of the pigs in the stillborn intra partum, weak, splayleg and trauma groups, respectively, as well as that of clinically normal splayleg littermates, differed significantly from that of the unaffected pigs. This was especially true for the levels of hemoglobin and the two macroglobulins. The importance of placental insufficiency causing chronic episodes of hypoxia which ultimately lead to a disturbance in organ development in the etiology of the mortality groups is discussed.

Lipoprotein and lipid profiles in the blood serum of the fetal, neonatal and adult pig.

As revealed by agarose gel electrophoresis and immunoassay, followed by staining with Sudan black, lipoproteins occur in the alpha-, pre-beta- and beta-regions of the serum of fetal, newborn, neonatal, juvenile and adult pigs. These lipoproteins undergo characteristic quantitative changes during development. After the onset of colostrum ingestion, a lipid-stainable zone appeared in the gamma-region of piglet's serum and sow colostrum/milk. The levels of total serum lipids, cholesterol, high and low density lipoproteins were low at birth in comparison to those of the adult pig, but increased to higher levels after the onset of colostrum and milk ingestion. After 3-8 weeks, the total lipids, cholesterol and low density lipoprotein values had decreased to adult level but the high density lipoprotein values remained high in comparison to the adult pig. It is discussed that the changes found in lipoprotein and lipid patterns in serum of the developing pig reflect changes in cellular metabolism and nutritional needs of the organism. It is suggested that the perinatal pig might be a useful model for studying the development of atherogenic diseases in man.

Enzymoblotting: a method for localizing proteinases and their zymogens using para-nitroanilide substrates after agarose gel electrophoresis and transfer to nitrocellulose.

A method--enzymoblotting--was developed for localizing various enzymes after electrophoretic separation, transfer to nitrocellulose, and incubation with specific substrates. As an application, the proteinases porcine trypsin (EC 3.4.21.4), bovine chymotrypsin (EC 3.4.21.1), porcine elastase (EC 3.4.22.11), and their zymogen forms from porcine pancreas homogenate were analyzed utilizing specific p-nitroanilide substrates. After agarose gel electrophoresis, transfer of the separated proteinases to a nitrocellulose membrane was performed by capillary diffusion for 30 min. After air-drying of the nitrocellulose membrane, it was incubated in the appropriate substrate solution for 60 min. N-alpha-Benzoyl-DL-arginine-para-nitroanilide HCl was used as a substrate for trypsin, N-benzoyl-L-tyrosine-para-nitroanilide and succinyl-L-phenylalanine-para-nitroanilide for chymotrypsin, and N-succinyl-L-alanyl-L-alanyl-L-alanine-para-nitroanilide for elastase. p-Nitroaniline, the product thus obtained, was diazotized with N-(1-naphthyl)ethylenediamine to a red azo dye, visible at the site of the proteinases on the nitrocellulose membrane. The results could be preserved at -18 degrees C. Zymogen forms of the pancreas proteinases were detected in a similar manner. They were converted to active proteinases in situ on the nitrocellulose membrane after preincubating the nitrocellulose membrane in the activation enzymes enteropeptidase or trypsin.

Identification and characterization of eight porcine pancreatic proteinases, carboxypeptidase A and amylase after electrophoretic separation using specific substrates.

Porcine pancreatic hydrolases in juice and homogenate surveyed by electrophoretic separation in agarose gel, at pH 8.6 and subsequently characterized using substrates of various specificity, either directly in the gel or after transfer to nitrocellulose (enzymoblotting) showed: Anodal and cathodal trypsin with Bz-Arg-pNA. Chymotrypsin A, B, and C with similar, but not identical, activities to Suc-Ala-Ala-Pro-Phe-pNA, Bz-Tyr-pNA, Suc-Phe-pNA and Ac-Phe-beta NE and with differences in their molecular weights and electrophoretical charges. Elastase I and protease E with Suc-(Ala)3-pNA and MeO-Suc-Ala-Ala-Pro-Val-pNA and elastase I also with elastin. Elastase II with the chymotrypsin substrates and with elastin. Carboxypeptidase A with CN-Phe. Amylase with blue starch polymer.

Immuno-cross-reactivity between alpha-macroglobulins from pig, dog, rat and man including human pregnancy-associated alpha 2-glycoprotein.

The antigenic relationships between the alpha-macroglobulins in human blood plasma and in serum from pig, dog and rat were investigated using ordinary immunoelectrophoresis and line immunoelectrophoresis. The results imply that these alpha-macroglobulins are more or less antigenically related. This is true also for human pregnancy-associated alpha 2-glycoprotein which is the only alpha-macroglobulin without reported proteinase-inhibiting properties. Among the alpha-macroglobulins a reciprocal cross-reactivity was observed for eight pairs, indicating a close structural relationship.

Intestinal absorption of proteins by the neonatal piglet fed on sow's colostrum with either natural or experimentally eliminated trypsin-inhibiting activity.

the intestinal absorption of total protein, IgG, albumin, beta-lactoglobulin, SCTI and trypsin was studied in newborn unsuckled piglets by feeding them either on normal colostrum or trypsin-inhibition-free colostrum. The results indicate a correlation between the molecular weights of the fed proteins and the time at which they reach their maximum concentration in the blood. Feeding of inhibition-free colostrum resulted in a significantly lower absorption. Evidently the trypsin inhibitors of sow's colostrum cause an increased efficiency in the absorption of undegraded colostrum proteins. However, as large quantities of proteins were absorbed even when inhibition-free colostrum was used for feeding, other factors must also be involved in the regulation of intestinal protein absorption.

Effects of systemic and local immunization on alveolar epithelial permeability to protein in the rat.

This study aimed to characterize the effect on alveolar epithelial permeability to protein as a result of immunization when animals were reexposed to antigen. Antigen (bovine serum albumin [BSA]) and a related bystander protein (human serum albumin [HSA]) were intratracheally instilled into lungs of immunized rats and their passages across the alveolar epithelium were measured as serum levels 16 h after instillation. Nonimmunized control rats showed similar passages of both BSA and HSA. In rats intradermally immunized against BSA, BSA was undetectable in serum, whereas serum levels of HSA were markedly increased compared with those in control rats. In rats immunized with BSA intratracheally, serum levels of both BSA and HSA were unchanged compared with those in control rats. Serum titers of specific IgG antibodies (anti-BSA) were measured and were higher in intradermally immunized animals than in intrapulmonary immunized animals, whereas no anti-BSA antibodies were detected in nonimmunized control rats. Anti-BSA antibodies were detected only in lavage fluid from intradermally immunized rats. These findings suggest that presence of specific antibodies locally in the lungs may increase alveolar epithelial permeability to protein. This finding may have clinical implications, e.g., for sensitive asthmatics, since increased nonspecific permeability caused by local immune-related inflammation may result in further allergies.

Synthesis of alpha-fetoprotein, albumin and total serum protein in neonatal pigs.

Studies on the incorporation of 3H-leucine established that synthesis of alpha-fetoprotein (AFP) does occur in the neonatal pig. This synthesis probably accounts for the increase in AFP concentration from 0.46 mg/ml on the day after birth to the maximum value of 1.02 mg/ml found 5 days after birth. After the 5th day, the concentration decreases successively and AFP cannot be detected by electroimmunoassay in the sera of 8-week-old pigs. During the first 3 weeks of extrauterine life, the albumin concentration in serum increases from 3 mg/ml to approximately 30 mg/ml due mainly to increased synthesis of albumin. The concentration of AFP in the fetal pig serum decreases from 3 mg/ml at 6.5 cm crown-to-rump (CR) length to 0.8 mg/ml at 28 cm CR length, whereas the albumin concentration increases from 0.2 mg/ml at 6.5 cm CR length to 1.0 mg/ml at 28 cm CR length. The total serum protein content in the fetus is approximately 20 mg/ml irrespective of gestational age. During the first day after birth there is a marked increase in both the synthesis and the concentration of total serum proteins. The concentration rises to 70 mg/ml during the first day and then slowly declines to 65 mg/ml during the 3 weeks following birth.