Recombinant human interleukin-1alpha increases serum albumin,Gc-globulin,and alpha1-antitrypsin levels in burned mice

The response to thermal injury is a complex physiologic process requiring communication between sites of injury and distant target organs. The liver, one of these target organs, synthesizes a family of secretory proteins, the acute phase proteins, that carries out specific immunoprotective functions. In this study we investigated the effects of daily recombinant human interleukin-1alpha (rhIL-1alpha) administration on the serum levels of negatively regulated, i.e., albumin and Gc-globulin and positively regulated, i.e., alpha1-antitrypsin, acute phase proteins in a murine model of thermal injury. Adult CF-1 female mice underwent a 6.5-seconds, 20% total burn surface area, full thickness steam injury, and received either intraperitoneal rhIL-1alpha (20 microg x kg(-1) x day(-1)) or diluent for 10 days. Seven and 14 days after injury, mice were sacrificed, and serum albumin, Gc-globulin and alpha1-antitrypsin levels were measured by crossed immunoelectrophoresis technique. Thermal injury significantly lowered serum albumin levels, tended to decrease Gc-globulin levels, and increased serum alpha1-antitrypsin levels. Daily rhIL-1alpha administration after burn injury prevented hypoalbuminemia, and increased serum levels of Gc-globulin and alpha1-antitrypsin. IL-1 therapy might be helpful to maintain the homeostasis and immunity of the host after thermal injury.     

Synthesis and secretion of α2-macroglobulin by human hepatocytes in culture

Hepatocytes were isolated by application of the two-step collagenase perfusion technique to pieces of human liver. The cells were incubated in serum-free medium or 10% FCS-medium supplemented with insulin, glucagon and dexamethasone, and kept in culture for more than 2 weeks. Seventy-five per cent of the medium was changed regularly and assayed for alpha 2-macroglobulin (alpha 2-M), pregnancy zone protein, alpha 1-antitrypsin and albumin by means of ELISA. Significant amounts of alpha 2-macroglobulin were present in all cultures. During incubation, alpha 2-M accumulated in the medium and the quantity of alpha 2-M released from the cells by far exceeded protein associated with hepatocytes prior to incubation. In 24 h 10(6) hepatocytes secreted 160.5 +/- 82.2 ng of alpha 2-M (mean +/- SD, n = 5). Cell-associated, as well as secreted alpha 2-M appeared to be on native form, as determined by immunoisolates from lysed cells and culture supernatants. Pregnancy zone protein was only detected in about 50% of the cultures and its rate of secretion was less than 2 ng 24 h-1 per 10(6) cells. In contrast, culture medium contained considerable quantities of alpha 1-antitrypsin and albumin. In 24 h, 10(6) hepatocytes released greater than 2 micrograms alpha 1-antitrypsin and greater than 5 micrograms albumin. The present study suggests the hepatocyte to be of major importance for the synthesis of intravascular alpha 2-M.     

Accumulation of PiZ alpha 1-antitrypsin causes liver damage in transgenic mice.

Circulating alpha 1-antitrypsin is synthesized primarily in the liver and secreted into the bloodstream, where it serves as the major protease inhibitor. The PiZ variant of alpha 1-antitrypsin is associated with decreased levels of the protein in sera as a result of its retention within hepatocytes. Homozygosity for the variant allele predisposes individuals to the development of pulmonary emphysema and an increased risk for liver disease. We and others have previously demonstrated that the normal PiM human alpha 1-antitrypsin gene can be properly expressed in the livers of transgenic mice. The PiZ variant of the human alpha 1-antitrypsin gene was introduced into the germline of mice to determine whether the mutant protein would accumulate in mouse hepatocytes and if such accumulation would result in the development of liver damage in an animal model. As expected, the mutant human protein was abundantly synthesized in the livers of the transgenic animals and accumulated within the rough endoplasmic reticulum of hepatocytes as it does in human patients. PiZ mice developed significantly more liver necrosis and inflammation than PiM transgenic mice or control littermates. The degree of liver damage was correlated with the amount of PiZ alpha 1-antitrypsin accumulated in the liver of the different pedigrees of mice. Although 40% of PiZ mice tested were seropositive for mouse hepatitis virus (MHV), the degree of liver damage was not influenced by the MHV seropositivity; rather, it was related only to the presence of accumulated PiZ protein.     

Variability of plasma proteins according to molecular size. Long-term and short-term intra-individual variation

The intra-individual variations in serum concentrations of alpha 1-antitrypsin, albumin and alpha 2-macroglobulin were determined using high precision analytical methods. The long-term (3 months) variations were 8.2% for alpha 1-antitrypsin and 2.9% for alpha 2-macroglobulin in five males and five females. The coefficients of variation for albumin were 1.5 and 3.4% for males and females, respectively. In males the long-term variations of albumin and alpha 2-macroglobulin were highly correlated. The short-term (2 days) intra-individual variations in six males were 2.5, 3.8 and 3.4% for alpha 1-antitrypsin, albumin and alpha 2-macroglobulin respectively (coefficients of variation). A diurnal variation was found for albumin with maximal concentrations at 18.00 hours. At 6.00 and 10.00 hours the fractional concentrations of alpha 1-antitrypsin and albumin were lower than for alpha 2-macroglobulin. The variations of the three proteins were positively correlated.     

Leucocyte-associated plasma proteins. Association of prealbumin, albumin, orosomucoid, alpha 1-antitrypsin, transferrin and haptoglobin with human lymphocytes, monocytes, granulocytes and a promyelocytic leukaemic cell line (HL-60)

The distribution of prealbumin, albumin, orosomucoid, alpha 1-antitrypsin, haptoglobin and transferrin, including their electrophoretic heterogeneous variants, was studied in isolated lymphocytes, monocytes and granulocytes and in a human promyelocytic cell line (HL-60) by crossed immunoelectrophoresis. Prealbumin, albumin and transferrin were present in lymphocytes, monocytes and granulocytes, whereas the cellular variants of orosomucoid and haptoglobin were present only in granulocytes. alpha 1-Antitrypsin was present in four electrophoretic variants which were differently distributed among the various cell types. Synthesis of alpha 1-antitrypsin by monocytes, granulocytes and HL-60 cells was demonstrated by 14C-leucine incorporation. The six plasma proteins could not be removed from intact cells by incubation with the respective antibodies at 0 degrees C, or iodinated by lactoperoxidase catalysed iodination at 23 degrees C. They were, however, readily solubilized by freeze-lysis of the cells, suggesting an intracellular localization. Compared to their plasma counterparts none of the proteins differed in their hydrophobic properties but the carbohydrate residues of orosomucoid, alpha 1-antitrypsin and haptoglobin were different. The pattern of disappearance of the proteins from the cells during incubation suggested that the localization of albumin and transferrin in relation to the cells differed from that of the other proteins.     

Development of an enzyme-linked immunosorbent assay with a monoclonal antibody prepared against alpha 1-antitrypsin for diagnostic screening of inflammatory disorders.

A monoclonal antibody, designated A2a18b8, of IgG1 class prepared against human alpha 1-antitrypsin, cross-reacts with alpha 1-antitrypsin in the serum of rat and baboon, but not with alpha 1-antitrypsin in serum of rabbit, pig, hamster, guinea pig, dog, or turtle. We used A2a18b8 in an enzyme-linked immunosorbent assay (ELISA) developed for human alpha 1-antitrypsin. Preliminary ELISA screening of 247 serum samples from patients with various inflammatory disorders indicated that the concentration of a specific epitope(s) on alpha 1-antitrypsin recognized by this monoclonal antibody was increased significantly in patients with active systemic lupus erythematosus, mixed connective tissue disease, and rheumatoid arthritis, but not in patients with sclerodermic disorders or Sj?gren's syndrome. Evidently, A2a18b8 has diagnostic value in that it selectively recognizes a specific epitope(s) on alpha 1-antitrypsin that is (are) apparently exposed during selective inflammatory disorders.     

Analysis of inflammatory response in human plasma samples by an automated multicapillary electrophoresis system.

A new automated multicapillary zone electrophoresis instrument with a new high-resolution (HR) buffer (Capillarys with HR buffer) for analysis of human plasma proteins was evaluated. Albumin, alpha(1)-antitrypsin, alpha(1)-acid glycoprotein, haptoglobin, fibrinogen, immunoglobulin (Ig)A, IgG and IgM were determined nephelometrically in 200 patient plasma samples. The same samples were then analyzed on the Capillarys system (Sebia, Paris, France). The albumin concentration from the nephelometric determination was used for quantification of the individual peaks in the capillary electrophoresis (CE) electropherogram. There was strong linear correlation between the nephelometric and electrophoretic determination of alpha(1)-antitrypsin (R(2) = 0.906), alpha(1)-acid glycoprotein (R(2) =0.894) and haptoglobin (R(2) = 0.913). There was also good correlation between the two determinations of gamma-globulins (R(2) = 0.883), while the correlation was weaker for fibrinogen (R(2) = 0.377). The Capillarys instrument is a reliable system for plasma protein analysis, combining the advantages of full automation, good analytical performance and high throughput. The HR buffer in combination with albumin quantification allows the simultaneous quantification of inflammatory markers in plasma samples without the need for nephelometric determination of these proteins.     

An arterio-venous study of serum alpha1-antitrypsin in patients with chronic bronchitis.

The effect of passage through the lung on alpha1-antitrypsin was studied in sixteen patients with chronic obstructive airways disease. The average mixed venous value of alpha1-antitrypsin (106.2% standard serum value) was not different from the the mean arterial value (105.6%). Where individual differences were found there was a similar difference in serum albumin. However, there was a small but significant rise (p less than 0.005) in serum alpha1-antitrypsin "complex" as blood traversed the lung (mixed value = 6.3%; mixed arterial value = 6.8%). The reason is not yet clear.     

Effects of posture on concentrations of serum proteins in healthy adults. Dependence on the molecular size of proteins

The effect of posture on changes in protein composition of serum and its dependence on molecular size has been investigated in forty individuals classified by age and sex. Blood specimens were drawn (A) at 0700 hours with persons still resting in bed after an overnight sleep; (B) at 0815 hours, in sitting position after 1 h of moderate exercise and again (C) at 0915 hours, lying in bed after 1 h of recumbency. Concentrations of alpha 1-antitrypsin, albumin and alpha 2-macroglobulin in serum were determined with high analytical precision. The mean fractional increases of concentrations from lying (A) to sitting (B) were 0.069 for alpha 1-antitrypsin and 0.063 for albumin, but lower, 0.040, for alpha 2-macroglobulin. Thus these increases, being related to molecular size, were not simply caused by posture induced changes of plasma volume. Considering the results of each individual we found a constant difference between the increase of alpha 1-antitrypsin and alpha 2-macroglobulin of about 0.030 for the whole range of increases (i.e. for alpha 1-antitrypsin 0-0.15). In contrast, the effects of recumbency were independent of molecular size. The mean, fractional decreases in concentration induced by changing of position from sitting (B) to lying (C) were approximately 0.065 for all three proteins.     

alpha 2-Macroglobulin in patients with obstructive lung disease, with and without alpha 1-antitrypsin deficiency

Serum alpha 2-macroglobulin concentrations were measured in 178 patients with emphysema and 115 control subjects of similar age and sex distribution. The study group included 59 PI type Z patients with alpha 1-antitrypsin deficiency, five with the rare alpha 1-antitrypsin null genotype (PI Q0 or --), and seven with alpha 1-antitrypsin deficiency of the rare PI types MmaltonZ or MduarteZ. Individuals with all types of alpha 1-antitrypsin deficiency were found to have significantly increased serum concentrations of alpha 2M (p less than 0.001). These increased concentrations were associated with all types of alpha 1-antitrypsin deficiency, not only with the PI type Z. The highest alpha 2-macroglobulin concentrations were found in the PI Q0 patients (5 with emphysema, 2 with no lung disease), and these patients had almost no circulating alpha 1-antitrypsin. Raised concentrations of serum alpha 2-macroglobulin were not due to emphysema: 86 patients with emphysema, of PI type M, and the normal control subjects had similar average concentrations of alpha 2-macroglobulin. One control subject with an average alpha 2-macroglobulin concentration of only 41% of normal was found.     

Nasal lavage as a tool for the assessment of upper-airway inflammation in adults and children

The prevalence of respiratory allergies has increased over the last 20 years, highlighting the need for a simple and noninvasive tool to investigate, in a clinical and epidemiological context, airway-inflammation mechanisms encountered in allergic and inflammatory processes. The nose, as the first region of the respiratory tract to come in contact with airborne pollutants, is easily explored with the use of nasal lavage (NL). We evaluated an NL method for adults and children, along with its reproducibility and capacity to separate different subgroups. NL reproducibility, assessed in 10 healthy, nonsmoking adults on three different occasions, was determined with the use of the intraclass coefficient of correlation for such inflammatory markers as total cell count, albumin, urea, neutrophil elastase, alpha(1)-antitrypsin, interleukin-6, and interleukin-8. Using this NL method, we analyzed nasal markers of 50 healthy adults (smokers and nonsmokers) and 12 healthy children. Our NL method demonstrated high reproducibility with regard to total cell count, albumin, urea, and alpha(1)-antitrypsin (intraclass correlation coefficient > 0.75). Compared with NL results in nonsmokers, NL in heavy smokers revealed significant increased concentrations of total cell counts and interleukin-8 and significant decreased concentrations of interleukin-6. These findings suggest that NL can be used as a tool in the assessment of inflammation because it has the correct reproducibility and can discriminate between heavy smokers and nonsmokers. Moreover, the use of this standardized method in children is feasible.     

Biochemical modifications in a case of analbuminemia (author's transl)]

A new case of analbuminemia is described for a six month old child of Algerian origin. The serum albumin concentration was 64 mg/l and its immunochemical action was identical to that of normal albumin. The system reacted by an increase of the synthesis of globulins. For the subject, the alpha1-antitrypsin, ceruloplasmin, haptoglobin, alpha2-macroglobulin, transferrin and immunoglobulins M contents were three times higher than the standard figures. However, it was possible to show that the presence of free bilirubin independent from proteins could be detected at a concentration of 17 mumol/l.     

Electrophoretic mobility, concentration, and activity of alpha 1-antitrypsin in serum of patients undergoing bone-marrow transplantation

We have observed an electrophoretically abnormal, nonfunctional species of alpha 1-antitrypsin in serum from patients who were receiving bone-marrow transplants for treatment of leukemia or aplastic anemia. Three of four patients in whose serum this protein appeared died soon after; the fourth recovered, and the disappearance of the abnormal alpha 1-antitrypsin paralleled his recovery. This suggests that the inability to maintain functional activity of alpha 1-antitrypsin predisposes patients to life-threatening complications during recovery from bone-marrow transplants.     

Alpha-1-antitrypsin-Pittsburgh. A potent inhibitor of human plasma factor XIa, kallikrein, and factor XIIf.

Alpha-1-antitrypsin-Pittsburgh is a human variant that resulted from a point mutation in the plasma protease inhibitor, alpha 1-antitrypsin (358 Met----Arg). This defect in the alpha 1-antitrypsin molecule causes it to have greatly diminished anti-elastase activity but markedly increased antithrombin activity. In this report, we demonstrate that this variant protein also has greatly increased inhibitory activity towards the arginine-specific enzymes of the contact system of plasma proteolysis (Factor XIa, kallikrein, and Factor XIIf), in contrast to normal alpha 1-antitrypsin, which has modest to no inhibitory activity towards these enzymes. We determined the second-order-inactivation rate constant (k') of purified, human Factor XIa by purified alpha 1-antitrypsin-Pittsburgh and found it to be 5.1 X 10(5) M-1 s-1 (23 degrees C), which is a 7,700-fold increase over the k' for Factor XIa by its major inhibitor, normal purified alpha 1-antitrypsin (i.e., 6.6 X 10(1) M-1 s-1). Human plasma kallikrein, which is poorly inhibited by alpha 1-antitrypsin (k' = 4.2 M-1 s-1), exhibited a k' for alpha 1-antitrypsin-Pittsburgh of 8.9 X 10(4) M-1 s-1 (a 21,000-fold increase), making it a more efficient inhibitor than either of the naturally occurring major inhibitors of kallikrein (C-1-inhibitor and alpha 2-macroglobulin). Factor XIIf, which is not inhibited by normal alpha 1-antitrypsin, displayed a k' for alpha 1-antitrypsin-Pittsburgh of 2.5 X 10(4) M-1 s-1. This enhanced inhibitory activity is similar to the effect of alpha 1-antitrypsin-Pittsburgh that has been reported for thrombin. In addition to its potential as an anticoagulant, this recently cloned protein may prove to be clinically valuable in the management of septic shock, hereditary angioedema, or other syndromes involving activation of the surface-mediated plasma proteolytic system.     

Sequential changes in the concentration of specific serum proteins during typhoid fever infection in man.

An automated immunoprecipitin system has been utilized to quantitate the concentration of 10 specific proteins in the plasma of man. Values obtained by this technique are in agreement with the published concentrations for these specific plasma proteins. This technique was utilized to determine the sequential change s in 10 individual plasma proteins of volunteers exposed to Salmonella typhi. In those volunteers who developed typical typhoid fever, plasma concentrations of the acute phase proteins, alpha1-acid glycoprotein, alpha1-antitrypsin, and haptoglobin, as well as C3 complement were significantly increased with the onset of febrile illness. In contrast, the concentration of plasma albumin and tranferrin were depressed while plasma IgM became elevated during early convalescence from this infection. No significant changes were observed in the plasma concentrations of alpha2-macroglobulin, IgG, or IgA. In the exposed volunteers who did not become ill, the only significant change was a brief depression of alpha1-antitrypsin. During typhoid fever the patterns of change for individual plasma acute-phase globulins were different from those reported for patients with hepatitis, myocaridal infarction, or surgery.     

Measles antibodies, anti-proteinase and plasminogen distribution in serum and plasma from patients affected with multiple sclerosis and patients affected with non-neurological diseases

Total protein content, alpha 1-antitrypsin, alpha 2-macroglobulin and plasminogen levels and measles antibody titers were determined in serum and plasma from patients affected with multiple sclerosis and patients affected with non-neurological diseases. The results were compared with those from a control group of healthy donors. Both multiple sclerosis patients and patients affected with non-neurological diseases differed from controls for the following parameters: total protein, plasminogen and measles antibody activity. However, when studied longitudinally the different parameters were not altered to the same degree in multiple sclerosis and non-neurological diseases, a fact which is translated in the difference of significance levels. Individual plasminogen values were very often higher in non-neurological diseases than in multiple sclerosis, whereas for increased measles antibody titers it was the reverse. Also, there were no notable changes in alpha 1-antitrypsin and alpha 2-macroglobulin values in multiple sclerosis, whereas in some non-neurological disease patients particularly high alpha 1-antitrypsin and alpha 2-macroglobulin values were observed. In the multiple sclerosis patients, no correlations existed between the duration of the disease and disturbed biochemical parameters, or between the disturbed parameters themselves.     

Electrophoretic variation of serum alpha1-antitrypsin treated with neuraminidase.

Purified or crude sera of several different phenotypes of alpha1-antitrypsin were treated with neuraminidase. The mobility of the alpha1-antitrypsin zone was delayed on starch gel electrophoresis following incubation. Treated, M-type alpha1-antitrypsin migrated in a manner similar to the untreated S type. Treated S migrated in a similar way to the untreated Z, and treated Z did so more slowly or cathodally. After exhaustive treatment, alpha1-acidglycoprotein bands appeared near the usual M or F positions. Treated samples still possessed the antigenic activity of alpha1-antitrypsin. The concentration and trypsin-inhibitory capacity were not changed by treatment. Several evaluations and considerations are discussed concerning the metabolism of some types of alpha1-antitrypsin.     

The role of macrophage inflammatory protein 1 alpha in Schistosoma mansoni egg-induced granulomatous inflammation

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is a 6-8-kD, lipopolysaccharide-inducible monocyte and neutrophil chemotactic protein that may be important in acute and chronic inflammation. The present study determined the sequential production, source, and in vivo contribution of murine MIP-1 alpha in synchronized Schistosoma mansoni egg pulmonary granuloma formation. Granulomas were examined under conditions of primary, secondary vigorous, and secondary immunomodulated immunity. Secreted MIP-1 alpha was measured in 24-h supernatants from intact granulomas (700/ml) cultured with or without soluble egg antigen (SEA). Primary granulomas isolated from naive mice over a 16-d period showed low spontaneous MIP-1 alpha production (< 1 ng/ml). However, when primary granulomas were challenged with SEA, significant MIP-1 alpha production was observed beginning at day 4 and peaking at day 16. Intact vigorous (isolated from 8-wk-infected mice) and modulated (isolated from 20-wk-infected mice) secondary pulmonary granulomas demonstrated comparable spontaneous MIP-1 alpha production. Addition of SEA to vigorous stage granulomas augmented expression of MIP-1 alpha at all time points, whereas stimulated modulated stage granulomas did not increase production. The latter observation is likely related to endogenous immunoregulatory mechanisms reported for modulated stage animals. Immunohistochemical localization of MIP-1 alpha in granuloma sections and cytocentrifuge preparations from vigorous lesions localized MIP-1 alpha production to macrophages within granulomas. Treatment of mice with rabbit anti-mouse MIP-1 alpha antibodies significantly decreased 8-d primary granuloma formation (> 40%) when compared with control mice. Anti-MIP-1 alpha sera also decreased vigorous (> 20%), but not modulated granuloma formation. These findings demonstrate that MIP-1 alpha contributes to cellular recruitment during schistosome egg granuloma formation.     

Plasma alpha 1-antichymotrypsin in liver disease

We studied 229 patients with biopsy verified liver disease and compared the plasma levels of alpha 1-antichymotrypsin and alpha 1-antitrypsin. We found a significant overall correlation between alpha 1-antichymotrypsin and alpha 1-antitrypsin levels (r = 0.50, p less than 0.001). The strongest correlations were found in patients with chronic active hepatitis (r = 0.76, p less than 0.0001) and alcohol hepatitis (r = 0.60, p less than 0.001). Several clinical subgroups lacked correlation. Unexpectedly high alpha 1-antichymotrypsin values were found in patients with venous congestion. We also used the alpha 1-antichymotrypsin/alpha 1-antitrypsin ratio as a tool to identify PiZ carriers (intermediate alpha 1-antitrypsin-deficiency, PiZ). The sensitivity and predictive values were low and did not exceed that obtained by the simple use of an isolated alpha 1-antitrypsin determination. A small subgroup with low alpha 1-antichymotrypsin/alpha 1-antitrypsin ratio included patients with chronic active hepatitis of unknown etiology. Hypo-alpha 1-antichymotrypsinemia may be secondary to the liver disease per se or be an expression of an abnormal genetic trait.     

Glycated proteins in serum: effect of their relative proportions on their alkaline reducing activity in the fructosamine test

The fructosamine test is considered clinically useful for assessing short-term integrated control of blood glucose, but there are few published data to support this hypothesis. We fractionated glycated and nonglycated proteins by affinity chromatography on phenylboronate columns and, with specific immunochemical methods, determined in the eluted fractions the following proteins, selected according to their biological half-lives and relative concentrations in serum: albumin, IgA, IgG, IgM, apolipoprotein B, haptoglobin, transferrin, alpha 1-antitrypsin, and alpha 2-macroglobulin. We found the following correlations between fructosamine (mmol/L) and, respectively, glycated albumin, IgG, and (albumin + IgG) (each in grams per liter): r = 0.901, 0.702, 0.878. IgM had the highest percentage of glycated molecules (range 11.1-37.5%, mean 22.4%), haptoglobin and alpha 1-antitrypsin the least. This result was almost independent of the proteins' molecular masses and fractional catabolic rate. Albumin evidently contributes most to results of the fructosamine test, confirming conclusions obtained in different ways by others.